Vol. 22 (2017)



DOI 10.1186/s11658-016-0030-0 Volume 22 (2017)
Title AVOIDING THE SIDE EFFECTS OF ELECTRIC CURRENT PULSE APPLICATION TO ELECTROPORATED CELLS IN DISPOSABLE SMALL VOLUME CUVETTES ASSURES GOOD CELL SURVIVAL
Authors Maciej Grys, Zbigniew Madeja* and Włodzimierz Korohoda*
Abstract Background: The harmful side effects of electroporation to cells due to local changes in pH, the appearance of toxic electrode products, temperature increase, and the heterogeneity of the electric field acting on cells in the cuvettes used for electroporation were observed and discussed in several laboratories. If cells are subjected to weak electric fields for prolonged periods, for example in experiments on cell electrophoresis or galvanotaxis the same effects are seen. In these experiments investigators managed to reduce or eliminate the harmful side effects of electric current application.
Methods: For the experiments, disposable 20 μl cuvettes with two walls made of dialysis membranes were constructed and placed in a locally focused electric field at a considerable distance from the electrodes. Cuvettes were mounted into an apparatus for horizontal electrophoresis and the cells were subjected to direct current electric field (dcEF) pulses from a commercial pulse generator of exponentially declining pulses and from a custom-made generator of double and single rectangular pulses.
Results: More than 80% of the electroporated cells survived the dcEF pulses in both systems. Side effects related to electrodes were eliminated in both the flow through the dcEF and in the disposable cuvettes placed in the focused dcEFs. With a disposable cuvette system, we also confirmed the sensitization of cells to a dcEF using procaine by observing the loading of AT2 cells with calceine and using a square pulse generator, applying 50 ms single rectangular pulses.
Conclusions: We suggest that the same methods of avoiding the side effects of electric current pulse application as in cell electrophoresis and galvanotaxis should also be used for electroporation. This conclusion was confirmed in our electroporation experiments performed in conditions assuring survival of over 80% of the electroporated cells. If the amplitude, duration, and shape of the dcEF pulse are known, then electroporation does not depend on the type of pulse generator. This knowledge of the characteristics of the pulse assures reproducibility of electroporation experiments using different equipment.
Keywords Avoiding side effects of electric current pulses, Disposable cuvettes, Reversible electroporation, Fluorescent dyes, Cell viability, Flow through electric field, Direct current electric field, Focused electric field
Address and Contact Information Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Cracow, Poland
* Corresponding author: z.madeja@uj.edu.pl; w.korohoda@uj.edu.pl
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DOI 10.1186/s11658-016-0032-y Volume 22 (2017)
Title MITF AND PU.1 INHIBIT ADIPOGENESIS OF OVINE PRIMARY PREADIPOCYTES BY RESTRAINING C/EBPβ
Authors ChongMei Ruan1†, Xiu Li2†, JunJie Hu1, Yong Zhang1 and XingXu Zhao1*
Abstract Background: PU box-binding protein (PU.1) is a master gene of hematopoietic lineage and an important specific transcription factor in osteoclast lineage. There is proof of its expression in adipose tissue, and it is known to significantly and negatively affect adipogenesis. However, it is unclear whether there are any other molecules involved in this process.
Methods: We wished to explore the effect of PU.1’s co-activator microphthalmia-associated transcription factor (MITF) on the adipogenic differentiation of ovine primary preadipocytes. The expression vectors pcDNA-MITF and pcDNA-PU.1, and MITF siRNA and PU.1 siRNA were transfected or co-transfected into ovine tail primary preadipocytes. Real-time PCR and western blot analysis were applied to investigate the expression levels of PU.1 and MITF. The morphologic changes in the cells were observed under a microscope at a magnification of × 200 after staining with Oil Red O. The triglyceride (TG) content in cells was also determined after transfection.
Results: MITF and its co-activator PU.1 synergistically exhibited an opposite expression pattern to that of CCAAT-enhancer-binding protein-β (C/EBPβ) during adipogenic differentiation of ovine primary preadipocytes. Before induction of differentiation, overexpression of MITF or PU.1 inhibited the expression of C/EBPβ and adipogenesis in the cells; and knockdown of MITF or PU.1 promoted the expression of C/EBPβ and adipogenesis in the cells. The inhibitory or promotive effect was enhanced when MITF and PU.1 were co-overexpressed or co-silenced. However, when MITF and/or PU.1 were overexpressed after day 2 of differentiation, no changes in adipogenesis of the cells were observed. Conclusions: MITF and its co-activator PU.1 inhibited adipogenesis of ovine primary preadipocytes by restraining C/EBPβ.
Keywords Adipogenesis, Lineage-specific transcription factor, Microphthalmia-associated transcription factor, PU box-binding protein, CCAAT-enhancer-binding protein-β
Address and Contact Information 1College of Veterinary Medicine, Gansu Agriculture University, Lanzhou 730070, China.
2College of Animal Science and Technology, Anhui Agriculture University, Hefei 230036, China.
* Corresponding author: zhaoxx@gsau.edu.cn
Equal contributors
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DOI 10.1186/s11658-017-0033-5 Volume 22 (2017)
Title MicroRNA-210 INDUCES ENDOTHELIAL CELL APOPTOSIS BY DIRECTLY TARGETING PDK1 IN THE SETTING OF ATHEROSCLEROSIS
Authors Ying Li1,2, Chunyan Yang1, Lili Zhang3 and Ping Yang1*
Abstract Background: Atherosclerosis is a chronically inflammatory disease and one of the leading causes of deaths worldwide. Endothelial cell apoptosis plays a crucial role in its development. Several microRNAs (miRNAs) are reportedly involved in atherosclerotic plaque formation, including miRNA-210 (miR-210). However, the underlying mechanism of its role in endothelial cell apoptosis during atherosclerosis is still largely unknown.
Methods: A mouse model with atherosclerosis induced by a high-fat diet (HFD) was built in ApoE (-/-) mice. The levels of endothelial cell apoptosis were determined via flow cytometry. The expressions of miR-210 and PDK1 in purified CD31+ endothelial cells from mouse aorta were measured via RT-qPCR and western blot. Binding between miR-210 and the 3′-untranslated region (UTR) of PDK1 mRNA was predicted using bioinformatics analyses and confirmed with a dual luciferase reporter assay. The effects of miR-210 were further analyzed in an in vitro model using human aortic endothelial cells (HAECs) treated with oxidized low-density lipoprotein (ox-LDL).
Results: We found that the HFD mice developed atherosclerosis in 12 weeks and had a significantly higher percentage of endothelial cell apoptosis. The upregulated level of miR-210 in the HFD mice and HAECs inversely correlated with the level of PDK1. Inhibiting miR-210 expression significantly reduced HAEC apoptosis, as evidenced by the results of the MTT and flow cytometry experiments. Further analysis identified PDK1 as the target of miR-210 and showed that PDK1 overexpression reversed the pro-apoptotic effect of miR-210 through mediation of the P13K/Akt/mTOR pathways.
Conclusion: Our study suggests a novel role for miR-210 in the progression of atherosclerosis through the regulation of endothelial apoptosis. This indicates that miR-210 might have potential in treatment of atherosclerosis.
Keywords Atherosclerosis, miR-210, PDK1, Endothelial cell apoptosis, ApoE (-/-)
Address and Contact Information 1Department of Cardiology, China-Japan Union Hospital of Jilin University, 130033 Changchun, China.
2Department of Neonatology, The First Hospital of Jilin University, 130021 Changchun, China.
3Department of Ultrasonography, Eastern Division of First Hospital of Jilin University, 130021 Changchun, China.
* Corresponding author: pingyangjida@163.com
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DOI: 10.1186/s11658-017-0035-3 Volume 22 (2017)
Title THE REGULATORY MECHANISMS OF NG2/CSPG4 EXPRESSION
Authors Emmanuel Ampofo*, Beate M. Schmitt, Michael D. Menger and Matthias W. Laschke
Abstract Neuron-glial antigen 2 (NG2), also known as chondroitin sulphate proteoglycan 4 (CSPG4), is a surface type I transmembrane core proteoglycan that is crucially involved in cell survival, migration and angiogenesis. NG2 is frequently used as a marker for the identification and characterization of certain cell types, but little is known about the mechanisms regulating its expression. In this review, we provide evidence that the regulation of NG2 expression underlies inflammation and hypoxia and is mediated by methyltransferases, transcription factors, including Sp1, paired box (Pax) 3 and Egr-1, and the microRNA miR129-2. These regulatory factors crucially determine NG2-mediated cellular processes such as glial scar formation in the central nervous system (CNS) or tumor growth and metastasis. Therefore, they are potential targets for the establishment of novel NG2-based therapeutic strategies in the treatment of CNS injuries, cancer and other conditions of these types.
Keywords NG2, CSPG4, Inflammation, Hypoxia, Methylation, Transcription MiRNA
Address and Contact Information Institute for Clinical & Experimental Surgery, Saarland University
* Corresponding author: emmanuel.ampofo@uks.eu
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DOI: 10.1186/s11658-017-0034-4 Volume 22 (2017)
Title A SIMPLE AND EFFECTIVE PROTOCOL FOR FAST ISOLATION OF HUMAN TENON’S FIBROBLASTS FROM A SINGLE TRABECULECTOMY BIOPSY – A COMPARISON OF CELL BEHAVIOUR IN DIFFERENT CULTURE MEDIA
Authors Agata Przekora1*, Tomasz Zarnowski2 and Grazyna Ginalska1
Abstract Background: Human Tenon’s fibroblasts (HTFs) play a crucial role in wound healing. They cause postoperative scarring of the filtering bleb and are thus responsible for trabeculectomy failure. This study aimed to find an effective and fast protocol for HTF isolation from trabeculectomy biopsies. The protocol was compared with the commonly recommended HTF isolation procedure, which uses Dulbecco’s modified Eagle’s medium (DMEM). We used Eagle’s minimum essential medium (EMEM) enriched with fibroblast growth factor (FGF), which selectively promoted the proliferation of HTF cells. A secondary goal was to compare HTF morphology, metabolism and growth during parallel cultivation of the isolated cells in FGF-enriched EMEM and DMEM.
Results: Standard procedures for HTF isolation from tissue biopsies require a 20- to 30-day culture of the explants to obtain the first monolayer. Our protocol yielded the first monolayer after approx. 15 days. More importantly, the majority of the cells were fibroblasts with only individual epithelium-derived cells present. Using FGF-enriched EMEM allowed 1.3 × 106 vimentin-positive fibroblasts to be obtained from a single biopsy within approx. 25 days. Using DMEM resulted in isolation failure and required exchange to FGF-enriched medium to recover the fibroblast culture. HTFs maintained in FGF-enriched EMEM also showed faster proliferation and a different type I collagen production ability compared to HTFs cultured in DMEM. Thus, FGF-enriched EMEM is recommended for fast propagation of HTFs unless the aim of the study is to assess the effect of a tested agent on proliferation ability or type I collagen production.
Conclusions: Our fast protocol for HTF isolation allows easy setup of cell banks by researchers under laboratory conditions and could be very useful during testing of novel ophthalmologic anti-fibrotic agents in vitro. Molecular analysis of HTFs isolated from patients with known treatment histories may provide valuable information on the effects of some medications taken before glaucoma surgery on the subsequent wound-healing process and potential for trabeculectomy failure.
Keywords Glaucoma Ocular disorders, Cell banking, Isolation protocol, Primary culture
Address and Contact Information 1 Department of Biochemistry and Biotechnology, Medical University of Lublin, Lublin, Poland
2 Department of Ophthalmology, Medical University of Lublin, Lublin, Poland
* Corresponding author: agata.przekora@umlub.pl
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DOI: 10.1186/s11658-017-0037-1 Volume 22 (2017)
Title PPARγ AGONIST THROUGH THE TERMINAL DIFFERENTIATION PHASE IS ESSENTIAL FOR ADIPOGENIC DIFFERENTIATION OF FETAL OVINE PREADIPOCYTES
Authors Yong Pu and Almudena Veiga-Lopez*
Abstract Background: Although the 3T3-L1 preadipocyte cell line represents an informative model for in vitro adipogenesis research, primary cultured cells are often needed to understand particular human or animal metabolic phenotypes. As demonstrated by in vitro cultured preadipocytes from large mammalian species, primary cultured cells require specific adipogenic differentiation conditions different to that of the 3T3-L1 cell line. These conditions are also species-specific and require optimization steps. However, efficient protocols to differentiate primary preadipocytes using alternative species to rodents are scarce. Sheep represent an amenable animal model for fetal biology and developmental origins of health and disease studies. In this work, we present with the first detailed procedure to efficiently differentiate primary fetal and adult ovine preadipocytes.
Methods: Fetal and adult ovine adipose and skin tissue harvest, preadipocyte and fibroblast isolation, proliferation, and standardization and optimization of a new adipogenic differentiation protocol. Use of commercial cell lines (3T3-L1 and NIH-3T3) for validation purposes. Oil red O stain and gene expression were used to validate adipogenic differentiation. ANOVA and Fisher’s exact test were used to determine statistical significance.
Results: Our optimized adipogenic differentiation method included a prolonged adipogenic cocktail exposure time from 2 to 8 days, higher insulin concentration, and supplementation with the peroxisome proliferator-activated receptor gamma (PPARγ) agonist, rosiglitazone. This protocol was optimized for both, fetal and adult preadipocytes.
Conclusions: Our protocol enables successful adipogenic differentiation of fetal and adult ovine preadipocytes. This work demonstrates that compared to the 3T3-L1 cell line, fetal ovine preadipocytes require a longer exposure to the differentiation cocktail, and the need for IMBX, dexamethasone, and/or the PPARγ agonist rosiglitazone through the terminal differentiation phase. They also require higher insulin concentration during differentiation to enhance lipid accumulation and similar to human primary preadipocytes, PPARγ agonist supplementation is also required for ovine adipogenic differentiation. This work highlights species-specific differences requirements for adipogenic differentiation and the need to develop standardized methods to investigate comparative adipocyte biology.
Keywords Sheep, Fetal, Preadipocyte, Adipogenic differentiation
Address and Contact Information Department of Animal Science, Michigan State University
* Corresponding author: veiga@msu.edu
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DOI: 10.1186/s11658-017-0036-2 Volume 22 (2017)
Title ABCC6 KNOCKDOWN IN HepG2 CELLS INDUCES A SENESCENT-LIKE CELL PHENOTYPE
Authors Rocchina Miglionico1, Angela Ostuni1, Maria Francesca Armentano1, Luigi Milella1, Elvira Crescenzi2, Monica Carmosino1* and Faustino Bisaccia1*
Abstract Background: Pseudoxanthoma elasticum (PXE) is characterized by progressive ectopic mineralization of elastic fibers in dermal, ocular and vascular tissues. No effective treatment exists. It is caused by inactivating mutations in the gene encoding for the ATP-binding cassette, sub-family C member 6 transporter (ABCC6), which is mainly expressed in the liver. The ABCC6 substrate (s) and the PXE pathomechanism remain unknown. Recent studies have shown that overexpression of ABCC6 in HEK293 cells results in efflux of ATP, which is rapidly converted into nucleoside monophosphates and pyrophosphate (PPi). Since the latter inhibits mineralization, it was proposed that the absence of circulating PPi in PXE patients results in the characteristic ectopic mineralization. These studies also demonstrated that the presence of ABCC6 modifies cell secretory activity and suggested that ABCC6 can change the cell phenotype.
Methods: Stable ABCC6 knockdown HepG2 clones were generated using small hairpin RNA (shRNA) technology. The intracellular glutathione and ROS levels were determined. Experiments using cell cycle analysis, real-time PCR and western blot were performed on genes involved in the senescence phenotype.
Results: To shed light on the physiological role of ABCC6, we focused on the phenotype of HepG2 cells that lack ABCC6 activity. Interestingly, we found that ABCC6 knockdown HepG2 cells show: 1) intracellular reductive stress; 2) cell cycle arrest in G1 phase; 3) upregulation of p21Cip p53 independent; and 4) downregulation of lamin A/C.
Conclusions: These findings show that the absence of ABCC6 profoundly changes the HepG2 phenotype, suggesting that the PXE syndrome is a complex metabolic disease that is not exclusively related to the absence of pyrophosphate in the bloodstream.
Keywords ABCC6, Cell cycle, Senescence, Reductive stress
Address and Contact Information 1Department of Sciences, University of Basilicata
2Institute of Experimental Endocrinology and Oncology (IEOS), National Research Council (CNR)
* Corresponding author: monica.carmosino@unibas.it or faustino.bisaccia@unibas.it
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DOI: 10.1186/s11658-017-0038-0 Volume 22 (2017)
Title SURVIVIN, A MOLECULAR TARGET FOR THERAPEUTIC INTERVENTIONS IN SQUAMOUS CELL CARCINOMA
Authors Zakir Khan†1,2*, Abdul Arif Khan†3, Hariom Yadav4, Godavarthi B. K. S. Prasad1 and Prakash Singh Bisen1*
Abstract Squamous cell carcinoma (SCC) is the most common cancer worldwide. The treatment of locally advanced disease generally requires various combinations of radiotherapy, surgery, and systemic therapy. Despite aggressive multimodal treatment, most of the patients relapse. Identification of molecules that sustain cancer cell growth and survival has made molecular targeting a feasible therapeutic strategy. Survivin is a member of the Inhibitor of Apoptosis Protein (IAP) family, which is overexpressed in most of the malignancies including SCC and totally absent in most of the normal tissues. This feature makes survivin an ideal target for cancer therapy. It orchestrates several important mechanisms to support cancer cell survival including inhibition of apoptosis and regulation of cell division. Overexpression of survivin in tumors is also associated with poor prognosis, aggressive tumor behavior, resistance to therapy, and high tumor recurrence. Various strategies have been developed to target survivin expression in cancer cells, and their effects on apoptosis induction and tumor growth attenuation have been demonstrated. In this review, we discuss recent advances in therapeutic potential of survivin in cancer treatment.
Keywords Squamous cell carcinoma (SCC), Survivin, Apoptosis, Cancer immunotherapy, YM155, Hsp90 inhibitors, CDK inhibitors
Address and Contact Information 1School of Studies in Biotechnology, Jiwaji University
2Department of Biomedical Sciences, Department of Pathology, Cedars-Sinai Medical Center
3Department of Pharmaceutics, College of Pharmacy, King Saud University
4National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health
†Contributed equally
* Corresponding author: zakirq.khan@gmail.com, zakir.khan@cshs.org or psbisen@gmail.com
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DOI: 10.1186/s11658-017-0039-z Volume 22 (2017)
Title SURVIVAL RATES OF HOMOZYGOTIC Tp53 KNOCKOUT RATS AS A TOOL FOR PRECLINICAL ASSESSMENT OF CANCER PREVENTION AND TREATMENT
Authors Damian Strzemecki, Magdalena Guzowska and Paweł Grieb*
Abstract Background: The gene that encodes tumor protein p53, Tp53, is mutated or silenced in most human cancers and is recognized as one of the most important cancer drivers. Homozygotic Tp53 knockout mice, which develop lethal cancers early in their lives, are already used in cancer prevention studies, and now Tp53 knockout rats have also been generated. This study assessed feasibility of using homozygous Tp53 knockout rats to evaluate the possible outcome of cancer chemoprevention.
Methods: A small colony of Tp53 knockout rats with a Wistar strain genetic background was initiated and maintained in the animal house at our institution. Tp53 heterozygotic females were bred with Tp53 homozygous knockout males to obtain a surplus of knockout homozygotes. To evaluate the reproducibility of their lifespan, 4 groups of Tp53 homozygous knockout male rats born during consecutive quarters of the year were kept behind a sanitary barrier in a controlled environment until they reached a moribund state. Their individual lifespan data were used to construct quarterly survival curves.
Results: The four consecutive quarterly survival curves were highly reproducible. They were combined into a single “master” curve for use as a reference in intervention studies. The average lifespan of untreated male Tp53 homozygous knockout rats was normally distributed, with a median of 133 days. Sample size vs. effect calculations revealed that confirming a 20% and 30% increase in the lifespan would respectively require a sample size of 18 and 9 animals (when assessed using the t-test with a power of 80% and alpha set at 0.05). As an example, the Tp53 homozygous knockout rat model was used to test the chemopreventive properties of carnosine, a dipeptide with suspected anticancer properties possibly involving modulation of the mTOR pathway. The result was negative.
Conclusion: Further evaluation of the Tp53 homozygous knockout male rat colony is required before it can be confirmed as a viable tool for assessing new methods of cancer prevention or treatment.
Keywords Tp53 knockout rats, Survival curve, Chemoprevention, Cancer
Address and Contact Information Department of Experimental Pharmacology, Mossakowski Medical Research Centre, Polish Academy of Sciences, 5 Pawińskiego Str., Warsaw 02-106, Poland
* Corresponding author: pgrieb@imdik.pan.pl
Equal contributors
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DOI: 10.1186/s11658-017-0040-6 Volume 22 (2017)
Title LncRNA H19 PROMOTES THE DIFFERENTIATION OF BOVINE SKELETAL MUSCLE SATELLITE CELLS BY SUPPRESSING Sirt1/FoxO1
Authors Xiaochun Xu1†, Shengyue Ji2†, Weili Li2, Bao Yi2, Hengxin Li2, Hongfu Zhang2* and Wenping Ma1*
Abstract Background: H19 is a well-characterized Long noncoding RNA (lncRNA) that has been proven to promote myoblast differentiation in humans and mice. However, its mechanism of action is still not fully interpreted.
Methods: Using RT-qPCR, we examined H19 RNA levels in various tissues from 1-week, 1-month, 6-month and 36-month old male cattle (i.e., newborn, infant, young and adult). The protein and mRNA levels of MyoG, MyHC, Sirt1 and FoxO1 in the satellite and C2C12 cells with an H19 silencing or overexpression vector were respectively detected using western blot and real-time qPCR.
Results: H19 was highly expressed in skeletal muscle at all the studied ages. High expression of H19 was required for the differentiation of bovine satellite cells. Knockdown of H19 caused a remarkable increase in the myoblast-inhibitory genes Sirt1/FoxO1, suggesting that H19 suppresses Sirt1/FoxO1 expression during myogenesis. Western blotting analysis of co-transfection of Sirt1 or FoxO1 expression vectors with pcDNA-H19 indicated that Sirt1/FoxO1 overexpression neutralized the promotion of myoblast differentiation through transfection of pcDNA-H19.
Conclusion: H19 promoted the differentiation of bovine skeletal muscle satellite cells by suppressing Sirt1/FoxO1.
Keywords H19 lncRNA, Myoblast differentiation, Sirt1, FoxO1
Address and Contact Information 1 College of Biology Sciences and Engineering, Beifang University of Nationalities, Yinchuan, Ningxia, People’s Republic of China
2 State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan West Road, Beijing 100193, People’s Republic of China
* Corresponding author: wtl505@126.com or petermama@163.com
† Equal contributors
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DOI: 10.1186/s11658-017-0042-4 Volume 22 (2017)
Title CELLS OF HUMAN BREAST MILK
Authors Malgorzata Witkowska-Zimny* and Ewa Kaminska-El-Hassan
Abstract Human milk is a complex fluid that has developed to satisfy the nutritional requirements of infants. In addition to proteins, lipids, carbohydrates and other biologically active components, breast milk contains a diverse microbiome that is presumed to colonize the infant gastrointestinal tract and a heterogeneous population of cells with unclear physiological roles and health implications. Noteworthy cellular components of breast milk include progenitor/stem cells. This review summarizes the current state of knowledge of breast milk cells, including leukocytes, epithelial cells, stem cells and potentially probiotic bacteria.
Keywords Human breast milk, Stem cells, Leukocytes, Microbiome, Probiotic bacteria
Address and Contact Information Department of Biophysics and Human Physiology, Medical University of Warsaw, Chalubinskiego 5, 02-004 Warsaw, Poland
* Corresponding author: mwitkowska@wum.edu.pl
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DOI: 10.1186/s11658-017-0041-5 Volume 22 (2017)
Title miR-378 SUPPRESSES THE PROLIFERATION, MIGRATION AND INVASION OF COLON CANCER CELLS BY INHIBITING SDAD1c
Authors Mingxi Zeng1, Linlin Zhu2, Liangping Li1 and Changming Kang1*
Abstract Background: MicroRNAs (miRNAs) play important roles in the growth and metastasis of colon cancer. It is known that one set of miRNAs are dysregulated in colon cancer cells, but the mechanism of their role in cancer development is still largely unknown. Our study focuses on the role of miR-378 in colon cancer cells.
Methods: Human colon cancer tissues and adjacent non-tumor tissues were collected from patients diagnosed in pathological examinations. In addition, human colon cancer cell lines LoVo, CaCo2, SW1116, SW480 and HCT-116, and a normal colonic mucosa cell line NCM460 were included. Quantitative RT-PCR was used to detect the miR-378 level in the clinical tissues and cell lines. In SW480 and HCT-116, miR-378 was artificially overexpressed or suppressed. Cell viability and proliferation were measured using MTT and colony formation assays, and apoptosis was detected via annexin V-PI staining and flow cytometry analysis. The transwell technique was applied to detect the migration and invasion of the colon cancer cells, and their epithelial–mesenchymal transition (EMT) was evaluated by detecting EMT-associated markers using Western blotting. Bioinformatics methods were used to predict the potential targets of miR-378, and luciferase reporter assays were performed to conform the direct binding between miR-378 and its target mRNA. The activity of the Wnt/β-catenin pathway was evaluated by detecting the key factors through Western blotting.
Results: We found that miR-378 expression was low in colon cancer tissues and cell lines. Overexpression of miR-378 not only inhibits the proliferation of colon cancer cells in vitro by inducing apoptosis, but also inhibits migration and invasion by inhibiting the EMT of colon cancer cells. SDAD1 is a direct target gene of miR-378, and knockdown of SDAD1 suppresses the proliferation, migration and invasion of colon cancer cells. We also confirmed that miR-378 alleviated the malignant phenotypes of colon cancer cells by inhibiting the Wnt/β-catenin pathway.
Conclusion: miR-378 inhibits the proliferation, migration and invasion of colon cancer cells by targeting SDAD1, defining miR-378 as a potential target for the diagnosis and treatment of colon cancer.
Keywords miR-378, Colon cancer, Proliferation, Migration, Invasion, SDAD1, Wnt/ β-catenin
Address and Contact Information 1 Department of Digestive Diseases, Sichuan Provincial People’s Hospital, No. 32 Yihuan Road, Qingyang District, Chengdu, Sichuan Province 610072, China.
2 Department of Digestive Diseases, West China Hospital of Sichuan University, Chengdu 610041, China.
* Corresponding author: kangchangming@126.com
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DOI: s11658-017-0043-3 Volume 22 (2017)
Title AGONIST-INDUCED ACTIVATION OF HUMAN FFA1 RECEPTOR SIGNALS TO EXTRACELLULAR SIGNAL-REGULATED KINASE 1 AND 2 THROUGH Gq- AND Gi-COUPLED SIGNALING CASCADES
Authors Jing Qian1#, Yuyang Gu1, Chun Wu2, Feng Yu1, Yuqi Chen1, Jingmei Zhu1, Xingyi Yao1, Chen Bei1 and Qingqing Zhu1
Abstract Background: FFA1 is abundantly expressed in the liver, skeletal muscle, monocytes and nervous system, but is particularly abundant in pancreatic β cells. It is widely believed that FFA1 exerts its regulatory roles in a variety of physiological and pathological functions. In response to oleic acid, FFA1 has been shown to induce the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) through a mechanism involving EGFR transactivation in a breast cancer cell line. However, the underlying molecular mechanism for ERK1/2 activation mediated by n-6 free fatty acid (LA) in HEK293 cells remains to be further elucidated.
Methods: A FLAG-FFA1 vector was stably expressed in HEK293 cells. Western blot analysis was applied to investigate the change in LA-induced ERK1/2 phosphorylation change in response to kinase inhibitors. Arrestin-2/3-specific siRNA was used to analyze the effect of arrestin-2/3 knockdown on FFA1-mediated ERK1/2 activation.
Results: We proved that activation of ERK1/2 by LA was rapid, peaking at 5 min. Further experiments proved that FFA1 couples to a Gq protein and activates PI-PLC, which induces the IP3/Ca2+ and DAG/PKC signal pathways, both of which are involved in ERK1/2 activation. We also showed that there is no EGFR transactivation, arrestin-2/3 or Gβγ pathway participation in ERK1/2 phosphorylation. Treating cells with PTX abolished ERK1/2 activation at a late time point (≥20 min), indicating a critical role for Gi subunits in FFA1-mediated ERK1/2 activation.
Conclusions: Our study provides a detailed delineation of the LA-mediated activation of ERK1/2 in HEK293 cells that are stably transfected with human FFA1. We also present evidence of Gi/Gq-induced synergism in the regulation of ERK1/2 phosphorylation. These observations may provide new insights into the pharmacological effects of FFA1 and the physiological functions modulated by FFA1-mediated activation of ERK1/2.
Keywords FFA1, Phosphatidylinositol-specific phospholipase C, Extracellular signal- regulated kinase 1 and 2, Gαq/11, Gαi/o
Address and Contact Information 1Huzhou University Schools of Nursing and Medicine, Huzhou University, HuZhou 313000, China.
2Institute of Biochemistry, College of Life Science, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, China.
# Corresponding author: 02300@zjhu.edu.cn
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DOI: 10.1186/s11658-017-0044-2 Volume 22 (2017)
Title SFRP2 ENHANCES THE OSTEOGENIC DIFFERENTIATION OF APICAL PAPILLA STEM CELLS BY ANTAGONIZING THE CANONICAL WNT PATHWAY
Authors Luyuan Jin†1,3, Yu Cao†1,3, Guoxia Yu†1,3,4, Jinsong Wang3,5, Xiao Lin1,2, Lihua Ge1, Juan Du1, Liping Wang1, Shu Diao1, Xiaomeng Lian6, Songlin Wang3,5, Rui Dong1 and Zhaochen Shan7*
Abstract Background: Exploring the molecular mechanisms underlying directed differentiation is helpful in the development of clinical applications of mesenchymal stem cells (MSCs). Our previous study on dental tissue-derived MSCs demonstrated that secreted frizzled-related protein 2 (SFRP2), a Wnt inhibitor, could enhance osteogenic differentiation in stem cells from the apical papilla (SCAPs). However, how SFRP2 promotes osteogenic differentiation of dental tissue-derived MSCs remains unclear. In this study, we used SCAPs to investigate the underlying mechanisms.
Methods: SCAPs were isolated from the apical papilla of immature third molars. Western blot and real-time RT-PCR were applied to detect the expression of β-catenin and Wnt target genes. Alizarin Red staining, quantitative calcium analysis, transwell cultures and in vivo transplantation experiments were used to study the osteogenic differentiation potential of SCAPs.
Results: SFRP2 inhibited canonical Wnt signaling by enhancing phosphorylation and decreasing the expression of nuclear β-catenin in vitro and in vivo. In addition, the target genes of the Wnt signaling pathway, AXIN2 (axin-related protein 2) and MMP7 (matrix metalloproteinase-7), were downregulated by SFRP2. WNT1 inhibited the osteogenic differentiation potential of SCAPs. SFRP2 could rescue this WNT1-impaired osteogenic differentiation potential.
Conclusions: The results suggest that SFRP2 could bind to locally present Wnt ligands and alter the balance of intracellular Wnt signaling to antagonize the canonical Wnt pathway in SCAPs. This elucidates the molecular mechanism underlying the SFRP2-mediated directed differentiation of SCAPs and indicates potential target genes for improving dental tissue regeneration.
Keywords SFRP2, Osteogenic differentiation, Stem cells from apical papilla (SCAPs), Wnt signaling, β-catenin
Address and Contact Information 1 Laboratory of Molecular Signaling and Stem Cells Therapy, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, No. 4 Tiantanxili, Dongcheng District, Beijing 100050, China.
2 Department of Implant Dentistry, Capital Medical University School of Stomatology, Beijing 100050, China.
3 Molecular Laboratory for Gene Therapy and Tooth Regeneration, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, No. 4 Tiantanxili, Dongcheng District, Beijing 100050, China.
4 Department of Stomatology, Beijing Children’s Hospital, Capital Medical University, No.56 Nanlishi Road, Xicheng District, Beijing 100045, China.
5 Department of Biochemistry and Molecular Biology, Capital Medical University School of Basic Medical Sciences, No. 10 Xitoutiao Youanmen, Fengtai District, Beijing 100069, China.
6 Department of Stomatology, Beijing Shijitan Hospital, Capital Medical University, Beijing 100045, China.
7 Oral and Maxillofacial Surgery Department, Capital Medical University School of Stomatology, No. 4 Tiantanxili, Dongcheng District, Beijing 100050, China.
* Corresponding author: shanzhch629@163.com
Equal contributors
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DOI: 10.1186/s11658-017-0046-0 Volume 22 (2017)
Title RELEVANCE OF THE NR4A SUB-FAMILY OF NUCLEAR ORPHAN RECEPTORS IN TROPHOBLASTIC BEWO CELL DIFFERENTIATION
Authors Sudha Saryu Malhotra and Satish Kumar Gupta*
Abstract Nur-77, a member of the NR4A sub-family of nuclear orphan receptors, is downregulated in the placentae of pre-eclamptic women. Here, we investigate the relevance of Nor-1, Nurr-1 and Nur-77 in trophoblastic cell differentiation. Their transcript levels were found to be significantly upregulated in BeWo cells treated with forskolin. The maximum increase was observed after 2 h, with a second peak in the expression levels after 48 h. The expression of NR4A sub-family members was also found to be upregulated in BeWo cells after treatment with hCG and GnRH. A similar significant increase was observed at the respective protein levels after 2 and 48 h of treatment with forskolin, hCG or GnRH. Silencing Nor-1, Nurr-1 or Nur-77 individually did not show any effect on forskolin-, hCG- and/or GnRH-mediated BeWo cell fusion and/or hCG secretion. After silencing any one member of the NR4A sub-family, an increase in the transcript levels of the other sub-family members was observed, indicating a compensatory effect due to their functional redundancy. Simultaneously silencing all three NR4A sub-family members significantly downregulated forskolin- and hCG-mediated BeWo cell fusion and/or hCG secretion. However, a considerable amount of cell death occurred after forskolin or hCG treatment as compared to the control siRNA-transfected cells. These results suggest that the NR4A sub-family of nuclear orphan receptors has a role in trophoblastic cell differentiation.
Keywords Nor-1, Nurr-1, Nur-77, Trophoblast differentiation, hCG, GnRH
Address and Contact Information Reproductive Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi -110 067, India
* Corresponding author: skgupta@nii.ac.in
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DOI: 10.1186/s11658-017-0045-1 Volume 22 (2017)
Title ADAM23 PROMOTES NEURONAL DIFFERENTIATION OF HUMAN NEURAL PROGENITOR CELLS
Authors Annett Markus-Koch1, Oliver Schmitt2, Susanne Seemann1, Jan Lukas1, Dirk Koczan3, Mathias Ernst4, Georg Fuellen4, Andreas Wree2, Arndt Rolfs1 and Jiankai Luo1*
Abstract Background: ADAM23 is widely expressed in the embryonic central nervous system and plays an important role in tissue formation.
Results: In this study, we showed that ADAM23 contributes to cell survival and is involved in neuronal differentiation during the differentiation of human neural progenitor cells (hNPCs). Upregulation of ADAM23 in hNPCs was found to increase the number of neurons and the length of neurite, while its downregulation decreases them and triggers cell apoptosis. RNA microarray analysis revealed mechanistic insights into genes and pathways that may become involved in multiple cellular processes upon up- or downregulation of ADAM23.
Conclusions: Our results suggest that ADAM23 regulates neuronal differentiation by triggering specific signaling pathways during hNPC differentiation.
Keywords ADAM23, Differentiation, Neuron, Neurite growth, hNPC
Address and Contact Information 1 Albrecht-Kossel-Institute for Neuroregeneration, Rostock University Medical Center, Gehlsheimer Straße 20, 18147 Rostock, Germany. 2 Institute of Anatomy, Rostock University Medical Center, Gertrudenstrsse 9, 18055 Rostock, Germany. 3 Institute for Immunology, Rostock University Medical Center, Schillingallee 70, 18055 Rostock, Germany. 4 Institute for Biostatistics and Informatics in Medicine and Ageing Research, Rostock University Medical Center, Ernst-Heydemann-Str. 8, 18057 Rostock, Germany.
* Corresponding author: jiankai.luo@uni-rostock.de
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DOI: 10.1186/s11658-017-0048-y Volume 22 (2017)
Title PIWI-LIKE PROTEIN, HIWI2 IS ABERRANTLY EXPRESSED IN RETINOBLASTOMA CELLS AND AFFECTS CELL-CYCLE POTENTIALLY THROUGH OTX2
Authors Suganya Sivagurunathan1,2, Jayamuruga Pandian Arunachalam3,4 and Subbulakshmi Chidambaram1,5*
Abstract Retinoblastoma (RB), a childhood cancer, is caused by biallelic mutation of the RB1 gene, but its development is not clearly understood. Furthermore, the presence of a cancer stem cell subpopulation in RB might impact its treatment. PIWI protein, known for its role in stem cell self-renewal, is aberrantly expressed in cancers. We examined the role of the PIWI-like protein HIWI2 in RB and its effect on the stem cell markers in cells of the RB line, Y79. The expression of HIWI2 is significantly increased in Y79 compared with its level in HeLa and ARPE19 cells. The stem cell markers Oct-3/4, Nanog and Sox-2 were not altered upon HIWI2 knockdown in Y79 cells. Interestingly, OTX2 was significantly downregulated in the absence of HIWI2. Otx2 transcripts also decreased in HIWI2-silenced Y79 and ARPE19 cells. Moreover, silencing HIWI2 in Y79 accumulated the cells at G2–M phase and reduced the levels of proliferating cell nuclear antigen (PCNA) and the tumor suppressor, p16. Our results demonstrate that HIWI2 is aberrantly expressed in Y79 cells and silencing of HIWI2 downregulates OTX2, suggesting that HIWI2 might play a role in the progression of RB.
Keywords Y79, ARPE19, PCNA, Stem cell markers
Address and Contact Information 1 RS Mehta Jain Department of Biochemistry and Cell Biology, Vision Research Foundation, Chennai, India.
2 School of Chemical and Biotechnology, SASTRA University, Thanjavur, India.
3 SN ONGC Department of Genetics and Molecular Biology, Vision Research Foundation, Chennai, India.
4 Central Inter-Disciplinary Research Facility (CIDRF), Sri Balaji Vidyapeeth University, Mahatma Gandhi Medical College and Research Institute Campus, Pondicherry, India.
5 Department of Biochemistry and Molecular Biology, Pondicherry University, Puducherry, India.
* Corresponding author: csubbulakshmi@gmail.com
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DOI: 10.1186/s11658-017-0050-4 Volume 22 (2017)
Title GENE FUNCTIONING AND STORAGE WITHIN A FOLDED GENOME
Authors Sergey V. Razin1,2* and Sergey V. Ulianov1,2
Abstract In mammals, genomic DNA that is roughly 2 m long is folded to fit the size of the cell nucleus that has a diameter of about 10 μm. The folding of genomic DNA is mediated via assembly of DNA-protein complex, chromatin. In addition to the reduction of genomic DNA linear dimensions, the assembly of chromatin allows to discriminate and to mark active (transcribed) and repressed (non-transcribed) genes. Consequently, epigenetic regulation of gene expression occurs at the level of DNA packaging in chromatin. Taking into account the increasing attention of scientific community toward epigenetic systems of gene regulation, it is very important to understand how DNA folding in chromatin is related to gene activity. For many years the hierarchical model of DNA folding was the most popular. It was assumed that nucleosome fiber (10-nm fiber) is folded into 30-nm fiber and further on into chromatin loops attached to a nuclear/chromosome scaffold. Recent studies have demonstrated that there is much less regularity in chromatin folding within the cell nucleus. The very existence of 30-nm chromatin fibers in living cells was questioned. On the other hand, it was found that chromosomes are partitioned into self-interacting spatial domains that restrict the area of enhancers action. Thus, TADs can be considered as structural-functional domains of the chromosomes. Here we discuss the modern view of DNA packaging within the cell nucleus in relation to the regulation of gene expression. Special attention is paid to the possible mechanisms of the chromatin fiber self-assembly into TADs. We discuss the model postulating that partitioning of the chromosome into TADs is determined by the distribution of active and inactive chromatin segments along the chromosome. This article was specially invited by the editors and represents work by leading researchers.
Keywords Active chromatin, TADs, Hi-C, Self-organization, Epigenetic regulatory mechanisms, Enhancers
Address and Contact Information 1 Institute of Gene Biology, Russian Academy of Sciences, Vavilov Street 34/5, 119334 Moscow, Russia
2 Lomonosov Moscow State University, Biological Faculty, Leninskie Gory 1, building 12, 119192 Moscow, Russia
* Corresponding author: sergey.v.razin@usa.net
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DOI: 10.1186/s11658-017-0047-z Volume 22 (2017)
Title LUTEOLIN, QUERCETIN, GENISTEIN AND QUERCETAGETIN INHIBIT THE EFFECTS OF LIPOPOLYSACCHARIDE OBTAINED FROM Porphyromonas gingivalis IN H9c2 CARDIOMYOBLASTS
Authors Gloria Gutiérrez-Venegas*, Alfredo Torras-Ceballos, Juan Arturo Gómez-Mora and Berenice Fernández-Rojas
Abstract Background: One of the microorganisms from dental plaque associated with severe inflammatory responses in infectious endocarditis is Porphyromonas gingivalis. It is a Gram-negative bacteria harvested from chronic periodontitis patients. Lipopolysaccharide (LPS) obtained from P. gingivalis promotes the expressions of interleukin-1 (IL-1), IL-6 and tumor necrosis factor alpha (TNF-α). Flavonoids are thought to participate in processes that control inflammation, such as the expression of cyclooxygenase-2 (COX-2).
Methods: We investigated the effects of luteolin, quercetin, genistein and quercetagetin on cardiomyoblasts treated with LPS alone or in combination with following inhibitors p38 (SB203580), ERK (PD98059), JNK (SP600125) and PKC (Calphostin C) for 1 h. The kinase activation and COX-2 expression levels were determined at the gene and protein levels.
Results: These flavonoids are considered to inhibit the activation of mitogen-activated protein kinase (MAPK) and the degradation of inhibitor of kappa B-alpha (IκB-α). They also play a role in COX-2 expression.
Conclusion: We conclude that the tested flavonoids inhibit inflammatory responses induced by LPS in H9c2 cells.
Keywords Cardiomyoblasts, Flavonoids, Mitogen-activated protein kinase, Lipopolysaccharide
Address and Contact Information Laboratorio de Bioquímica de la División de Estudios de Posgrado de la Facultad de Odontología, Universidad Nacional Autónoma de México Ciudad Universitaria, 04510 México DF, Mexico
* Corresponding author: gloria@fo.odonto.unam.mx
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DOI: 10.1186/s11658-017-0049-x Volume 22 (2017)
Title ARE ONLINE PREDICTION TOOLS A VALID ALTERNATIVE TO GENOMIC PROFILING IN THE CONTEXT OF SYSTEMIC TREATMENT OF ER-POSITIVE BREAST CANCER?
Authors Umar Wazir*, Kinan Mokbel, Amtul Carmichael and Kefah Mokbel
Abstract Background: Clinicians use clinical and pathological parameters, such as tumour size, grade and nodal status, to make decisions on adjuvant treatments for breast cancer. However, therapeutic decisions based on these features tend to vary due to their subjectivity. Computational and mathematical algorithms were developed using clinical outcome data from breast cancer registries, such as Adjuvant! Online and NHS PREDICT. More recently, assessments of molecular profiles have been applied in the development of better prognostic tools.
Methods: Based on the available literature on online registry-based tools and genomic assays, we evaluated whether these online tools could be valid and accurate alternatives to genomic and molecular profiling of the individual breast tumour in aiding therapeutic decisions, particularly in patients with early ER-positive breast cancer.
Results and conclusions: Early breast cancer is currently considered a systemic disease and a complex ecosystem with behaviour determined by the complex genetic and molecular signatures of the tumour cells, mammary stem cells, microenvironment and host immune system. We anticipate that molecular profiling will continue to evolve, expanding beyond the primary tumour to include the tumour microenvironment, cancer stem cells and host immune system. This should further refine therapeutic decisions and optimise clinical outcome.

This article was specially invited by the editors and represents work by leading researchers.
Keywords Breast cancer, Molecular biotechnology, Genomic assay, Prognosis, Gene expression regulation
Address and Contact Information The London Breast Institute, Princess Grace Hospital, 45 Nottingham Place, London W1U 5NY, UK
* Corresponding author: umar.wazir@rcsed.ac.uk
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DOI: 10.1186/s11658-017-0051-3 Volume 22 (2017)
Title THE NOVEL PROTEIN C3orf43 ACCELERATES HEPATOCYTE PROLIFERATION
Authors Chunyan Zhang1,2, Cuifang Chang2, Deming Li2, Fuchun Zhang1* and Cunshuan Xu2*
Abstract Background: Our previous study found that single-pass membrane protein with coiled-coil domains 1 (C3orf43; XM_006248472.3) was significantly upregulated in the proliferative phase during liver regeneration. This indicates that C3orf43 plays a vital role in liver cell proliferation. However, its physiological functions remains unclear.
Methods: The expressions of C3orf43 in BRL-3A cells transfected with C3orf43-siRNA (C3-siRNA) or overexpressing the vector plasmid pCDH-C3orf43 (pCDH-C3) were measured via RT-qPCR and western blot. Cell growth and proliferation were determined using MTT and flow cytometry. Cell proliferation-related gene expression was measured using RT-qPCR and western blot.
Results: It was found that upregulation of C3orf43 by pCDH-C3 promoted hepatocyte proliferation, and inhibition of C3orf43 by C3-siRNA led to the reduction of cell proliferation. The results of qRT-PCR and western blot assay showed that the C3-siRNA group downregulated the expression of cell proliferation-related genes like JUN, MYC, CCND1 and CCNA2, and the pCDH-C3 group upregulated the expression of those genes.
Conclusion: These findings reveal that C3orf43 may contribute to hepatocyte proliferation and may have the potential to promote liver repair and regeneration.
Keywords Novel protein, C3orf43, Cell proliferation, siRNA, Overexpression vector
Address and Contact Information 1 Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China
2 State Key Laboratory Cultivation Base for Cell Differentiation Regulation and Henan Engineering Laboratory for Bioengineering and Drug Development, College of Life Science, Henan Normal University, Xinxiang, Henan 453007, China
* Corresponding author: zfcxju@sina.com; cellkeylab@126.com
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DOI: 10.1186/s11658-017-0053-1 Volume 22 (2017)
Title TGF-Β1-INDUCED EPITHELIAL–MESENCHYMAL TRANSITION IN LUNG CANCER CELLS INVOLVES UPREGULATION OF miR-9 AND DOWNREGULATION OF ITS TARGET, E-CADHERIN
Authors Hui Wang, Qian Wu, Ying Zhang, Hua-Nan Zhang, Yong-Bin Wang and Wei Wang*
Abstract Background: TGF-β1 plays an important role in the epithelial–mesenchymal transition (EMT) of epithelial cancers, including non-small cell lung cancer (NSCLC). While the full underlying mechanism remains unclear, miR-9 is known to play a critical role in the regulation of NSCLC cell invasion. We tested whether miR-9 targets E-cadherin and thus affects TGF-β1-induced EMT in NSCLC cells by assessing the expression levels of miR-9 and E-cadherin for NSCLC patients and then verifying the targeting of E-cadherin by miR-9 using the dual luciferase reporter system.
Results: MiR-9 was significantly upregulated in NSCLC tissues compared with its level in adjacent normal tissues. The expression of E-cadherin in NSCLC tissues was significantly decreased. In addition, we found that TGF-β1 significantly upregulated the expression of miR-9 and downregulated the expression of E-cadherin. E-cadherin was confirmed as a direct target gene of miR-9. Using an miR-9 inhibitor reversed the TGF-β1-mediated inhibition of E-cadherin expression and upregulation of the mesenchymal marker α-SMA. TGF-β1 significantly induced cell invasion, and this effect was significantly inhibited by miR-9 inhibitors.
Conclusions: TGF-β1 induced EMT in NSCLC cells by upregulating miR-9 and downregulating miR-9’s target, E-cadherin.
Keywords MiR-9, E-cadherin, Invasion, NSCLC
Address and Contact Information Department of Respiratory Medicine, The Second Hospital of Shandong University, 247 Beiyuan Street, Jinan, Shandong 250033, China
* Corresponding author: weumwc@sina.com
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DOI: 10.1186/s11658-017-0054-0 Volume 22 (2017)
Title miR-200b DOWNREGULATES CFTR DURING HYPOXIA IN HUMAN LUNG EPITHELIAL CELLS
Authors Sylwia Bartoszewska1, Wojciech Kamysz1, Bogdan Jakiela2, Marek Sanak2, Jarosław Króliczewski3, Zsuzsa Bebok4,Rafal Bartoszewski5*† and James F. Collawn4†
Abstract Background: Hypoxic conditions induce the expression of hypoxia-inducible factors(HIFs) that allow cells to adapt to the changing conditions and alter the expression of a number of genes including the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a low abundance mRNA in airway epithelial cells even during normoxic conditions, but during hypoxia its mRNA expression decreases even further.
Methods: In the current studies, we examined the kinetics of hypoxia-induced changes in CFTR mRNA and protein levels in two human airway epithelial cell lines, Calu-3 and 16HBE14o-, and in normal primary bronchial epithelial cells. Our goal was to examine the posttranscriptional modifications that affected CFTR expression during hypoxia. We utilized in silico predictive protocols to establish potential miRNAs that could potentially regulate CFTR message stability and identified miR-200b as a candidate molecule.
Results: Analysis of each of the epithelial cell types during prolonged hypoxia revealed that CFTR expression decreased after 12 h during a time when miR-200b was continuously upregulated. Furthermore, manipulation of the miRNA levels during normoxia and hypoxia using miR-200b mimics and antagomirs decreased and increased CFTR mRNA levels, respectively, and thus established that miR-200b downregulates CFTR message levels during hypoxic conditions.
Conclusion: The data suggest that miR-200b may be a suitable target for modulating CFTR levels in vivo.
Keywords CFTR, HIF-1, micro-RNA 200b, hsa-miR-200b-3p
Address and Contact Information 1Department of Inorganic Chemistry, Medical University of Gdansk, Gdansk, Poland.
2Department of Medicine, Jagiellonian University Medical College, Krakow, Poland.
3Department of Chemical Biology, Faculty of Biotechnology,University of Wroclaw, Wroclaw, Poland.
4Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, USA.
5Department of Biology and Pharmaceutical Botany, Medical University of Gdansk, Hallera 107, 80-416 Gdansk, Poland.
* Corresponding author: rafalbar@gumed.edu.pl
This article was specially invited by the editors and represents work by leading researchers.
Equal contributors
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DOI: 10.1186/s11658-017-0056-y Volume 22 (2017)
Title HEAT SHOCK PROTEIN 27 PLAYS A PROTECTIVE ROLE IN THORACIC AORTIC DISSECTION BY PROMOTING CELL PROLIFERATION AND INHIBITING APOPTOSIS
Authors Sili Zou1†, Mingfang Liao1†, Junlin Yang1, Tong Huang1, Mark Green2, Jianjin Wu1 and Lefeng Qu1*
Abstract Background: Thoracic aortic dissection (TAD) is one of the most severe aortic diseases.The study aimed to explore the potential role of heat shock protein 27 (HSP27) in the pathogenesis of TAD using an in vitro model of oxidative stress in vascular smooth muscle cells (VSMCs).
Methods: HSP27 was analyzed in aortic surgical specimens from 12 patients with TAD and 8 healthy controls. A lentiviral vector was used to overexpress HSP27 in rat aortic VSMCs. Cell proliferation and apoptosis were measured under oxidative stress induced by H2O2.
Results: HSP27 expression was significantly higher in aortic tissue from patients with TAD and VSMCs in the aortic media were the main cell type producing HSP27. Elevated oxidative stress was also detected in the TAD samples. Overexpression of HSP27 significantly attenuated H2O2-induced inhibition of cell proliferation. Furthermore, HSP27 was found to decrease H2O2-induced cell apoptosis and oxidative stress.
Conclusions: These results suggest that HSP27 expression promotes VSMC viability, suppresses cell apoptosis, and confers protection against oxidative stress in TAD.
Keywords Aortic dissection, Heat shock protein 27, Oxidative stress, Thoracic aortic dissection, Vascular smooth muscle cells
Address and Contact Information 1Department of Vascular and Endovascular Surgery, Changzheng Hospital, the Second Military Medical University, 415 Fengyang Road, Shanghai, People’s Republic of China.
2DICAT Biomedical Computation Centre, Vancouver, BC, Canada.
* Corresponding author: qulefeng@smmu.edu.cn
Equal contributors
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DOI: 10.1186/s11658-017-0059-8 Volume 22 (2017)
Title APOLIPOPROTEIN E POLYMORPHISM AND THE RISK OF ANEURYSMAL SUBARACHNOID HEMORRHAGE IN A SOUTH INDIAN POPULATION
Authors Arati Suvatha1, Sibin Madathan Kandi1, Dhananjaya Ishwara Bhat2, Narasinga Rao2, Vikas Vazhayil2 and Chetan Ghati Kasturirangan1*
Abstract Background: The rupture of a brain aneurysm causes bleeding in the subarachnoid space. This is known as aneurysmal subarachnoid hemorrhage (aSAH). We evaluated the association of apolipoprotein E (APOE) polymorphism and the risk of aSAH in a South Indian population.
Methods: The study was performed on 200 subjects with aSAH and 253 healthy control subjects. Blood samples (5 ml) were used to isolate DNA and genotyping was performed for rs7412 and rs429358 using a Taqman allelic discrimination assay. Statistical software R.3.0.11 was used to statistically analyze the data and a p value < 0.05 was considered as statistically significant.
Results: We found a significant association with the risk of aSAH in ε3/ ε4 genetic model (OR = 1.91, 95% CI = 1.16–3.14, p = 0.01). However, in the other genetic models and allele frequency, there was no significant association with the risk of aSAH. In subtyping, we found a significant association of ε2 allele frequency with posterior communicating artery (PCOM) aneurysm (OR = 3.59, 95% CI = 1.11–11.64, p = 0.03).
Conclusion: Our results suggest that APOE polymorphism has an influence on the risk of aSAH in this South Indian population, specifically in the PCOM subtype.
Keywords Aneurysmal subarachnoid hemorrhage, Apolipoprotein E, Polymorphism, Posterior communicating artery aneurysm
Address and Contact Information 1Department of Human Genetics, National Institute of Mental Health and Neuro Sciences, Bangalore, Karnataka 560029, India.
2Department of Neurosurgery, National Institute of Mental Health and Neuro Sciences, Bangalore 560029, India.
* Corresponding author: drchetangk@gmail.com
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DOI: 10.1186/s11658-017-0057-x Volume 22 (2017)
Title IFN-λ1 IN CHO CELLS: ITS EXPRESSION AND BIOLOGICAL ACTIVITY
Authors Wu-mei Yuan1,2†, Wan-ju Zhang3†, Fen-lian Ma1, Jin-song Li1, Qian Zhang1 and Li-shu Zheng1*
Abstract Background: Many studies have investigated the characteristics and biological activities of type III interferon (IFN), finding that it has similar features to type I IFN but also unique actions because it is recognized by a different receptor.
Results: A full-length recombinant human IFN-λ1 (rhIFN-λ1) cDNA was cloned into the pDF expression vector and stably expressed in Flp-In-CHO cells. After four purification steps (ammonium sulfate precipitation, SP Sepharose chromatography, Blue Sepharose 6 fast flow affinity chromatography and molecular sieve chromatography), the rhIFN-λ1 had a purity of about 90% and was found to have the predicted biological activities. The anti-viral activity of rhIFN-λ1 was determined as 106 IU/mg using the vesicular stomatitis virus (WISH-VSV) assay system. The anti-proliferation activity of rhIFN-λ1 was measured using the MTS method and the growth inhibition ratio was 57% higher than that for recombinant human IFN-α2b (rhIFN-α2b) when the rhIFN-λ1 concentration was 1000 IU/ml. rhIFN-λ1 had lower natural killer cell cytotoxicity than rhIFN-α2b.
Conclusion: The Flp-In-CHO system is suitable for stably expressing rhIFN-λ1 that possesses the predicted anti-viral, anti-proliferation and natural killer cell cytotoxicity-promoting activities.
Keywords Recombinant human IFN-λ1(rhIFN-λ1), Flp-in-CHO cell, Protein expression, Purification, Biological activity
Address and Contact Information 1Key Laboratory for Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, China CDC, 100 Ying-Xin St., Xi-Cheng District, Beijing 100052, China.
2Key Laboratory of Xinjiang Ethnic Diseases, Department of Biochemistry, School of Medicine, Shihezi University, Shihezi, Xinjiang 830002, China.
3Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China.
* Corresponding author: zhenglishu2000@sina.com
Equal contributors
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DOI: 10.1186/s11658-017-0060-2 Volume 22 (2017)
Title COLLAGEN-DERIVED DIPEPTIDE PROLYL-HYDROXYPROLINE PROMOTES OSTEOGENIC DIFFERENTIATION THROUGH Foxg1
Authors Yoshifumi Kimira1*, Haruka Odaira1, Kaho Nomura1, Yuri Taniuchi1, Naoki Inoue2, Sachie Nakatani1, Jun Shimizu1, Masahiro Wada1 and Hiroshi Mano1
Abstract Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. We previously reported that Pro-Hyp promotes the differentiation of osteoblasts by increasing Runx2, osterix and Col1α1 mRNA expression levels. Here, to elucidate the mechanism of Pro-Hyp promotion of osteoblast differentiation, we focus on the involvement of Foxo1 in osteoblast differentiation via Runx2 regulation and the role of Foxg1 in Foxo1 regulation. The addition of Pro-Hyp had no effect on MC3T3-E1 cell proliferation in Foxo1- or Foxg1-knockdown cells. In Foxo1-knockdown cells, the addition of Pro-Hyp increased ALP activity, but in Foxg1-knockdown cells, it had no effect on ALP activity. An enhancing effect of Pro-Hyp on the Runx2 and osterix expression levels was observed in Foxo1-knockdown cells. However, no enhancing effect of Pro-Hyp on osteoblastic gene expression was observed when Foxg1 was knocked down. These results demonstrate that Pro-Hyp promotes osteoblastic MC3T3-E1 cell differentiation and upregulation of osteogenic genes via Foxg1 expression.
Keywords Prolyl-hydroxyproline, Collagen peptide, Osteoblast differentiation, Foxo1, Foxg1
Address and Contact Information 1Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado-shi, Saitama 350-0295, Japan.
2Nitta Gelatin Inc., Peptide Division, 2-22 Futamata, Yao, Osaka 581-0024, Japan.
* Corresponding author: kimira@josai.ac.jp
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DOI: 10.1186/s11658-017-0061-1 Volume 22 (2017)
Title BIO- AND CHEMOCATALYSIS CASCADES AS A BRIDGE BETWEEN BIOLOGY AND CHEMISTRY FOR GREEN POLYMER SYNTHESIS
Authors Aleksandra Marszałek-Harych, Dawid Jędrzkiewicz and Jolanta Ejfler*
Abstract The development and integration of bio- and chemocatalytic processes to convert renewable or biomass feedstocks into polymers is a vibrant field of research with enormous potential for environmental protection and the mitigation of global warming. Here, we review the biotechnological and chemical synthetic strategies for producing platform monomers from bio-based sources and transforming them into eco-polymers. We also discuss their advanced bio-application using the example of polylactide (PLA), the most valuable green polymer on the market.
Keywords Polylactide, Lactic acid, Ring opening polymerization
Address and Contact Information Faculty of Chemistry, University of Wrocław, 14 F. Joliot-Curie, 50-383 Wrocław, Poland
* Corresponding author: jolanta.ejfler@chem.uni.wroc.pl
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DOI: 10.1186/s11658-017-0062-0 Volume 22 (2017)
Title KNOCKDOWN OF Rab21 INHIBITS PROLIFERATION AND INDUCES APOPTOSIS IN HUMAN GLIOMA CELLS
Authors Jian Ge, Qianxue Chen*, Baohui Liu, Long Wang, Shenqi Zhang and Baowei Ji
Abstract Background: Gliomas are commonly malignant tumors that arise in the human central nervous system and have a low overall five-year survival rate. Previous studies reported that several members of Rab GTPase family are involved in the development of glioma, and abnormal expression of Rab small GTPases is known to cause aberrant tumor cell behavior. In this study, we characterized the roles of Rab21 (Rab GTPase 21), a member of Rab GTPase family, in glioma cells.
Methods: The study involved downregulation of Rab21 in two glioma cell lines (T98G and U87) through transfection with specific-siRNA. Experiments using the MTT assay, cell cycle analysis, apoptosis assay, real-time PCR and western blot were performed to establish the expression levels of related genes.
Results: The results show that downregulation of Rab21 can significantly inhibit cell growth and remarkably induce cell apoptosis in T98G and U87 cell lines. Silencing Rab21 resulted in significantly increased expression of apoptosis-related proteins (caspase7, Bim and Bax) in glioma cells.
Conclusions: We inferred that Rab21 silencing can induce apoptosis and inhibit proliferation in human glioma cells, indicating that Rab21 might act as an oncogene and serve as a novel target for glioma therapy.
Keywords Glioma, Rab21, RabGTPases, Proliferation, Apoptosis
Address and Contact Information Department of Neurosurgery, Renmin Hospital of Wuhan University, No.9 Zhangzhidong Road, Wuchang District, Wuhan, Hubei 430060, People’s Republic of China
* Corresponding author: xueqiansky@sohu.com
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DOI: 10.1186/s11658-017-0055-z Volume 22 (2017)
Title THE ROLE OF BONE MARROW MONONUCLEAR CELL-CONDITIONED MEDIUM IN THE PROLIFERATION AND MIGRATION OF HUMAN DERMAL FIBROBLASTS
Authors Yolanda Menéndez-Menéndez1, Jesús Otero-Hernández1, Jose Antonio Vega2,3, Marcos Pérez-Basterrechea1, Silvia Pérez-López1, María Álvarez-Viejo1*† and Amaia Ferrero-Gutiérrez1†
Abstract Background: Several recent studies have demonstrated the great potential of bone marrow cells in regenerative medicine, not only for their ability to differentiate to match a damaged cell type, but also because they synthesize and release various growth factors and cytokines. We examined the effect of bone marrow cell-conditioned medium in the healing process, especially in terms of fibroblast proliferation and migration.
Methods: These in vitro studies consisted of co-culture (without direct contact) of dermal fibroblasts with mononuclear bone marrow cells and the use of conditioned medium obtained from these cultures in a scratch wound model.
Results: Mononuclear cells were found to increase the proliferation of fibroblasts, and the conditioned medium showed a stimulatory effect on the migration of fibroblasts.
Conclusion: When considered together with the observed increase in growth factor levels in conditioned medium, it appears that these cells act through a paracrine mechanism.
Keywords Bone marrow mononuclear cells, Cell migration and proliferation, Human dermal fibroblasts, Paracrine interactions, Wound repair
Address and Contact Information 1Unidad de Coordinación de Trasplantes, Terapia Celular y Medicina Regenerativa, Hospital Universitario Central de Asturias, Oviedo, Spain.
2Departamento de Morfología y Biología Celular, Universidad de Oviedo, Oviedo, Spain.
3Facultad de Ciencias de la Salud, Universidad Autónoma de Chile, Temuco, Chile.
* Corresponding author: maria.alvarezv@sespa.es
Equal contributors
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DOI: 10.1186/s11658-017-0066-9 Volume 22 (2017)
Title The mechanism of (+) taxifolin’s protective antioxidant effect for •OH-treated bone marrow-derived mesenchymal stem cells
Authors Xican Li1,2*† , Hong Xie1,2†, Qian Jiang1, Gang Wei1,2, Lishan Lin1, Changying Li1, Xingmei Ou1, Lichan Yang1, Yulu Xie1,2, Zhen Fu3,4, Yamei Liu3,4 and Dongfeng Chen4*
Abstract The natural dihydroflavonol (+) taxifolin was investigated for its protective effect on Fenton reagent-treated bone marrow-derived mesenchymal stem cells (bmMSCs). Various antioxidant assays were used to determine the possible mechanism. These included •OH-scavenging, 2-phenyl-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide radical-scavenging (PTIO•-scavenging), 1, 1-diphenyl-2-picryl-hydrazl radical-scavenging (DPPH•-scavenging), 2, 2′-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) radical-scavenging (ABTS+•-scavenging), Fe3+-reducing, and Cu2+-reducing assays. The Fe2+-binding reaction was also investigated using UV-Vis spectra. The results revealed that cell viability was fully restored, even increasing to 142.9 ± 9.3% after treatment with (+) taxifolin. In the antioxidant assays, (+) taxifolin was observed to efficiently scavenge •OH, DPPH• and ABTS+• radicals, and to increase the relative Cu2+- and Fe3+-reducing levels. In the PTIO•-scavenging assay, its IC50 values varied with pH. In the Fe2+-binding reaction, (+) taxifolin was found to yield a green solution with two UV-Vis absorbance peaks: λmax = 433 nm (ε =5.2 × 102 L mol−1 cm −1 ) and λmax = 721 nm (ε = 5.1 × 102 L mol−1 cm −1). These results indicate that (+) taxifolin can act as an effective •OH-scavenger, protecting bmMSCs from •OH-induced damage. Its •OH-scavenging actionconsists of direct and indirect antioxidant effects. Direct antioxidation occurs via multiple pathways, including ET, PCET or HAT. Indirect antioxidation involves binding to Fe2+.
Keywords (+) Taxifolin; bmMSCs, •OH damage, Antioxidant mechanism, Electron transfer, Fe2+ binding
Address and Contact Information 1 School of Chinese Herbal Medicine, Guangzhou University of Chinese Medicine, Waihuang East Road No. 232, Guangzhou Higher Education Mega Center, Guangzhou 510006, China.
2 Innovative Research & Development Laboratory of TCM, Guangzhou University of Chinese Medicine, Guangzhou 510006, China.
3 School of Basic Medical Science, Guangzhou University of Chinese Medicine, Guangzhou 510006, China.
4 The Research Center of Integrative Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, China.
* Corresponding author: lixican@126.com; lixc@gzucm.edu.cn; CDF27212@21cn.com; chen888@gzucm.edu.cn
Equal contributors
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DOI: 10.1186/s11658-017-0064-y Volume 22 (2017)
Title The crucial role of SEMA3F in suppressing the progression of oral squamous cell carcinoma
Authors Yi Liu* , Ronghua Li, Kai Yin, Gang Ren and Yongdong Zhang
Abstract Background: Oral squamous cell carcinoma (OSCC) is one of the most common types of malignancy. Semaphorin 3F (SEMA3F) is highly conserved but present at a lower level in various cancers than in healthy tissues. While it has been reported that SEMA3F is involved in cancer cell proliferation, migration and invasion, its function in OSCC remains unknown.
Methods: The expression of SEMA3F in OSCC tissues and OSCC-derived cells was analyzed using qRT-PCR and western blotting. Using SAS and HSC2 cells, we also monitored the effect of SEMA3F on OSCC cell proliferation, migration and invasion using MTT, colony formation and transwell assays. The function of SEMA3F in OSCC tumor formation was also assessed in vivo.
Results: SEMA3F was significantly downregulated in OSCC tissues and OSCC-derived cells. SEMA3F shows growth inhibitory activity in SAS and HSC2 cells and may act as a tumor suppressor. It can inhibit the migration and invasion potential of OSCC cells. Our results also demonstrate that SEMA3F can suppress the growth of OSCC cells in vivo.
Conclusions: This study revealed that SEMA3F plays a role as a tumor suppressor in OSCC cell proliferation, migration and invasion. Our finding provides new insight into the progression of OSCC. Therapeutically, SEMA3F has some potential as a target for OSCC treatment, given sufficient future research.
Keywords Oral cancer, SEMA3F, Cell proliferation, Migration, Invasion, MTT assay, Transwell assay, Xenograft model
Address and Contact Information Department of Stomatology, Tianjin First Center Hospital, Tianjin 300192, People’s Republic of China
* Corresponding author: 13821075639@163.com
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DOI: 10.1186/s11658-017-0065-x Volume 22 (2017)
Title Ectopic expression of TWIST1 upregulates the stemness marker OCT4 in the esophageal squamous cell carcinoma cell line KYSE30
Authors Mohammad Hossein Izadpanah1, Mohammad Reza Abbaszadegan1, Yasaman Fahim1 and Mohammad Mahdi Forghanifard2*
Abstract Background: The transcription factor TWIST1 plays an important role in the epithelial–mesenchymal transition (EMT) process and in the migration, invasion and metastasis of cancer cells. OCT4, which is a homeobox transcription factor, has an important role in the self-renewal potential of cancer cells. Our aim here is to elucidate impact of ectopic expression of TWIST1 on OCT4 gene expression in esophageal squamous cell carcinoma (ESCC).
Methods: The ESCC line was KYSE30. GP293T cells were transfected with purf-IRES-GFP and pGP plasmids to produce recombinant viral particles. A semi-confluent KYSE30 culture was transduced with the prepared retroviral particles. mRNA extraction and cDNA synthesis were performed from normal KYSE30 cells and those ectopically expressing TWIST1. Expressional analysis of TWIST1 and OCT4 were performed with relative comparative real-time PCR.
Results: Ectopic expression of TWIST1 in KYSE30 cells was related to its significant overexpression: nearly nine-fold higher in GFP-hTWIST1 KYSE-30 cells than in control GFP cells. This induced expression of TWIST1 caused significant upregulation of OCT4 in GFP-hTWIST1 KYSE-30 cells: nearly eight-fold higher. In silico analysis predicted the correlation of TWIST1 and OCT4 through ETS2.
Conclusions: Overexpressed TWIST1 can be correlated with upregulation of the cancer stem cell marker OCT4 and the protein may play critical regulatory role in OCT4 gene expression. Since OCT4 is involved in the self-renewal process, the results may suggest a new linkage between TWIST1 and OCT4 in the cell biology of ESCC, highlighting the probable role of TWIST1 in inducing self-renewal.
Keywords Esophageal squamous cell carcinoma (ESCC), Cancer stem cells (CSCs), TWIST1, OCT4
Address and Contact Information 1 Division of Human Genetics, Immunology Research Center, Avicenna Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran.
2 Department of Biology, Damghan Branch, Islamic Azad University, P.O.Box: 3671639998, Cheshmeh-Ali Boulevard, Sa’dei square, Damghan, Islamic Republic of Iran.
* Corresponding author: forghanifard@gmail.com
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