MicroRNAs 200/205 as Gene Therapy Candidates in Inhibiting the Production of Gastric Cancer Stem Cells Through EMT Pathway

سال انتشار: 1397
نوع سند: مقاله کنفرانسی
زبان: انگلیسی
مشاهده: 375

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شناسه ملی سند علمی:

NSCMRMED03_130

تاریخ نمایه سازی: 30 دی 1397

چکیده مقاله:

Background and Aim: Epithelial-Mesenchymal Transition (EMT) couldbe described in two different contexts of normal and tumor cells. EMTtransforms cancer cells to cancerous stem cells which result in metastasesand drug resistance. Preventing the EMT phase or reversing the EMTphase to MET can be effective in controlling cancer disease. This studydeliberates the potential of miR-200/205 in controlling the EMT processin gastric cancer cells and its application as a candidate for gene therapyin the metastatic cancer field.Methods: To induce EMT pathway, Gastric adenocarcinoma cell linewas treated with serum-free RPMI 1640 containing 10 ng/mL TGF-β.After 48 hours treatment, total RNA (including miRNA) of treated anduntreated AGS were extracted. To confirm the induction of EMT process,RNA expression level of Mesenchymal markers (Vimentin), markerinvolves in migration-invasion (β-catenin), E-cadherin a key marker ofepithelial phenotype, ZEB1/ZEB2 and Snail were analyzed by RotorGenq 5plex HRM. Western blot technique used to consider protein levelexpression change of EMT markers. Specific monoclonal antibodies forE-cadherin, Vimentin, and β-catenin in wet blotting system were applied.To characterize miR-200a expression, specific primers were designedand synthesized. The changes in the expression level of miR-200a werestudied by using the real-time PCR technique. We evaluated comparativeanalysis results according to GAPDH and U6 as housekeeping genes forEMT markers and miRNA respectively.Results: In this study, we stabilized the in vitro model of TGF-β-inducedEMT process in AGS cell line. The results of our Real-Time PCR inconfirming the EMT model showed that the expression level of E-cadherinas a marker in the cell surface binding molecule was significantlydecreased (P<0.05), and other mesenchymal and cancer stem cellsmarkers including Vimentin, B-catenin, Snail, ZEB1/2 were significantlyincreased (P<0.05). The western blot results also confirmed the changesin E-cadherin, Vimentin, and B-catenin at protein levels. The results of the western blot were in line with the comparative analysis of real-time PCR.After the induction of EMT in AGS cells and its approval, the expressionlevel of miR-200 and 205 was studied. Based on the results obtainedfrom real-time PCR, there was a significant decrease (P<0.05) in the levelexpression of those miRs after the transformation of AGS from cancer cellto cancer stem cells.Conclusion: Current study demonstrated that in the in vitro model ofEMT, this process in gastric cancer cells decreases transcription level ofmiR200/205. There are some studies on non-GI cancers which identifiedthe role of miR200/205 on the repression of EMT and inducing MET byaffecting on ZEB1/2. Based on our results, miR200/205 as suppressors ofEMT could be used as a candidate for gene therapy research purposes onpreventing a metastatic form of Gastric cancer, which is one of the mostaggressive cancers.

نویسندگان

Sepideh Mirzaei

Department of Biology, Faculty of Science, Islamic Azad University, Science and Research Branch, Tehran, Iran

Kaveh Baghaei

Basic and Molecular Epidemiology of Gastrointestinal Disorder Research Center, Research Institute for Gastroenterology andLiver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Kazem Parivar

Department of Biology, Faculty of Science, Islamic Azad University, Science and Research Branch, Tehran, Iran

Fatemeh Yarian

Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran