| OBJECTIVE:Gastric cancer is one of the most common carcinomas and is the second leading cause of cancer-related death in China. In spite of the improvement in surgical and multimodal therapy, the prognosis of advanced gastric cancer still remains poor in recent years. Better understanding the molecular mechanism of gliomagenesis will certainly lead to the development of novel targeted therapies.An important event in the metastatic cascade is epithelial to mesenchymal transition (EMT), EMT is an evolutionarily conserved program of gene expression during which epithelial cells adopt characteristics of mesenchymal cells EMT is an important pivotal cellular program thatinduces rapid changes in the shape and motility of epithelial cells. During this process, cells lose their cell-cell and cell-matrix contacts and epithelial polarity, gain the disseminated cell migration ability and become motile mesenchymal cells. To explore the molecular mechanism of EMT, also help to find new prognostic factors to gastric cancer.Recently, the group of non-coding RNA (ncRNA) such as microRNA, have been proven to play an essential role in the regulation of many physiological and pathological processes such as cancer. Approximately22nucleotides in length,microRNAs bind to sequences with partial complementarity on target RNA transcripts, called microRNA response elements (MREs), usually resulting in the repression of target gene expression or translation. Remarkably, microRNA-RNA function have identified profound awareness, a reversed RNA-microRNA function have rarly been reported by now. Many experimental techniques normally neglect UTRs and limit functional studies to gene coding regions. Thus, by limiting their focus or scope to coding region, several noncoding regions including UTRs in the human transcriptome have been found to be regulators,either as competition endogenous RNA or exogenous RNA that regulate the progress of the tumor.However,little is known about the mechanism and effects involved in this progress.METHODS:In the first partTo design and synthesize siRNA targeting ZEB2and ZEB23’UTR, then transfect it into SGC7901gastric adenocarcinoma cells with lipofectamine2000. The realtime PCR and Western blotting were were performed to evaluate the expression levels of miR-200a/b/c and ZEB2mRNAs and the protein expression level of ZEB2, vimentin, and E-cadherin respectively. Finally, the effects of ZEB2siRNA and on EMT and tumor proliferation, migration, invasive ability in gastric carcinoma cells in vitro were also analyzed, scratch test transwell cell invasion, and the cell cloning were used to detect the migratory, invasive,and proliferative activity of the cells.In the second partSynthetic ZEB23’UTR and miR-200b micmics were transfected into GES-1cell line by lipofectamine2000. Real-time quantitative PCR was performed to evaluate the expression levels of miR-200a/b/c and ZEB1/ZEB2mRNAs after transfection. The protein expression levels of ZEB1, ZEB2,MMP2/9and PCNA were detected by Western blot assay. The invasion and migration capability were analyzed by transwell assay and wound healing test. MTT assay was used to detect the proliferation ability.RESULTS:In the first partReal-time PCR and Western blotting assay indicated that the expressions of ZEB2mRNA and the protein in SGC7901cells interfered by ZEB2siRNA were significantly lower than those of other groups. The invasion and migration ability of SGC7901cells were also degraded after siRNA interference of ZEB2gene,and the expression of E-cadherin, miR-200familys especially miR-200b increased significantly than those of control group (P<0.05), while vimentin are decreased significantly at both m RNA and protein levels. The mobility, invasion, migration capability and the expression of MMP-9, vimentin proteins of the cells after transfected ZEB23’UTR were much higher than those of other groups (P<0.05), while the differences were not significant between control group and mutation group. Compared with siZEB2group,the expression of miR-200familys was higher and the invasion and migration ability was lower in combined group.In the second partCompared with control group and mutation group,the expressions of miR-200a/b/c especially for miR-200b was significantly decreased and the ZEB1/ZEB2were significantly increased at both mRNA and protein levels after transfected with the ZEB23’UTR and the capability of migration, invasion, proliferation were much higher (P<0.05). Compared with ZEB23’UTR group, the capability of proliferation, invasion, migration in combined group were significantly lower.CONCLUTION:ZEB23’UTR can regulates EMT dependent on the miR-200family members in gastric carcinoma,consequently inhibiting the proliferation and migratory as well as invasion ability of carcinoma cells. ZEB23’UTR can increase the ability of cell proliferation, invasion and metastasis through regulating the levels of miR-200a/b/c and then influence the regulation of transcription of the target gene,which could lead to malignant transformation of GES-1cells. which may provide a new way of gastric cancer chemotherapy and a potential gene therapeutic target. |
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