| Background and objective:Gastric cancer is a high invasive cancer and is among the most prevalent malignancies in china, and is the second cause of cancer related death worldwide. It has caused serious damage to people healthy. Many factors play roles in the occur and development of gastric cancer by mediating lots of genes and other molecules. microRNAs are small non-protein-coding RNAs, consists of approximately 22 nucleotides. Through specific binding to the complementary 3’or 5’un-translation region sequence of the target genes, microRNA can repress the expression of the targeting genes or degrade the genes according the complementary ratio. microRNAs can regulate many cellular process such as cell proliferation, differentiation, cell senescence and apoptosis. Prior evidences have already demonstrated that many microRNAs were abnormal expressed in gastric cancer, and many of them were involved in the tumorigenesis, prognosis, progression, metastasis, invasion and resistance to drug and radioactive therapy. Emerging evidences showed that microRNA can function as early biomarker to detect and diagnose gastric cancer. This study is based on our previous research, in this study, we constructed the microRNA-4754 over-expression lentivirus and transfected it into the SGC-7901 cell, then we tested the biology effects of microRNA-4754 exerted on cell proliferation, colony formation, migration and metastasis ability and cell apoptosis of human gastric cancer cell SGC-7901,moreover, we also explored its potential mechanism.Methods:The real-time PCR was employed to test the expression of microRNA-4754 in six human gastric cancer cell line:AGS, SGC-7901, BGC-823, NCI-N87, MKN-28, MGC-803,according to the results, we chosen one cell line of the lowest microRNA-4754 expression level to do the following tests. We constructed the microRNA-4754 over-expression lentivirus and transfected it into the SGC-7901 cell, then we still use the real-time PCR to detect the expression level of microRNA-4754 after transfected. Cell Counting Kit-8(CCK-8) and colony formation assay were chosen to detect cellular viability; Transwell and wound healing assay were employed to test cellular migration and metastasis ability; Flow Cytometry and Hoechst33342/PI were both used to test cell apoptosis. We further explored the mechanism of microRNA-4754 through Western Blot. The experiments were divided into three groups, control group:normal SGC-7901 cell; negative group:SGC-7901 cell transfected with blank lentivirus; experiment group:SGC-7901 cell transfected with microRNA-4754over-expression lentivirus.Data analysisBoth data were analyzed used the Graphpad Prism5 software. All data were reported as the mean ± standard deviation. Differences between groups were analyzed by t-test and one way-ANOVA test. All values were presented based on two-tailed statistical analysis, p<0.05 were considered as statistically significantly.Results:1.The results of real time PCR showed that the expression of microRNA-4754 can be detected in all of the six gastric cancer cell lines, and it was lowest expressed in SGC-7901 cell; the expression level of microRNA-4754 in SGC-7901 cell was significantly raised after transfected with the over-expression lentivirus of microRNA-4754;2. The results of CCK-8 and colony formation experiments indicated the proliferation and colony formation rate of SGC-7901 cell was significantly inhibited after transfected with over-expression lentivirus of microRNA-4754 compared to the negative and control group, cell viability was significantly inhibited;3. The migration and metastasis experiments were both tested by Trans well experiments, and the results showed the number of cells invaded the membrane were significantly decreased after transfected with the over-expression lentivirus of microRNA-4754 when compared to the negative and control group, cell migration and metastasis ability were both inhibited by over-expression of microRNA-4754;4. The wound healing results showed after cultured for 48h, SGC-7901 cell transfected with microRNA-4754over-expression lentivirus moved slowly to the centre of the healing compared to the negative and control group;5. The Flow Cytometry results showed the apoptosis rate of SGC-7901 cell transfected with microRNA-4754over-expression lentivirus was higher than the negative and control group;6. Hoechst33342/PI double staining results confirmed that in the same culture condition, the rate of PI positive cell (apoptosis cell) in SGC-7901cell transfected with microRNA-4754over-expression lentivirus was higher than it in the negative and control group;7.Western Blot results showed that after transfected with microRNA-4754 over-expression lentivirus, the expression level of Phosphorylation rapamycin target protein was significantly decreased compared to the negative and control group.Conclusions:1. The construction of microRNA-4754over-expression lentivirus was effective and it can stable transfected into the human gastric cancer cell line SGC-7901;2. microRNA-4754 can inhibit the proliferation, colony formation, migration and metastasis ability and can induce the apoptosis of SGC-7901 cell, indicated that microRNA-4754 can exert anti-cancer bioactivity in human gastric cancer cell line SGC-7901;3. microRNA-4754 can exerts anti-cancer bioactivity in human gastric cancer cell line SGC-7901 through mediating the expression of Phosphorylation rapamycin target protein. |
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