Expression And Biological Functions Of MicroRNA-100 In Gastric Cancer

Part ? Expression of microRNA-100 in Gastric Cancer and Its Effect on Biological Functions of Gastric Cancer CellsObjective:To investigate the expression of microRNA-100 in gastric cancer cells and tissues and to analyze the biological functions of microRNA-100 in gastric cancer cells.Methods:1.Reverse transcriptional quantitative polymerase chain reaction(RT-PCR)was used to detect the expression of microRNA-100 in BGC823 and normal gastric cell line GES-1 and the difference between them was analyzed.2.RT-PCR was used to detect microRNA-100 in 39 paired gastric cancer tissues and para-cancerous tissues.The expression level of microRNA-100 in cancer tissue and para-cancerous tissues was compared.The correlation between microRNA-100 and clinicopathological features of gastric cancer was analyzed by Chi-square test and Pearson correlation analysis.Kaplan Meier survival function was used to analyze the relationship between the expression of microRNA-100 and the survival rate of patients.Finally,an independent risk factor for the prognosis of GC patients was analyzed by COX multivariate analysis.3.To evaluate the effect of microRNA-100 on the tumor phenotype of gastric cancer cells,microRNA-100 inhibitor and microRNA-100 mimic were transfected in BGC823 cells respectively.The proliferation,invasion and migration of gastric cancer cells in each group were evaluated by MTT,scratch and transwell experiments.Results:1.The results of in vitro cell RT-PCR showed that the expression of microRNA-100 in BGC823 cells was significantly lower than that of normal gastric cell line GES-1(P<0.01).2.RT-PCR for GC tissues and adjacent tissues of 39 GC patients,the results showed that the expression of microRNA-100 in GC tissues was significantly lower than that in adjacent tissues(P<0.01).3.Chi-square test and Pearson correlation analysis showed that microRNA-100 expression was closely related to lymph node metastasis(r=0.387,P=0.047),tumor diameter(r=-0.351,P=0.034)and tumor stage(r=0.347,P=0.008).Kaplan Meier survival function analysis showed that the 5-year overall survival rate of patients with significantly decreased expression of microRNA-100 was lower than that of patients with high expression,and the difference was statistically significant(P<0.05).COX multivariate analysis showed that microRNA-100,tumor size,tumor stage,and lymph node metastasis were independent risk factors for the prognosis of GC patients.4.In vitro cell experiments,microRNA-100 inhibitor and microRNA-100 mimic were transfected into BGC823 cells.Compared with the control group,the expression of microRNA-100 in the microRNA-100 inhibitor group was lower,while the expression of microRNA-100 in the microRNA-100 mimic group was significantly higher.MTT proliferation,scratches and transwell invasion and metastasis experimental results showed that compared with the control group,microRNA-100 mimic group BGC823 cell proliferation,invasion and metastasis ability significantly reduced,microRNA-100 inhibitor group BGC823 cell proliferation,invasion and metastasis ability enhanced,the difference between groups was statistically significant(P<0.05).Conclusions:The expression of microRNA-100 is significantly decreased in GC patients and BGC823 cell lines.The low expression of microRNA-100 is closely related to lymph node metastasis,tumor diameter,tumor stage and prognosis in GC patients,and promotes the proliferation,invasion and migration ability of BGC823 cells.The low expression of microRNA-100 is an independent risk factor for the prognosis of GC patients.Part ? Molecular Mechanism of microRNA-100 Regulating the Biological Functions of Gastric Cancer CellsObjective:To investigate the biological targets of microRNA-100 and analyze the possible mechanisms of microRNA-100 influence on the proliferation,migration and invasion of gastric cancer cells.Methods:1.To identify the target gene of microRNA-100,the bio-informatics software target-scan was used to predict the target gene of microRNA-100.The dual luciferase reporter gene experiments identified whether microRNA-100 binds to CXCR73'UTR.RT-PCR method was used to detect the effect of miRNA on the expression of CXCR7 in vitro.2.The expression of CXCR7 in BGC823 and GES-1 was detected by RT-PCR and Western blot.3.RT-PCR was used to detect CXCR7 expression in 39 paired gastric cancer tissues and para-cancerous tissues.The expression level of CXCR7 in cancer tissue and para-cancerous tissues was compared.The correlation between CXCR7 expression and clinicopathological features of gastric cancer was analyzed by Chi-square test and Pearson correlation analysis.Kaplan Meier survival function was used to analyze the relationship between the expression of miRNA and the survival rate of patients.4.In combination with miRNA expression levels,Pearson correlation was used to analyze the correlation between miRNA and CXCR7 expression levels.5.In vitro rescue experiments were performed to restore CXCR7 expression levels in transfected miRNA inhibitor BGC823 cells using RNAi technology,and then MTT,scratches,and transwell experiments were used to observe the effect of restoring CXCR7 expression on proliferation,invasion,and migaration of BGC823 cells.Results:1.The dual lueiferase reporter assay results showed that there was no significant change in luciferase activity in the blank control group and the negative control group,but after transfection of miRNA mimic,the activity of the luciferase enzyme vector of CXCR7-3'UTR-WT was significantly decreased.The activity of CXCR7-3'UTR-MU luciferase enzyme vector did not change significantly and the difference between groups was statistically significant(P<0.05).2.The results of RT-PCR showed that compared with the control group,the expression of CXCR7 in the miRNA mimic group was significantly decreased,and the expression of CXCR7 was significantly increased in the microRNA-100 inhibitor group BGC823 cells.The difference between the groups was statistically significant(P<0.05).3.RT-PCR experiments showed that compared with normal normal gastric cell line GES-1,the expression of CXCR7 in BGC823 cells was significantly increased,the difference was statistically significant(P<0.01).Western blot results showed that the expression of CXCR7 protein in BGC823 cells was significantly higher than that of normal gastric cell line GES-1(P<0.01).4.RT-PCR results showed that CXCR7 was up-regulated in GC tissues compared with adjacent tissues,and the difference was statistically significant(P<0.01).Pearson correlation analysis showed that the expression of CXCR7 and microRNA-100 was negatively correlated in 39 GC tissues(r=-0.923,P=0.000).5.Chi-square test and Pearson correlation analysis showed CXCR7 expression was closely related with lymph node metastasis(r=-0.485,P=0.002),tumor diameter(r=-0.399,P=0.013)and tumor stage(r=-0.332,P=0.038).Kaplan Meier survival function analysis showed that the 5-year overall survival rate of patients with significantly increased expression of CXCR7 was lower than that of patients with low expression,and the difference was statistically significant(P<0.05).COX multivariate analysis showed that microRNA-100,tumor size,tumor stage,and lymph node metastasis were independent risk factors for the prognosis of GC patients.6.In vitro rescue experiments,after silencing the expression of CXCR7 in microRNA-100 inhibitor transfected BGC823 cells by RNAi technology,compared with the microRNA-100 inhibitor group,the expression of CXCR7 in the silencing group BGC823 cells was significantly reduced and close to the level of the control group,the difference was statistically significant(P<0.05);compared with the microRNA-100 inhibitor group,the proliferation,invasion,and migration ability of the BGC823 cells in the silencing group was significantly decreased(P<0.05)and was similar to the control group.Recovering CXCR7 expression levels reversed the effects of microRNA-100 on the biological phenotype of BGC823 cells.Conclusions:CXCR7 is a biological target for miRNA100 and microRNA-100 negatively regulates CXCR7 expression in GC cells.The expression of CXCR7 in GC tissues and BGC823 cells was up-regulated.The high expression of CXCR7 was closely related to lymph node metastasis,tumor diameter,tumor stage and prognosis in GC patients,and promoted the proliferation,invasion and migration ability of BGC823 cells.Recovering CXCR7 expression can reverse the effects of microRNA-100 on the biological phenotype of BGC823 cells.microRNA-100 regulates the tumor biological function of gastric cancer cells by down-regulating CXCR7 expression.