| BackgroundGastric cancer(GC)is one of the most common malignant tumors in the world.Since GC represents characteristics of high morbidity,high metastasis rate,high invasion rate and low cure rate,it brings a great threat to human life and health.China is one of the countries with the highest morbidity of GC.Because of its insidious onset and rapid advance,the 5-year survival rate is less than 40%among Chinese patients.Therefore,a searching for effective methods that improve the early diagnosis rate of GC and novel molecular targeting therapies as well as early warning indicators for metastasis and recurrence become a hot and difficult research for GC.miR-630,a member of the miRNA family,plays an important role in the development and progression of various malignant tumors.It exhibits different regulatory effects in tumors from different tissues.In some tumors,it functions as an oncogene.However,in other tumors,it functions as an anti-oncogene.The function of miR-630 in the development and progression of GC remains unclear.High aggressiveness and metastasis are one of the major features of GC.Invasion and metastasis are involved in the regulation of multiple gene expression and multiple signal transduction pathways,which is very complicated.Epithelial-mesenchymal transition(EMT)is one of the key mechanisms involoved in tumor cell invasion and metastasis.FoxMl plays an important role in a series of physiological processes such as cell cycle-specific gene regulation,cell proliferation and differentiation as well as organ development.Numerous studies have shown that FoxMl,which functions as an oncogene,is highly expressed in a variety of solid malignancies.It was reported that FoxMl was abnormally expressed in GC and closely related to EMT.However,the related regulatory mechanism is not fully elucidated and worth of doing futher study.ObjectiveIn the present study,we detected the expression level of miR-630 in tumor tissues from patients with GC,and analyzed its correlation with clinicopathological parameters.In addition,the target gene of miR-630 was predicted by bioinformatics software,and the molecular mechanism of miR-630 expression in the biological behavior and EMT of GC cells were investigated.The collective information will be a cornerstone for getting more messages about the mechanism of the occurrence and development of GC,and provide a reliable basis for clinical diagnosis and treatment of GC.Method(1)In the present study,80 cases of GC tissue and adjacent tissue were randomly selected from patients with GC from March 2007 to March 2010,respectively.The expression level of miR-630 in the above tissue samples was detected by quantitative real-time PCR.The correlation between miR-630 and clinical parameters and prognosis of GC patients was also analyzed.(2)The human GC cell line SGC7901 was divided into three groups;control group,miR-630 mimic group and miR-630 inhibitor group.The expression levels of miR-630 in three groups were detected by quantitative real-time PCR.The proliferation and apoptosis of the cells were observed by CCK-8 and flow cytometry,respectively.The invasive and migration abilities of the cells were measured by transwell chamber and cell scratches,respectively.(3)The target gene of miR-630 was predicted by bioinformatics analysis.The expression levels of luciferase in miR-630 mimic group,miR-630 + FoxMl-WT group and miR-630 + FOxM1-Mut group were detected.To comfirm that FoxMl was the target gene of miR-630 and participated in the process of EMT,the expression levels of FoxMl,Vimentin and E-cadherin in the control group,miR-630 mimic group and miR-630 inhibitor group were analyzed by western blot.In addition,to investigate the effect of miR-630 or FoxMl over-expression on the EMT,the expression levels of FoxM1,Vimentin and E-cadherin in the control group,miR-630 mimic group and miR-630 mimic + pcDNA3.1(+)-FoxM1 group were detected by western blot.The invasion and migration ability of each group was also analyzed by transwell chamber method.To investigate the effect of miR-630 or FoxM1 low-expression on the EMT,the expression levels of FoxM1,Vimentin and E-cadherin in the control group,miR-630 inhibitor group and miR-630 inhibitor +si-FoxMl group were detected by western blot.The invasion and migration ability of each group was also analyzed by transwell chamber method.To analyze the related molecular mechanism,the expression levels of GTP-Racl,Rac,p-PI3K,PI3K,pAKT and AKT in the control group,mimic NC group,miR-630 inhibitor group,pcDNA3.1(+)-FoxMl group and miR-630 mimic + pcDNA3.1(+)-FoxM1 group were detected by western blot.Results(1)Compared with normal tissues,the expression level of miR-630 in GC was significantly declined(P<0.05).The expression of miR-630 was correlated with tumor size,differentiation,degree of invasion,lymph node metastasis and TNM staging(P<0.05),but not with age and sex(P>0.05).The overall survival(OS)and disease-free survival(DFS)of patients with high expression of miR-630 were significantly longer than those of low expression of miR-630(P<0.01),indicating that miR-630 over-expression is associated with a good prognosis in patients with gastric cancer.Multivariate analysis showed that miR-630 was an independent predictor of OS and DFS(OS:HR = 2.46,95%CI = 1.18-3.76,P = 0.017;DFS:HR=2.54,95%CI=1.25-4.69,P = 0.0010).(2)Compared with control group,the expression level of miR-630 in miR-630 mimic group was significantly higher(P<0.05),and the expression level of miR-630 in miR-630 inhibitor group was significantly lower(p<0.05).The above results showed that miR-630 over-expression and inhibition of expression of human GC SGC7901 cell line was successfully constructed and could be used in the next experimental study.Compared with control group,the proliferative activity of miR-630 mimic cells was significantly decreased(P<0.05),while the proliferation activity of miR-630 inhibitor group was significantly increased(P<0.05).These results suggest that miR-630 inhibits the proliferation of gastric cancer SGC7901 cells.Compared with control group,the apoptosis rate of miR-630 mimic cells was significantly increased(P<0.05),while it in miR-630 inhibitor group was significantly lower(P<0.05).The above results suggest that miR-630 can promote the apoptosis of human GC SGC7901 cells.Compared with control group,the number of invasive cells in miR-630 mimic group was significantly decreased(P<0.05),while the number of invasive cells in miR-630 inhibitor group was significantly increased(P<0.05).The above results show that miR-630 has an inhibitory effect on the invasion of human GC SGC7901 cells.Compared with control group,the mobility of miR-630 mimic cells was significantly decreased(P<0.05),while the mobility of miR-630 inhibitor group was significantly increased(P<0.05).The above results show that miR-630 has an inhibitory effect on the migration of human GC SGC7901 cells.(3)FoxMl is predicted to be a target gene for miR-630,and the position of miR-630 bound to FoxMl is located in its 3'UTR region.Compared with miR-630 mimic group,the expression of luciferase in miR-630 + FoxMl-WT group was significantly lower than that in miR-630 mimic group(P<0.05).Compared with miR-630 mimic group,the expression level of luciferase in the miR-630 +FoxM1-Mut group did not change significantly(P>0.05).The results showed that FoxMl was the target gene of miR-630.The expression of E-cadherin protein in miR-630 mimic group was significantly higher than that in control group(P<0.05),conversely,the expression of FoxMl and Vimentin protein was significantly decreased(P<0.05).The expression of E-cadherin protein in miR-630 inhibitor group was significantly decreased(P<0.05),and the expression of FoxMl and Vimentin protein showed declined trend(P<0.05).Compared with the control group,the expression of E-cadherin protein in miR-630 mimic group was significantly higher than that in control group(P<0.05),and the expression of FoxM1 and Vimentin protein was lower(P<0.05).Compared with the control group,the level of E-cadherin protein in miR-630 mimic + pcDNA3.1(+)-FoxM1 group was significantly decreased(P<0.05),while the expression of FoxM1 and Vimentin protein was significantly increased<0.05).Compared with the control group,the invasion and migration ability in miR-630 mimic cells was significantly decreased(P<0.05),while it in miR-630 mimic + pcDNA3.1(+)-FoxMl cells was significantly increased(P<0.05).Compared with the control group,the expression of E-cadherin protein in miR-630 inhibitor group was significantly decreased(P<0.05),while the expression of FoxMl and Vimentin protein was significantly increased(P<0.05).Compared with the control group,the expression of E-cadherin protein in miR-630 inhibitor + si-FoxMl group was significantly higher than that in the control group(P<0.05),and the expression of FoxM1 and Vimentin protein was significantly decreased(P<0.05).The invasion and migration ability in miR-630 mimic + si-FoxM1 group was significantly decreased(P<0.05),while it in miR-630 inhibitor group was significantly increased(P<0.05).Compared with the control group,significantly,the expression of GTP-Racl,p-PI3K and p-AKT in miR-630 mimic cells were decreased(P<0.05).More importantly,the expression levels of GTP-Rac1,p-PI3K and p-AKT in pcDNA3.1(+)-FoxMl cells were significantly higher than those in the control group(P<0.05).Conclusion(1)The expression level of miR-630 in GC tissues was decreased,which was related to tumor size,differentiation degree,infiltration degree,lymph node metastasis and TNM staging in GC patients.miR-630 can be used as an independent predictor of 5-year overall survival and disease-free survival in GC patients.(2)miR-630 inhibits the progression of GC by inhibiting the proliferation,invasion and migration of GC cells and promoting apoptosis of GC cells.(3)miR-630 inhibits the expression of FoxMl by targeting Ras/PI3K/AKT signaling pathway,and ultimately inhibits the expression of EMT in GC cells. |
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