MicroRNA-429 Inhibits Proliferation And Invasion Of Gastric Cancer By SIX1 Gene

Objective: In China,the mortality of gastric cancer is second,only after lung cancer.Although in recent years the morbidity of gastric cancer has declined in developing countries and early diagnosis and treatment have been improved,the prognosis of gastric cancer is also poor.Metastasis and recurrence are two of the most important reasons of the mortality of gastric cancer.Therefore,it is very necessary to find the molecular and discuss the molecular pathogenesis and signal paths involved in the development of gastric cancer,which has an important significance in detection,diagnosis and treatment of gastric cancer.As one of the sine oculis homeobox(SIX)family,SIX1 exists in many animals and human.SIX1 can control the cell cycle progression,promote cell proliferation and migration,and inhibit cell apoptosis.SIX1 plays an important role in embryonic cell development and differentiation of many organs.Recent researches have shown that SIX1 is an important oncogene,which is overexpressed in many tumors of genital system,digestive system and hematological system,and is related with the prognosis of some tumors.Micro RNAs are small RNAs with non-coding.Recent researches have shown that mi RNA can control cell differentiation,proliferation,apoptosis,migration and invasion of many tumors,including gastric cancer.As one of the mi RNA,mi R-429 also involves in the development of malignant tumors.There are researches on mi R-429 inhibiting the development and invasion of liver cancer and prostatic cancer.But the effect of mi R-429 in human gastric cancer has not been clarified yet.In this study,we examined SIX1 protein expression in gastric cancer tissues to discuss its relationship with clinical pathological parameters and prognosis.We also discuss the pathogenesis involving in cell proliferation and invasion of gastric cancer in molecular level.Also,we detected mi R-429 expression in gastric cancer tissues and analyze the mechanism of mi R-429 in gastric cancer.Methods: In our study,SIX1 protein expression in gastric cancer tissues was examined by immunohistochemistry.We used RT-PCR and Western blot to detect the SIX1 expression in gastric cancer cells.The effect of SIX1 on malignant behavior of gastric cancer cells was assessed by growth curves and transwell experiment.Western blot was used to detect the related proteins of SIX1.Also,we examined mi R-429 expression in gastric cancer by RT-q PCR.The effect of mi R-429 on gastric cancer cells was assessed by CCK8 and transwell experiment.Western blot was used to detect the related proteins of mi R-429.Moreover,we constructed the system of mi RNA target gene detection to clarify if SIX1 was the target gene of mi R-429 in gastric cancer.Results: 1.SIX1 was over expressed in gastric cancer tissues.In 208 gastric cancer tissues,49.5%(103/208)SIX1 was over expressed.2.The expression of SIX1 was related with TNM stage and lymph node metastasis(p<0.05).Survival analysis by Kaplan-Meier survival curve and log-rank test demonstrated that high expression of SIX1 in cancer tissue predicted worse overall survival.3.MTT showed that SIX1 promoted cell proliferation of SGC-7901 cells,and SIX1 si RNA could inhibit cell proliferation of HGC-27 cells.Transwell showed that SIX1 promoted cell invasion(p<0.05).4.SIX1 could up-regulate the expression of cyclin D1 and MMP2 in SGC-7901 cells.In HGC-27 cells SIX1 interference could inhibit these proteins.5.Overexpression of SIX1 could up-regulate the phosphorylation level of ERK.6.SIX1 could down-regulate the expression of E-cadherin in SGC-7901 cells,and SIX1 interference could up-regulate the expression of E-cadherin.7.The expression of mi R-429 was lower in gastric cancer tissues compared to normal mucosa tissues.8.mi R-429 inhibited cell proliferation and invasion.CCK8 showed that cell proliferation decreased after mi R-429 mimic transfection.Transwell showed that after mi R-429 mimic transfection,the number of SGC-7901 cells decreased(p<0.05).9.mi R-429 could inhibit the expression of MMP2 and ERK pathway.After mi R-429 mimic transfection the expression of MMP2 decreased and mi R-429 could inhibit ERK pathway.10.SIX1 was a direct target gene of mi R-429 in gastric cancer cells.Western blot showed that mi R-429 could inhibit the expression of SIX1.mi RNA target gene report system showed 3’ non-code area of SIX1 gene was the direct binding site with mi R-429.Conclusion: 1.SIX1 was over expressed in human gastric cancer tissues.Overexpression of SIX1 has relationship with TNM stage and lymph node metastasis,which could predict worse overall survival.2.Overexpression of SIX1 promoted cell proliferation and invasion of gastric cancer cells.3.SIX1 promoted cell invasion and metastasis through ERK-MMP2.4.mi R-429 was lower expressed in gastric cancer tissues and inhibited cell proliferation and invasion of gastric cancer cells.5.micro RNA-429 inhibited cell proliferation and invasion of gastric cancer by SIX1 regulating MMP2.