The Role Of MiR-152 And DNMTs Family In The Carcinogenesis And Progression Of Gastric Cancer

Objective:To study the role of miR-152 and DNMTs family in the occurrence and development of gastric cancer,and to explore the possible molecular mechanism,so as to provide the experimental basis for predicting the prognosis of gastric cancer.Methods:miR-152 was detected by using in situ hybridization method in 80 gastric cancer tissues and corresponding adjacent normal tissues,and the expression level of miR-152 was detected by qRT-PCR in different degree of differentiation of gastric cancer cell lines.Then the effects of miR-152 overexpression on cell proliferation,invasion and cell anchorage dependent growth were observed in gastric epithelial cell line MKN-45 and HGC-27,which by miR-152 mimic transfection into in vitro.Immunohistochemistry and qRT-PCR were used to detect the expression of DNMTs in gastric cancer tissues and cells,as well as the correlation between DNMT1 protein expression and miR-152 expression.Bioinformatics software prediction,qRT-PCR binding luciferase reporter gene analysis were used to identified the target genes of miR-152.The expression of DNMT1 protein in cells was detected by overexpression of miR-152 and DNMT1 3 'UTR deletion mutant,and the proliferation,invasion and the ability of cell anchorage dependent growth were observed.Results:The results of in situ hybridization and qRT-PCR showed that,compared to the normal tissues,downregulation of miR-152 expression in 80%(64/80)patients with gastric cancer and gastric carcinoma.And there was significant correlation between the miR-152 expression and the depth of invasion and TNM staging of gastric cancer.Compared with early gastric cancer,the expression level of miR-152 was significantly decreased in advanced gastric cancer.The degree of differentiation was lower,the expression level of miR-152 was lower.Human gastric epithelial cell line,miR-152 expression level were significantly lower in poorly differentiated or undifferentiated gastric epithelial cell line.Gastric cancer cell lines transfected miR-152 mimic showed high expression of miR-152,the proliferation and invasion of gastric cancer cells could be significantly suppressed,as well as the ability of cell anchorage dependent growth.Immunohistochemistry and qRT-PCR results showed that the positive expression of DNMT1 was significantly higher than that of DNMT3a and DNMT3b,and the positive expression of DNMT1 in gastric cancer and the depth of invasion,lymph node metastasis and TNM stage were significantly related,while DNMT3a and DNMT3b were not found similar results.The expression of DNMT1 in gastric cancer cells was significantly higher than that in normal gastric epithelial cells,while the expression of DNMT3a and DNMT3b were contrary.Moreover,the expression level of DNMT1 was related to the degree of differentiation of gastric cancer cells.Pearson's correlation test showed that there was a negative correlation between miR-152 expression and DNMT1 protein expression in gastric cancer and gastric cancer cell lines.Luciferase reporter gene analysis and Western blot analysis shows that DNMT1 is the target gene of miR-152.By transfection of DNMT1-wtUTR(or DNMT1-mtUTR)and miR-152(mimic),found gastric cancer cells transfected miR-152 mimics,DNMT1 protein expression was inhibited by miR-152 through it's 3'UTR.It was also found that the mutation of DNMT1 3 'UTR could reverse the inhibitory effect of miR-152 overexpression on cell proliferation,invasion and tumor cell anchorage dependent growth.Conclusion:miR-152 can inhibit the proliferation,growth and invasion of gastric cancer cells,and the low expression of miR-152 is a key event in the invasion and metastasis of gastric cancer.The low expression of miR-152 in gastric cancer cells,which lead to the increase of expression of target protein DNMT1,thus affecting the relevant tumor suppressor gene hypermethylation,and promote the occurrence and development of gastric cancer,and ultimately affect the prognosis of gastric cancer.