MiR-30c-5p Suppresses Gastric Cancer Cell Growth And Metastasis By Directly Targeting ADAM12 Expression

Background: Globally,gastric cancer（GC）is one of the most common malignant tumors in the digestive system.Despite decades of development,the mortality of patients with GC has decreased,but the burden and costs of medical rehabilitation have significantly increased.Because of the high complexity of the mechanisms involved in the development and progression of gastric cancer,current treatments cannot yet achieve satisfactory results.Micro RNAs（miRNAs）are small non-coding RNAs that can play regulatory roles in most cell types by degrading or repressing the expression of m RNAs.In addition,miRNAs can participate in various biological processes,including cell proliferation,apoptosis,differentiation,etc.Numerous studies have reported that the aberrant expression of miRNAs could promote or inhibit tumor progression.Mi R-30c-5p（miR-30c）is one of the miR-30 family members,and multiple experiments have shown that miR-30 c might play a suppressive role in a variety of tumors.As one of the target genes of miR-30 c,ADAM metallopeptidase domain 12（ADAM12）acts as an oncogene in various tumors.However,the roles of miR-30 c and ADAM12 in GC remain unclear.Meanwhile,whether miR-30 c regulates GC progression by targeting ADAM12 is a mechanism to investigate.Objective: In order to investigate the development mechanism of gastric cancer and improve the prognosis of patients with gastric cancer,this study explored the effect of miR-30c-5p（miR-30c）and ADAM12 on GC and verified the target relationship between them.Methods: The Gene Expression Omnibus（GEO）and the Cancer Genome Atlas（TCGA）databases were used to assess the expression levels of miR-30 c and ADAM12 in GC.In contrast,the Kaplan Meier plotter database was used to assess the prognostic value of miR-30 c and ADAM12.TCGA gastric cancer expression data were combined,and Targetscan,DIANA,and miRwalk databases were used to predict and screen miR-30 c target genes.Gastric cancer mucosal epithelial cells（GSE-1）and two gastric cancer cells（MGC-803 and BGC-823）were used as research objects.The expression levels of miR-30 c and ADAM12 were detected using a real-time fluorescence polymerase chain reaction.Dual-luciferase reporter assay validated the targeting relationship of miR-30 c and ADAM12.The expression of miR-30 c was up-regulated or suppressed in cells by synthetic miR-30 c mimics（mimics）or inhibitor set（inhibitor）and their corresponding controls using Lipofectamine transfection.Lentiviral infection was utilized to construct ADAM12 cell lines that were overexpressed or suppressed.Plate cell clonogenic assay,CCK8 assay,and transwell assay were performed to evaluate the effect of miR-30 c / ADAM12 on proliferation,metastasis,and invasion of gastric cancer cells.The effects of miR-30 c / ADAM12 on cell cycle distribution and apoptosis were assessed by flow cytometry,and the corresponding protein levels were determined by Western blotting.In order to verify whether ADAM12 over-expression reverses the inhibitory effect of miR-30 c on ADAM12,a co-transfection method was employed to transfect miR-30 c mimics into ADAM12 lentivirus stable gastric cancer cells.protein-protein interaction network was used to evaluate the relationship between ADAM12 and cyclins and apoptosis proteins.Results:1.miR-30 c expression was down-regulated in gastric cancer cell lines.GEO and TCGA data analysis results showed that miR-30 c expression was down-regulated in gastric cancer tissues and associated with a good prognosis.2.Overexpression of miR-30 c was able to inhibit gastric cancer cell proliferation,migration,invasion,promote apoptosis（such as BCL2 down-regulated and Bax /Cleaved Caspase-3 up-regulated）,and arrest the transformation of gastric cancer cells from G0 / G1 to S phase（such as Cyclin D1 downregulated and P21CIP1 / P27KIP1 /P53 up-regulated）.3.The results of bioinformatics analysis indicated ADAM12 was the target gene of miR-30 c,and the dual-luciferase reporter assay showed that there was a target relationship between them.4.ADAM12 expression was up-regulated in gastric cancer cell lines.The results of GEO and TCGA data analysis showed that ADAM12 expression was up-regulated in gastric cancer tissues,and a higher ADAM12 expression was associated with a poor prognosis.Moreover,ADAM12 has the potential as a diagnostic marker for GC.5.Knockdown of ADAM12 inhibited GC cell proliferation,migration,invasion,promoted apoptosis（related proteins,such as BCL2 down-regulation,Bax/cleaved caspase-3 up-regulation）,and arrested gastric cancer cell transformation from G0/G1 to S phase（related proteins,such as Cyclin D1 down-regulation,P21CIP1 / P27KIP1 / P53up-regulation）.Conclusion: In vitro miR-30 c could inhibit gastric cancer cell proliferation,migration,invasion,inducing apoptosis and arrest gastric cancer cell transition from G0/G1 to S phase by targeting ADAM12.miR-30 c expression is decreased,and the prognosis is good in gastric cancer;conversely,ADAM12 expression was increased,and the prognosis was poor in gastric cancer.The oncogene ADAM12 was valuable for the diagnosis of gastric cancer.