The Role Of Hsa-miR-204-HDAC9 Pathway On Gastric Carcinogenesis And Progression

Gastric cancer is one of the common malignant tumors in the digestive system.The carcinogenesis,development and prognosis of gastric cancer is a comprehensive process affected by multiple factors in different pathways.Non-coding RNAs,such as microRNA(miRNA),can regulate genes expression at the post-transcriptional level,which is included in epigenetical regulation,thereby participating in cancer cells proliferation,migration,autophagy,senescence and so on.This process is also closely covallent to the tumorigenesis and development of gastric cancer.The abundance,specificity,and stability of endogenous miRNA have the potential to be effective molecular markers in the diagnosis,treatment,and prognosis of gastric cancer.Therefore,it is of great significance to explore the role and mechanism of specific miRNAs and the corresponding target genes in Gastric carcinogenesis for the prevention and treatment of gastric cancer.Objective:To explore the mechanism of hsa-miR-204 targeting HDAC9 to regulate the expression of CDK2 and Cyclin E involved in the gastric carcinogenesis.Methods and results:1.HDAC9 is highly expressed in gastric cancerHuman epithelial tissue samples obtained by microdissection were screened on Microarray.The results showed that the expression level of HDAC9 increased in the process of the gastric malignant transformation,from superficial gastritis,atrophic gastritis,intestinal metaplasia to gastric cancer.QRT-PCR and IHC were examined to detect the expression levels of HDAC9 in 18 pairs of gastric cancer and adjacent normal tissue specimens,as well as four types of gastric cancer cells and the gastric immortalized epithelial cells GES-1.The results showed that HDAC9 was highly expressed in gastric cancer tissues compared with the adjacent normal ones,and it was overexpressed in gastric cancer cells compared with GES-1.2.HDAC9 promotes the proliferation of gastric cancer cellsGastric cancer cells were transfected with specific HDAC9 siRNA.The results of Western blotting detected that the inhibiton of HDAC9 expression resulted in the decreased expression of CDK2 and Cyclin E.The proliferation ability of cells was checked by CCK8 assay,clony formation assay and Edu assay.It was found that inhibition of HDAC9 expreesion significantly slowed down the proliferation of gastric cancer cells.3.Decreased expression of HDAC9 inhibits tumoregenesis of gastric cancer cells in nude mice modelsSGC-7901 cells that HDAC9 expression was inhibited by specific siRNAs were injected into nude mice models for subcutaneous injection tumorigenic experiment.The experimental results showed that the tumoregenesis of gastric cancer cells in nude mice models was retarded with the expression of HDAC9 down regulated.4.Hsa-miR-204 regulates the proliferation of gastric cancer cellsGastric cancer cells were transfected with hsa-miR-204 inhibitor and mimics respectively.The proliferation ability of the cells was detected by CCK8 assay,clony formation assay and Edu assay.The results showed that inhibition of hsa-miR-204 promoted the proliferation of gastric cancer cells,while overexpression of hsa-miR-204 inhibited the proliferation of gastric cancer cells.5.Hsa-miR-204 inhibits tumoregenesis of gastric cancer cells in nude mice modelsIt was injected of SGC-7901 cells that successfully overexpressed hsa-miR-204 into nude mice models subcutaneously for tumorigenic experiment.The experimental results showed that tunoregensis of gastric cancer cells in nude mice models slowed down with overexpression of hsa-miR-204.6.Hsa-miR-204 affects the expression of CDK2 and Cyclin E by regulating HDAC9 expression level in gastric cancer cellsGastric cancer cells were transfected with hsa-miR-204 inhibitor and mimics respectively.The results of qRT-PCR and Western blotting showed that inhibition of the hsa-miR-204 expression increased the expression of HDAC9,CDK2 and Cyclin E,while overexpression of hsa-miR-204resulted in the opposite.7.Hsa-miR-204 binds to the 3’-UTR of HDAC9 directly to regulate the proliferation of gastric cancer cellsThe result of the dual luciferase activity assay indicated that hsa-miR-204 could directly bind to the 3’-UTR region of HDAC9.By co-transfecting hsa-miR-204 inhibitor and HDAC9 siRNAs in gastric cancer cells,it was showed with Western blotting that the increased expression of CDK2 and Cyclin E induced by hsa-miR-204 inhibition could be partially restored with inhibition of HDAC9 expression.The results of CCK8 assay,clony formation assay and Edu assay suggested that inhibition of HDAC9 expression could partially restore the promotion of hsa-miR-204 inhibition on cell proliferation.Conclusion:The hsa-miR-204-HDAC9 pathway might regulate the expression of CDK2 and Cyclin E to participate in the proliferation of gastric cancer cells.