| Background and ObjectiveGastric cancer(GC)is one of the three common malignant tumors worldwide.Although therapy of surgery,chemotherapy and radiation has made GC a great progress the mortality rate caused by GC is still in the forefront.The 5-year survival rate is hardly improved,and the prognosis is still not ideal.Risk factors associated with GC include HP infection,dietary factors,smoking,heavy physical labor,obesity and etc.The pathogenesis of GC remains to be fully elucidated.Therefore,we must explore the pathogenesis of GC,find molecular biomarkers for early detection of GC,and effective treatment of molecular targets.Micro RNAs(miRNA,miR)are a highly conserved endogenous non-coding small RNAs of 17-25 nucleotides in length,consisting of 2-7 nucleotides on their 5’untranslated region."Seed sequence" completely or incompletely binds to the target gene 3’UTR negatively regulates target gene translation,which affects a variety of cellular biological processes,including cell development,differentiation,proliferation,apoptosis,and cell cycle.But The mechanism miR remains to be elucidated,and abnormal expression of miR leads to a range of human diseases including cancers.MiR-192 and miR-215 are homologous miRNAsand negatively regulates target gene by binding the “seed sequence”.For example,in the research report,miR-192 was significantly up-regulated in lung cancer,esophageal cancer,and ovarian cancer,and miR-215 was significantly up-regulated in ovarian cancer and hepatocellular carcinoma.However,the expression of miR-192 and-215 was significantly down-regulated in colon cancer and renal cell carcinoma,suggesting that miR expression is tissue-specific and acts as a carcinogenic or tumor suppressor in different cancers,suggesting a correlation between its presence and tumorigenesis.In our previous research,we have confirmed that miR-192 and-215 are significantly up-regulated in GC,and play a role in promoting GC cell proliferation and cell migration.SET8 is called Histone lysine methylation transferase(HKMTs),was discovered in 2002,and was also named as KMT5 A,SETD8,and PR-SET7.It is a cell cycleregulated monomethyltransferase with a SET domain that specifically methylates histone H4K20 in eukaryotes to H4K20 mel for transcriptional regulation,except for the methylation mechanism.SET8 can also participate in histone modification,chromatin condensation,regulation of cell cycle,genome stability,DNA replication and other biological functions,which play an important role in regulating the growth and metabolism of organisms.SET8 plays different roles in different types of tumors.SET8 is currently considered to be an oncogene,and most of it is concentrated in breast cancer,hepatocellular carcinoma and non-small cell lung cancer.However,its specific mechanism of action is still unclear.MethodsTransfection of miR-192/-215 mimics(mims)or inhibitors(inhibitors,inhs)in gastric cells,expression of miR-192/-215 were up-regulated or down-regulatedto search for the differentially expressed genes by using gene expression profile.At the same time,using Quantitative real time polymerase chain reaction(qRT-PCR)and Western Blot confirmed the regulatory relationship between miR-192/-215 and SET8 after up-regulating or down-regulating miR-192/-215 expression in gastric cells Construction of dual luciferase plasmid determined the direct binding of SET8 to miR-192/-215;EdU detection,scratch test,apoptosis experiment verified the effect the biological function of SET8 in gastric cells;Nude mice tumor exnograft test and metastasis test of tail vein injection detected the effect of SET8 on tumor formation and GC lung metastasis.ResultsSET8 was identified as a miR-192/-215 potential target gene by gene expression profile,qRT-PCR and Western Blot.The dual luciferase activity assays sconfirmed that miR-192/-215 directly targets SET8.Western Blot and qRT-PCR showed that the levels of SET8 protein and mRNA in GC cells were significantly lower than those in normal gastric mucosal epithelial cells(P<0.01).qRT-PCR was also used to detect the expression of SET8 mRNA in 22 matched gastric tissues.The results showed that the expression level of SET8 mRNA in normal tissues was significantly higher than that in GC tissues(P<0.05).EdU detection,scratch test,apoptosis experiment confirmed that down-regulation of SET8 gene expression can affect the biological function of gastric cells;Assays in vivo confirmed that SET8 can affect tumor formation and GC lung metastasis.ConclusionMiR-192/-215 is highly expressed in GC tissues.SET8 is lowly expressed in human GC cell lines and GC tissues.MiR-192/-215 directly targets SET8 to negatively regulate its expression,and SET8 may be inhibited in GC.SET8 can inhibit cell proliferation,migration,tumor formation in vivo and Gastric metastasis,and promote apoptosis. |
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