Hypoxia-induced Mir-214 Expression Promotes Tumour Cell Proliferation And Migration By Enhancing The Warburg Effect In Gastric Carcinoma Cells

Objective:microRNAs(miRNAs)are a class of endogenously expressed,single-stranded,non-coding RNAs with the length about 22 nucleotides.Through base-pairing with 3’untranslated region of messenger RNA,miRNA depress expression of target genes at the post-transcriptional level by inhibiting translation of mRNA or by destabilizing mRNA.miRNA showed a duality in the initiation and progression of cancers(GC).Namely,miRNAs play a role as oncogene in kinds of tumors while serve as tumor suppressor genes in others.miR-214 was demonstrated to be up-regulated in the gastric cancer and serve as an oncogene in kinds of cancers such as Melanoma,Osteosarcoma,T-cell lymphoma,Glioma,Hepatocellular,Stomach,Uterus cervix,Bladder,Colorectal,Ovary and Breast cancers.However,how it was upregulated and came into play in the gastric cancer was not clear.PRDM16(PR/SET domain 16)was known since it can regulate brown/beige fat identity and function while A2AR(Ado A2A receptor)was of importance in the immunotherapy.But the roles they play in the gastric cancer and the relationship between miR-214 and PRDM16,A2AR is not clear.Methods:miR-214 level was checked by PCR.A2AR and PRDM16 expression were estimated by western blotting and PCR.Luciferase reporter assay to identify the target of miR-214.Transwell chambers(8-μM pore size;Costar)were used in the in vitro migration assay.Cell Counting Kit(Dojindo;Japan)was used to evaluate the growth ability.The CellTiter-Glo(?)Luminescent Cell Viability Assay(Promega;USA),Lactic and Glucose(Biovision;USA)Assay Kit were used to detect the Warburg effect.Results:miR-214 was upregulated due to the environment of hypoxia and it was of high expression in the human gastric cancer tissues.A2AR and PRDM16 was down-regulated in the gastric cancers,which was antiparallel with the upexpression of miR-214.Luciferase reporter assay proved that miR-214 can specifically target the 3’UTR of A2AR,PRDM16 and regulate their expression.Upregulation of miR-214 in the environment of both hypoxia or normoxia can effectively enhance migration,proliferation and the Warburg effect of gastric cancer cell lines while downregulation of miR-214 reduced migration,proliferation and the Warburg effect.So,we can see that miR-214 serve as an oncogene in the gastric cancer.When knocking down the expression of A2AR and PRDM16 by the siRNA,we come to the result that down-regulation of A2AR or PRDM16 enhanced proliferation,migration capacities and the Warburg effect of gastric cancer cells.Animal experiments confirmed that miR-214 can promote tumor growth and the effect of miR-214 can be recovered by up-regulation of A2AR and PRDM16.Conclusions:Our study demonstrated that hypoxia induced miR-214 can specifically target the 35 UTR of A2AR and PRDM16 and reduced the mRNA and protein level of them.miR-214,by down-regulation A2AR and PRDM16,enhances the Warburg effect and thus promotes proliferation and migration of GC cells.