MiR-139-5p Regulates Hepatocellular Carcinoma Aerobic Glycolysis And Proliferation Through A ETS1-modulated Positive Feedback Loop

ObjectiveTo identify the role of miR-139-5p in regulation of hepatocellular carcinoma aerobic glycolysis and proliferation and then to find out the molecular mechanism of these roles.Contents and methods1.To identify the effect of miR-139-5p on hepatocellular carcinoma aerobic glycolysis and proliferation.1)Use of packaging lentivirus constracted by company and mimics to transfect hepatocellular carcinoma cells of SK-Hep-1 and SMMC-7721,establishing cell lines with over-expression of miR-139-5p;2)Activity of aerobic glycolysis in hepatocellular carcinoma cells was detected by measurement of the glucose consumption and lactate production of culture media and the expression of genes involving in aerobic glycolysis was detected by RT-PCR and Western blot after overexpressing miR-139-5p;3)Capacities of proliferation in hepatocellular carcinoma cells were detecd by CCK-8 and tumorigenicity assays in nude mice after overexpressing miR-139-5p.2.To find out the molecular mechanism underlying the regulation of miR-139-5p on hepatocellular carcinoma aerobic glycolysis and proliferation.1)The expression of transcription factors was detected by RT-PCR and Western blot after overexpressing miR-139-5p;2)Use of dual luciferase reporter assays to identify the direct target gene of miR-139-5p;3)Use of RT-PCR and Western blot assays to detect whether ETS1,the target gene of miR-139-5p,regulate the expression of HK1 and PFKFB3;4)The rescue exprements of ETS1 were performed by using RT-PCR and Western blot;5)Use of ChIP and dual luciferase reporter experiments to explore the regulatory mechanism of ETS1 on HK1,PFKFB3;6)Use of RT-PCR,Western blot,ChIP and dual luciferase reporter experiments to explore the regulatory mechanism of ETS1 on miR-139-5p.3.To identify the express feature of miR.139-5p and ETS1 in hepatocellular carcinoma tissues.1)The expression of miR-139-5p in hepatocellular carcinoma tissues was detected by RT-PCR,the expression of miR-139-5p in hepatocellular carcinoma tissues and the association with the prognosis of hepatocellular carcinoma patients was assessed by using TCGA and GEO data set;2)The expression of ETS1 in hepatocellular carcinoma tissues and the association with the expression of miR-139-5p was detected by RT-PCR.Results1.miR.139-5p suppresses aerobic glycolysis and proliferation of hepatocellular carcinoma cell.1)SK-Hep-1 and SMMC-7721 cells with overexpression of miR-139-5p had attenuated activity of aerobic glycolysis and downregulated expression of HK1 and PFKFB3 compared with scramble ones;2)HCC cells with overexpression of miR-139-5p showed compromised cell proliferation ability.2.miR-139-5p directly targets ETS1 to downregulate its expression,consequently inhibiting the target gene of ETSI including HKI and PFKFB3;Meanwhile,down-regulated ETS1 can induce miR-139-Sp expression in turn through a Drosha-dependent post-transcriptional pathway.1)The mRNA and protein levels of ETS1 were downregulated by miR-139-5p and dual luciferase reporter experiments showed miR-139-5p can directly target ETS1;2)ETS1 can promote the expression of HK1 and PFKFB3 as showed by RT-PCR and Western blot.Restoration of ETS1 protein in HCC cells remarkably abolished the inhibitory effects of miR-139-5p on the mRNA and protein levels of HK1 and PFKFB3.ChIP and dual luciferase reporter experiments showed ETS1 directly binds to the promoter regions of both HK1 and PFKFB3 to promote their expression;3)ETS1 can suppress the expression of miR-139-5p but not affect the expression of Pri-miR-139 and PDE2A,this suggests that ETS1 modulate miR-139-5p in post-transcriptional pathway.RT-PCR and Western blot showed ETS1 modulate miR-139-5p involved by the Drosha complex,ETS1 directly binds to the promoter regions of Drosha to inhibit its expression.3.miR-139-Sp was down-regulated in HCC tissues and patients with low expression mir-139 suffered poor prognosis after analysising the TCGA cohort.ETS1 was up-regulated in HCC tissues and negatively associated with the expression of miR-139-5p.ConclusionmiR-139-5p suppresses aerobic glycolysis and proliferation of hepatocellular carcinoma cell and downregulates the expression of HK1 and PFKFB3;Mechanistically,miR-139-5p can directly target ETS1 to down-regulate its expression,consequently inhibiting the target gene of ETS1 including HK1 and PFKFB3.Chromatin immunoprecipitation(ChIP)and dual-luciferase reporter assays showed ETS1 bound directly to the promoter region of HK1 and PFKFB3 to promote their expression.Meanwhile,down-regulated ETS1 can induce miR-139-5p expression in turn through a Drosha-dependent post-transcriptional pathway.Further study showed that the expression of miR-139-5p was down-regulated in HCC tissues.Importantly,patients with low expression of miR-139-5p had poorer outcome by analyzing the TCGA cohort.While ETS1 was up-regulated in HCC tissues and negatively associated with the expression of miR-139-5p.