MiR-885-5p Regulates Glycolysis By Silencing Hexokinase 2 In Hepatocellular Carcinoma

Objective Growing tumor cells possess a distinct metabolic phenomenon that allows them to preferentially utilize glucose through aerobic glycolysis,which is referred to as the“Warburg effect.”Accumulating evidences suggest micro RNAs(miRNAs)could regulate such metabolic reprogramming.This study aims to explore the role of miR-885-5p in the development and progression of hepatocellular carcinoma(HCC)and whether it can regulate the"warburg effect"under hypoxia conditions.Primary study on the mechanism of miR-885-5p in glycolysis under hypoxia,evaluating its targeted gene function and prognostic value.related gene function analysis.Methods Agilent human miRNA array was used to analyze the expression of mir-885-5p in seven liver cancer cell lines and three normal primary hepatocytes(PHHC).Further,quantitative Real-Time PCR(qRT-PCR)was used to verify the expression of mir-885-5p in eight liver cancer cell lines and four PHHC.Different types of hypoxia models were constructed and appropriate hypoxia modes and conditions were selected for subsequent cell experiments.Cationic liposome was constructed to deliver miR-885-5p and its transfection efficiency was evaluated.The role of miR-885-5p in the development of HCC was investigated through cell proliferation,invasion,migration assay,cell apoptosis and cycle analysis after hypoxia induction and transfection with a miR-885-5p mimic or inhibitor and its corresponding control.The subcutaneous transplanted tumor model of liver cancer was constructed to study the effect in vivo.The regulation of miR-885-5p on glycolysis under hypoxia was studied by glucose uptake,lactic acid generation experiments and detection of glycolysis related key enzyme by western blot.Target genes of miR-885-5p were found by targetscan and other miRNA target gene prediction softwares,and verified by qRT-PCR,western blot and luciferase assay.Si RNA interference technique was used to downregulate hypoxia-inducible factor 1?(HIF-1?),and western blot was used to detect the expression of HK2 and other key enzymes in glucose metabolism to clarify the molecular mechanism of miR-885-5p regulating glycolysis under hypoxia.A variety of databases were used to collect clinical data of patients with HCC for bioinformatics analysis of the expression levels of miR and its target genes,to explore the correlation between expression and clinical characteristics of patients,to conduct protein interaction,GO and GSEA functional enrichment analysis of gene expression,and to evaluate the prognostic value of miR-885-5p and its target genes.Results Our microarray analysis and qRT-PCR validation revealed miR-885-5p was strongly down-regulated in HCC tissues and cell lines relative to the paired para-carcinoma normal tissues and PHHC.The expression of miR-885-5p in different anoxic models(anoxic incubator,anoxic chemical inducer Co Cl₂and DFX)was generally down-regulated compared with normoxia.Liposomes own a high transport efficiency,which can be absorbed rapidly by HCC cells and completely release miR-885-5p.Exogenous overexpression of miR-885-5p inhibited the proliferation,migration and induced apoptosis of HCC cells in vitro.Compared with normoxia,the inhibition of the above malignant phenotypes of HCC cells was more significant under hypoxia.Therapeutic application of miR-885-5p significantly inhibited the growth of subcutaneous tumors in nude mice.Forced expression of miR-885-5p in HCC cells significantly reduced glucose uptake and lactate production by repressing several key enzymes related to glycolysis.HK2 was found to be the direct target gene of miR-885-5p.The role of miR-885-5p in regulating glycolysis by targeting HK2may be partially mediated through HIF-1?under hypoxia.Western blot and immunohistochemical analysis showed that the expression of HK2 in HCC cell lines and tissues was significantly higher than that in PHHC and normal tissues.HK2interaction proteins are all key proteins in the glycolysis pathway.GSEA(Gene Set Enrichment Analysis)of the high-expression group are enriched in the aspects of cytokine production,protein secretion,response to biological stimuli,GTPase activity,MAPK pathway regulation,immune defense regulation,movement,cell adhesion and so on.Both miR-885 and HK2 have prognostic value in HCC patients.Conclusions We found that the anoxic regulation of miR-885-5p can regulate glycolysis in HCC,which plays an important role in the occurrence and progress of HCC.The regulation by targeting HK2 may be partially mediated through HIF-1?.Both miR-885-5p and HK2 are closely related to the clinical prognosis of HCC patients.A comprehensive analysis of the regulatory axis of miR-885-5p/HK2 may contribute to exploring new therapeutic targets for HCC patients and accurately evaluate their prognosis.