Functional Mechanism Of HK1 MRNA M6A Demethylation Modification Mediated By RNA M6A Demethylase ALKBH5 Promoting Metabolic Reprogramming Of Hepatocellular Carcinoma

Background and objectiveHepatocellular carcinoma(HCC)is one of the most common malignant tumors in the world.It is a kind of primary liver cancer with high mortality.At present,surgical resection is the main treatment of primary liver cancer.However,due to the lack of special clinical symptoms and the rapidly course of disease,most liver cancer patients are in the middle or late stage at the time of diagnosis,and the best opportunity for surgery has been lost,resulting in a high postoperative recurrence rate,a poor prognosis and a short survival time.In addition,the pathogenesy of hepatic carcinoma is very complex,and there is still a lack of effective molecule-targeted drugs.Finding out effective molecular therapeutic targets is essential for the treatment of HCC patients.Glycolysis is the main energy metabolism feature of tumor cells,and N6-methyladenosine(m6A)is the most common internal modification of mRNA.In recent years,it has been found that m6 A modification is closely related to the occurrence and development of tumor,but its role in HCC,especially the effect on glycolysis of tumor,has not been reported.In this study,we found that the expression of m6 A demethylase ALKBH5 was up-regulated in HCC,and the up-regulated expression of ALKBH5 was closely related to the poor prognosis of HCC patients,suggesting that ALKBH5 plays an important role in HCC.This paper aims to study the functional mechanism and clinical significance of the regulation of m6 A demethylase ALKBH5 on glycolysis metabolism and occurrence and development of HCC,and to provide new target clues for improving the prognosis of HCC patients.Methods1.The mRNA and protein levels of ALKBH5 in HCC tissues were detected by RT-PCR and Western blot assays.2.We studied the relationship between ALKBH5 and the clinicopathological features and prognosis of HCC patients by analyzing the data of HCC clinical samples.3.Western Blot and RNA dot blot were used to detect the effects of ALKBH5 on the methylation of RNA in HCC cells.4.Glucose uptake and lactate production were measured to study the effect of ALKBH5 on the glycolysis of HCC cells.5.The effects of ALKBH5 on the migration,invasion,cell proliferation and colony formation of HCC cells were detected by Transwell,cell growth and colony formation assays.6.ALKBH5-Knockout-SMMC7721 cells were injected into nude mice to detect the effect of ALKBH5 on subcutaneous tumor formation.7.Bioinformatics analysis of m6 A high-throughput sequencing data,real time PCR and Western blot were preformed to filter the downstream molecules regulated by ALKBH5 demethylation,identified the site of ALKBH5 demethylation.8.Through Western Blot,cell function experiments and detection of glucose uptake and lactate production,the effect of HK1 on the glycolysis and malignant phenotype of hepatocarcinoma cells and the role of ALKBH5 in it were detected.9.mRNA half-life and mRNA stability assay and RIP were preformed to detect the effect of ALKBH5 on HK1 mRNA stability.10.Bioinformatics prediction,Luciferase,Mutation,Ch IP-q PCR and EMSA were preformed to detect and verify the expression of ALKBH5 activated by ATF2.Results1.Both the mRNA and protein levels of ALKBH5 were significantly up-regulated in HCC tissues.2.The up-regulated expression of ALKBH5 was positively correlated with tumor recurrence and clinical stage of HCC patients,and ALKBH5 was an independent predictor of prognosis in patients with HCC.3.ALKBH5 decreased the total RNA methylation levels in HCC cells.ALKBH5 promotes the glycolysis,migration,invasion,cell proliferation and colony formation of HCC cells.It also promotes the subcutaneous tumor formation of HCC cells.4.The hexokinase 1(HK1)mRNA was identified as one of the downstream targets of ALKBH5 demethylation.In HCC,ALKBH5 promotes the expression of HK1,and the levels of HK1 were positively correlated with the levels of ALKBH5.The site of ALKBH5 demethylation on HK1 mRNA was identified.5.We found that ALKBH5 promoted the expression of HK1 and that promoted the glycolysis,migration and invasion,cell proliferation and colony formation of HCC cells.On the contrary,ALKBH5 silencing or knockout inhibited the expression of HK1,the glycolysis and the malignant phenotype of HCC.6.ALKBH5 enhances the stability and expression of HK1 mRNA by promoting the combination of HK1 mRNA and RNA stabilizing factor Hu R.7.In HCC,ALKBH5 is regulated by transcription factor ATF2,which promotes the transcription of ALKBH5.ConclusionsIn this study,we found that the expression of ALKBH5 was up-regulated in HCC,and the up-regulated expression of ALKBH5 was closely related to the poor prognosis of HCC.ALKBH5 promotes the glycolysis and malignant phenotype of HCC.We elucidated the molecular mechanism that ALKBH5 promoted the metabolic reprogramming,occurrence and development of HCC through demethylation of a methylation site(A3014)on HK1 mRNA.We demonstrated that ATF2,as a transcription factor,induces ALKBH5 transcription and promotes its expression.We also elucidated the mechanism of the upregulation of ALKBH5 in HCC.