LncRNA WAC-AS1 Promotes Glycolysis And Tumor Progression In Hepatocellular Carcinoma

PART Ⅰ Identification of novel glycolysisrelated long non-coding RNA WAC-AS1 and its effect on the prognosis of HCC patientsBackground and objective Hepatocellularcarcinoma(HCC)is a common malignant tumor in human.Its morbidity and mortality ranks the 4th and 2th among malignant tumors in China.The overall prognosis of HCC is still not satisfactory,mainly due to the complications of the pathogenesis,the difficulty in early diagnosis and easy recurrence after surgery.Studies have shown that energy metabolic reprogramming is an important biological behavior of tumors,and long non-coding RNAs(lncRNAs)play an important role in regulating tumor metabolism.So in this part we identified lncRNAs associated with glycolysis and prognosis in HCC and analyzedthe correlation between lncRNAs and prognosis of HCC patients..Methods We analyzed the co-expression of glycolysis-related genes and lncRNAs in The Cancer Genome Atlas(TCGA)database to screen glycolysis-related lncRNAs.Further prognostic analysis and differential expression analysis were performed.We further analyzed the relationship between lncRNAs and clinicopathologic features of patients.Results We screened 502 lncRNAs that have co-expression relationships with glycolytic genes based on co-expression analysis.Among them,112 lncRNAs were abnormally expressed in HCC,and 40 lncRNAs were related to the prognosis of patients.Eight lncRNAs(WAC-AS1,SNHG3,SNHG12,MSC-AS1,MIR210HG,PTOV1-AS1,AC145207.5 and AL031985.3)that were closely related to the prognosis of patients werescreened out bymultivariate COX regression analysis.Among all the screened lncRNAs,lncRNA WAC-AS1 had the greatest prognostic impact(P<0.05,HR>1).The analysis of clinical samples from Zhongnan Hospital verified that the high expression of lncRNA WAC-AS1 correlates with the patients’prognosis(P=0.007,HR=1.613),tumor diameter(P=0.001)and lymph node infiltration(P=0.021).Conclusion We identified a novel lncRNA WAC-AS1 associated with glycolysis and prognosis in hepatocellular carcinoma(HCC)based on co-expression analysis and prognostic analysis of glycolysis related genes.Part Ⅱ Effects of long non-coding RNA WAC-AS1 on the tumor biological behavior of HCCObjective To investigate the expression of long non-coding RNA WAC-AC1 in HCCcell lines and its effect on thebiological behavior of HCC invivoand in vitro.Methods RT-qPCR was used to detect the expression difference of lncRNA WAC-AS1 in liver cancer cell lines and normal liver cell lines.After knockdown or overexpression of lncRNA WAC-AS 1,plate cloning assay and CCK-8 assay were used to detect cells proliferative capacity and cell viability.For the detection of glycolysis levels,we used Glucose Colorimetric Assay Kit Ⅱ to measure changes in the rate of cellular glucose uptake,and lactate assay kit to detect Lactic acid production rate.Extracellularacidification rate was detected by extracellular flux analyzer.A nude mouse model of subcutaneous transplanted tumor was established for in vivo experiments.The cells were inoculated subcutaneously into nude mice,and the effect of lncRNA WAC-AS1 on tumor proliferation was detected according to the growth curve and size of the subcutaneous tumor.Results The expression of lncRNA WAC-AS1 in HCC cell lines was significantly higher than that in HCC cell lines(P<0.05).The results of plate colony formation assay showed that the knockdown or overexpression of lncRNA WAC-AS1 could significantly inhibit or promote the proliferation of tumor cells(P<0.01).CCK-8 assay showed that the viability of HCC cells was significantly regulated by the knockdown or overexpression of lncRNA WAC-AS1(P<0.001).The overexpression lncRNA WAC-AS1 can promote glucose uptake and lactic acid production of HCC cells(P<0.01).Animal experiments showed that the overexpression of lncRNA WAC-AS1 could promote the growth of HCC cells(P<0.01).Conclusions LncRNA WAC-AS1 promotes progression of hepatocellular carcinoma in vitro and in vivo.LncRNA WAC-ASlis anovel biomarker forprognosis of HCC patients.Part Ⅲ Study on the mechanism of long non-coding RNA WAC-AC1 in promoting the progression of HCCobjective To explore the mechanism of long non-coding WAC-AS1 in promoting the progression ofHCC.Methods RT-qPCR analysis and western blot analysis were performed to verify the co-expression relationship between WAC-AS1 and glycolysis related genes at RNA and protein levels.The correlation between the expression of WAC-AS1 and ARPP19 was analyzed in TCGA database,and the mechanism of the WAC-AS1 regulation on ARPP19 was speculated.Bioinformatics techniques were used to predict the binding sites among WAC-AS1,miENA-320d and ARPP19.The dual luciferase assay was used to verify the binding site between WAC-AS1,miRNA-320d and ARPP19.The regulation of WAC-AS1 on the expression of ARPP19 as endogenous RNA competitively binding miR-320d was analyzed by RT-qPCR.The regulation of WAC-AS1 and miR-320d on the expression of ARPP19 protein was detected by RT-qPCR and western blotting analysis.The effects of WAC-AS1 and miR-320d on glucose uptake and lactic acid production of hepatocellular carcinoma cells were evaluated under hypoxia conditions.Results LncRNA WAC-AS1 and glycolysis related genes have co-expressed relationship at RNA and protein levels,and the knockdown or overexpression of WAC-AS1 could affect the expression of several glycolysis-related genes(ARPP19,CHST12,MED24 and KIF2A)(P<0.01).The expression of the markers of glycolysis LDHA and TPI1 were regulated by WAC-AS1 too(P<0.01).Through bio informatics analysis,miR-320d and ARPP19 were identified as downstream molecules of lncRNA WAC-AS1.The expression of ARPP19 were down-regulated at mRNA and protein levels after the knocked down of lncRNA WAC-AS1,and this regulation could be reversed by miR-320d mimics.The specific binding sites between lncRNA WAC-AS1,miR-320d and ARPP19 were further verified by dual luciferase assay.The results of salvage experiment showed that miR-320d could reverse the regulation of lncRNA WAC-AS 1 on the expression of ARPP19,and the glucose uptake and lactate production of HCC cells was also reversed.Through the above experimental results,we proved that the glycolysis of HCC was regulated by lncRNA WAC-AS1 through miR-320d/ARPP19 axis.Conclusion LncRNAWAC-AS1 acts as endogenous competitive RNA to bind miR-320d,thereby upregulates the expression of ARPP19 and eventually promotes glycolysis and cell proliferation in HCC.