| Objective:Hepatocellular carcinoma(HCC)is one of the most common malignant tumour,ranking fourth in the global cancer-related mortality rate,and the first in the cancer-related mortality rate among men under 60 years old in China.Hypoxia is one of the characteristics of most solid tumors,including hepatocellular carcinoma.Hypoxic microenvironment promotes the metastasis,enhances radioresistant and chemoresistant in tumor cells,and changes the gene expression level.Hypoxia up-regulates the expression of key glycolysis enzymes to enhance cell glycolysis to provide energy and substances necessary for the cells survival,adapting to hypoxic environment.However,the molecular mechanism of glycolysis in hepatocellular carcinoma is still unclear Therefore,investigating the regulatory mechanism of glycolysis in hepatocellular carcinoma cells under hypoxic conditions is worthwhile.Yes association protein(YAP)is an important transcriptional co-activator and also is a key protein shuttles between cell cytoplasm and nuclear YAP is highly expressed in many malignant tumors,including hepatocellular carcinoma.And it plays a key role in transcriptional regulation in the nucleus.The regulation mechanism of YAP in tumor metabolism reprogramming has become a new research hotspot in the field of cancer therapy.Enhanced glycolysis can promote the expression of YAP protein by up-regulating the expression of glycolysis key enzymes,vice versa,YAP activation can also increase the expression of glycolysis key enzymes to promote glycolysis in cancer cells.In addition,YAP also inhibits gluconeogenesis in tumor cells and reduces the level of glucose expression in serum.However,the potential regulatory mechanism of YAP and glycolysis in HCC in hypoxic microenvironment remains unclear.Therefore,this study aims to explore the molecular mechanism of YAP in regulating glycolysis of hepatocellular carcinoma cells under hypoxia condition.And it will provide potential targets for the clinical treatment strategies in hepatocellular carcinoma.Methods:(1)1.The expression of YAP in 54 cases of hepatocellular carcinoma and adjacent tissues was detected by real-time PCR and western blotting.2.Immunohi stochemi stry,immunofluorescence and subcellular fractionation were performed to detect the localization of YAP in hepatocellular carcinoma.3.GSEA gene enrichment analysis was used to analyze the Hippo-YAP enrichment in hypoxic tissues of hepatocellular carcinoma.4.The correlation between YAP and HIF-1α expression was analyzed by real-time PCR and western blotting(2)HepG2 and Huh7 hepatocellular carcinoma cells were cultured under normal oxygen(20%O2)and hypoxia(1%O2)for 24 hours,respectively.1.Western blotting was used to detect the expression of HIF-la,LATS1,p-LATS1(Ser 909),YAP and p-YAP(Ser 127);2.The expression and localization of YAP were detected by immunofluorescence and subcellular fractionation;3.Real-time PCR was used to examine the expression of YAP downstream target genes CTGF,CYR61,AREG and EDN1;4.siRNA was transfected to knock down the expression of HIF-la and western blotting was used to detect the expression of YAP,p-YAP(Ser127)protein.and the localization of YAP was detected by immunofluorescence.5.Glucose uptake was detected by glucose detection kit and lactate production was detected by lactate detection kit.6.Extracellular acidification rate(ECAR)was detected by Sea horse XFe96.7.The expression levels of Glutl,PKM2,LDHA and PGK1 were detected by real-time PCR and Western blotting.8.Cell invasion and migration were detected by Transwell(3)The expression of YAP was knocked down by siRNA transfection and cultured under hypoxia(1%O2)for 24 hours.1.Glucose uptake was detected by glucose detection kit,lactate production was detected by lactate detection kit;2.Extracellular acidification rate(ECAR)was detected by Sea horse XFe96;3.The expression levels of HIF-1α and PKM2 were detected by real-time PCR and Western blotting;4.Cell invasion and migration were detected by Transwell(4)1.We use the HitPredict website(http://hintdb.hgc.jp/htp/)to predict the interaction between YAP and HIF-1α protein;2.Immunofluorescence was used to detect the expression and localization of YAP and HIF-1α;3.Immunoprecipitation(Co-IP)was performed to detect the binding of YAP and HIF-1α;4.Cell transfection was used to knock down the expression of YAP(siRNA)or activate the expression of YAP(YAP5SA),then cells were cultured in hypoxia for 24 hours.The expression of HIF-1α was detected by real-time PCR and Western blotting.5.Actinomycin D was used to inhibit intracellular protein synthesis and detect the expression of HIF-1α protein.6.YAP-HIF-1α binding to PKM2 promoter was detected by Chromatin immunoprecipitation(ChIP).7.Luciferase reporter gene was used to detect the binding sequence at PKM2 promoter regionResults:(1)1.YAP expression was high in 54 cases of HCC tissues as compared with the adjacent noncancerous tissues;2.HCC tissues showed stronger nuclear YAP staining as opposed to cytoplasmic staining;3.GSEA showed an enriched expression of Hippo-YAP in hypoxic HCC tissues from TCGA database.Moreover,after analyzing 369 HCC cases from TCGA database,we found a positive relationship between YAP mRNA and HIF-1αmRNA expression;4.We then analyzed the expression of YAP and HIF-1α in 54 cases of HCC tissues by real-time PCR and western blotting,it showed a positively relationship between YAP mRNA and HIF-1α mRNA expression(2)Compared with normoaxia condition(20%O2),1.The expression of HIF-1α protein was increased significantly in hypoxia condition(1%O2),while the expression of LATS1,p-LATS1(Ser909),p-YAP(Ser127)protein were decreased.The total YAP was not changed;2.Besides,hypoxia triggered YAP nuclear translocation in the HCC cell under hypoxia condition;3.And the expression of CTGF,CYR61,AREG and EDN1 were also increased significantly in hypoxia condition(1%O2);4.However,silencing HIF-1α by siRNA did not change the expression of YAP and p-YAP(Ser127),as well as the nuclear translocation of YAP in hypoxia condition(1%O2);5.In addition,hypoxia increased cells glucose uptake and lactate production rates significantly,respectively;6.Hypoxia also increased ECAR;7 The expression of Glutl,PKM2,LDHA and PGK1 were also upregulated by hypoxia,especially PKM2;8.And the cell migration and invasion abilities were also enhanced by hypoxia.(3)Under hypoxia condition(1%O2),1.cells glucose uptake and lactate production rates were decreased by YAP siRNA,2.as well as the ECAR;3.Silencing YAP also inhibited the expression of PKM2 mRNA,PKM2 protein and HIF-1α protein;4.In addition,the cell migration and invasion abilities were also inhibited by YAP siRNA(4)1.Bioinformatics predicted the interaction between YAP and HIF-1α protein;2.Then we found a co-translocation in the cells nuclear of YAP and HIF-la under hypoxia condition(1%O2);3.And Co-IP showed that YAP could bind HIF-1α in the cells nuclear under hypoxia condition(1%O2);4.After silencing YAP or activating YAP(YAP5SA),the expression of HIF-1α protein was significantly changed,however,the HIF-la mRNA level was not changed;5.Furthermore,silencing YAP promoted HIF-la protein degradation;6 Finally,we found YAP bound and sustained HIF-1α stability to bind PKM2 at its promoter(5’-ACGTG-3’)and directly activates its transcriptionConclusion:Our findings indicate that YAP forms a complex with HIF-1α in the nucleus and sustains HIF-1α stability to bind to PKM2 gene in its promoter anddirectly activates its transcription to accelerate glycolysis in HCC cells under hypoxia.The HIF-1α-YAP regulatory axis may provide a better understanding of the molecular mechanism of HCC glycolysis and progression,and YAP may be a potential therapeutic target in HCC... |
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