| Hepatocellular carcinoma(HCC)is one of the most common malignant tumors,and its mortality rate ranks second all over the world.Liver cancer stem cells(LCSCs)are a small group of heterogeneous cell populations with self-renewal ability and multi-directional differentiation potential,which play an important role in drug resistance and recurrence of liver cancer.Hepatitis B virus(HBV)infection is closely related to the development of HCC.And hepatitis B virus x protein(HBx)is essential for the replication and spread of HBV,which involved in the regulation of genome stability,signaling pathway and cell cycle.However,the relationship between HBx-mediated cellular stress response and LCSCs in the process of liver carcinogenesis remains to be elucidated.Mitochondria play an important role in cellular energy metabolism,and their function and quantity integrity are closely related to cell outcomes.Mitophagy is one of the important ways to degrade damaged mitochondria for mitochondrial homeostasis.However,the role of mitophagy in energy metabolism remains to be studied.Metabolic reprogramming refers to the changes in metabolic patterns of cells during malignant transformation,including glycolysis,electron transport chain(ETC),oxidative phosphorylation and pentose phosphate pathway.Metabolic reprogramming has been one of the markers represented tumor development.However,the relationship between mitophagy and reprogramming of glycolysis,which regulated HBx-mediated LCSCs in HCC cells,remains to be studied.Objectives:To establish a cancer sternness phenotypes model of HCC cells induced by HBx expression,to elucidate HBx-induced BNIP3L-dependent mitophagy involved in glycolysis metabolism reprogramming,mediating the outcome of cancer sternness phenotypes.To investigate the effects of BNIP3L intervention on mitophagy,glycolysis metabolism reprogramming,and cancer sternness phenotypes.The results provide a basis for further elucidating the regulation mechanism of cancer sternness phenotypes induced by HBx expression and exploring potential targets for the prevention and control of hepatocarcinogenesis.Methods:(1)GEO analysis:The GEO database(GSE83148)was used to analyze the differences of cancer sternness-related genes,glycolysis-related genes and autophagy-related genes MAP1LC3B in liver tissues of chronically HBV-infected patients and normal humans.And to analyze the correlation between MAP1LC3B and glycolysis-related genes.(2)In vivo:The pcDNA3.1-HA or pcDNA3.1-HBX-HA(1 μg/mL)were transiently transfected into human hepatoma Huh7 and MHCC-97H cells to construct a HBx-expressing hepatoma cell model.Then,the cells were inoculated subcutaneously into the right lower limb of BALB/c nude mice to establish a nude mouse xenograft animal model.①The effect of HBx on tumor growth was detected:the growth of tumors was observed every 3 days.After 24 days,the nude mice were sacrificed,and the excised xenografts were used for the following tests after measuring the volume.2 The effect of HBx on sternness phenotypes were detected:the sternness-related genes(ABCG2,BMI1,NANOG,KLF4,OCT4)and ACTB were detected by qRT-PCR;The cancer stemness-rclatcd proteins(ABCG2,Oct4,Bmi-1,CD44)was detected by WB and IHC.3 The effect of HBx on BNIP3L-dependent mitophagy was detected:the expression of BNIP3L-dependent mitophagy-related proteins(HIF-1α,Beclinl,BNIP3L,LC3B)was detected by WB and IHC.④The effect of HBx on glycolysis metabolism reprogramming was detected:the glycolysis-related genes(SLC2A1,HK2,PFKL,LDHA)was detected by qRT-PCR,and the intracellular ATP content and extracellular lactate secretion was detected by chemiluminescence.(3)In vitro:①In this study,using a low-ratio side population Huh7 cell line and a high-ratio side population MHCC-97H cell line.Then two HCC cell lines were transiently transfected with pcDNA3.1-HBX(1 μg/mL)to establish a HBx expressing model.The levels of cancer sternness-related genes,glycolysis-related genes,ETC-related genes(ND4L,NDUFA4,AND2,CytB,ATP6,ATP8)and ACTB were detected by qRT-PCR.The expression of cancer stemness and mitophagy-related proteins were detected by WB.The self-renewal ability of cells was detected by clonal formation assay and anchored non-dependent growth assay.The co-localization of BNIP3L,LC3B and mitochondria and the intracellular autophagy flux were detected by Confocal;the proportion of SP cells and the glucose uptake capacity were detected by flow cytometry(FCM).The intracellular ATP content and extracellular lactate secretion were detected by chemiluminescence.②The two HCC cell lines were transiently transfected with pGEM、pGEM-HBV and pGEM-HBV X null(1 μg/mL).The expression of cancer stemness-related proteins was detected by WB.③LCSCs were enriched by Sphere-formation assay.And the detection index was the same as that of HBx expressing group.④The Huh7 cells were pretreated with CCCP(15 μmol/L)for 3 h to establish a mitophagy positive model,and HBx-expressing MHCC-97H cells were treated with BNIP3L small interfering RNA(siBNIP3L)(50 nmol/L)for 8 h to establish a mitophagy inhibition model.The BNIP3L-dependent mitophagy and cancer sternness-related indicators were detected.⑤The STF-31 and 2-DG were used to establish a glycolysis inhibition model in two HCC cell lines.The cancer sternness-related indicators were detected.⑥The HBx-expressing Huh7 cells were transiently transfected with pTT5 or pTT5-9D11(200 ng/mL).The BNIP3L-dependent mitophagy,glycolysis and cancer sternness-related indicators were detected.Results:(1)GEO analysis:The GEO database(GSE83148)of HBV-infected liver tissues(n = 122)and normal liver tissues(n = 6)was screened.The mRNA levels of cancer sternness and glycolysis-related genes in HBV-infected liver tissues were significantly higher than those in normal liver tissues.The mRNA level of MAP1LC3B was significantly higher than that in normal tissues.And M4P1LC3B and glycolysis-related genes showed significant positive correlation.(2)In vivo:The transformed Huh7 cells had a tumor-forming volume of about 2-3 cm3 for 3 weeks.The texture was loose and a large number of blood vessels are formed.The transformed MHCC-97H cells had a tumor-forming volume of about 0.1-0.2 cm3 for 3 weeks,and the tumors had a smooth surface.①Compared with the Ctrl group,the HBx-expressing group had a faster tumor growth and a significant increase in tumor mass and volume.②Excision of tumor tissues revealed that the expression of sternness-related proteins and genes in HBx-expressing group was significantly higher than that of Ctrl group.③Excision of tumor tissues revealed that the BNIP3L-dependent mitophagy-related proteins in total and mitochondrial proteins in HBx-expressing group was significantly higher than Ctrl group.④Compared with Ctrl group,the intracellular ATP content was decreased in HBx-expressing group,and the lactic acid content and the levels of glycolysis-related genes were increased.(3)In,vitro:Firstly,1.25%and 12.8%of side population cells were presented in hepatoma Huh7 and MHCC-97H cells,respectibely.①Compared with the MP cells,the cancer stemness-related genes and proteins were increased in SP cells.②Compared with Ctrl group,the cancer sternness-related genes and proteins were increased in HBx-expressing group,the ability of clonal formation and anchored non-dependent growth and proportion of SP cells were upregulated;the intracellular autophagic flow,autophagosome,and the expression of BNIP3L-dependent mitophagy-related proteins were increased.And the glucose uptake,lactate secretion and the levels of glycolysis-related genes were increased,but the intracellular ATP content and the levels of ETC-related genes were decreased.③Compared with HBV-expressing group,cancer sternness and BNIP3L-dependent mitophagy-related protein were decreased both in HBx knockdown and deletion groups.④The levels of cancer sternness-related genes and proteins were increased in LCSCs.The ability of clonal formation was also enhanced.And the proportion of SP cells were upregulated.The expression of BNIP3L-dependent mitophagy-related proteins were increased,and the co-localization of BNIP3L,LC3B and mitochondria were also enhanced.Besides the glucose uptake,lactate secretion and the levels of glycolysis-related genes were increased,but the intracellular ATP content and the level of ETC-related genes were decreased.Then,⑤Compared with HBx-expressing cells,the BNIP3L-dependent mitophagy were decreased in HBx-expressing MHCC-97H cells treated with siBNIP3L.Compared with HBx-expressing cells,the indicators of cancer stemness-related genes and proteins were decreased in HBx-expressing MHCC-97H cells treated with siBNIP3L.The ability of clonal formation and the proportion of SP cells were decreased.Besides the glucose uptake,lactate secretion and the levels of glycolysis-related genes were increased,but the intracellular ATP content were decreased.Compared with Ctrl group,the expression of BNIP3L-dependent mitophagy-related proteins and the co-localization of BNIP3L,LC3B and mitochondria were increased in HBx-expressing or CCC.P treated Huh7 cells.Compared with Ctrl group,the levels of cancer sternness-related genes and proteins were increased in HBx-expressing and CCCP treated Huh7 cells.The ability of clonal formation and the proportion of SP cells were increased.Besides glucose uptake,lactate secretion and the levels of glycolysis-related genes were deereased but the intracellular ATP content were increased.⑥In the STF-31 intervention model,compared with the HBx-expressing group,the expression of sternness-related genes and proteins was decreased in the HBx+STF-31 group,and the ability to clonal formation was weakened,and the SP cells ratio was decreased;the same result also observed in the 2-DG intervention model.In the LCSCs,the expression of stcmness-related proteins were decreased in the STF-31 or 2-DG intervention group compared with Ctrl group.⑦The HBx-expressing Huh7 cells were transient transfected with pTT5 or pTT5-9D1 1 to construct the HBx knockdown model.Compared with the HBx-expressing group,the cancer sternness and BNIP3L-dependent mitophagy-related proteins were decreased in the HBx knockdown group.And glycolytic metabolism processes were also inhibited.Conclusion:The in vitro experment model of HBV,HBx expression and HBx knockdown HCC cells and the xenografted tumor model of HBx-expressing HCC cells in nude mice were established to induce glycolysis metabolism reprogramming and mediate cancer sternness of liver cancer cells.Upregulation of cancer sternness phenotype can induce the expression and mitochondrial translocation of BNIP3L in hepatocarcinoma cells and LC3B interaction are related to mitophagy and mitochondrial ETC dysfunction.Targeted intervention of BNIP3L expression can regulate glycolytic metabolism and cancer sternness in HCC cells induced by HBx-expressing.Reprogramming and cancer sternness phenotype.The results provide a basis for further elucidating the regulation mechanism of HBx-expressing in inducing the cancer sternness and exploring potential targets for the prevention and control of hepatocarcinogenesis. |
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