The Role Of GTP Cyclohydrolase 1 In The Proliferation Of Hepatoma Cells And The Underlying Mechanism

Section 1 The expression and clinical significance of GTP cyclohydrolase 1(GCH1)in hepatocellular carcinoma tissues and hepatoma cellsBackground&Aims:Hepatocellular carcinoma(HCC)is a major form of primary liver cancer.Metabolic reprogramming is a hallmark of cancer,including HCC.Thus,identifying novel metabolic regulators of HCC is essential for a better understanding of the role of metabolic reprogramming in HCC.GTP cyclohydrolase 1(GCH1)is the first rate-limiting enzyme in tetrahydrobiopterin(BH4)de novo biosynthesis.In this section,we aimed to investigate the expression and clinical significance of GCH1 in HCC.Methods:(1)We used RT-qPCR and Western blotting assays to detect GCH1 expression levels in HCC tissues and paired peri-tumor tissues as well as immortalized liver cell line MIHA and hepatoma cell lines.Moreover,we used methylation-specific PCR to investigate the methylation status of GCH1 promoter.(2)We examined the association of GCH1 expression with clinicopathologic characteristics and overall and recurrence-free survival by performing tissue microarray-based immunohistochemistry analyses of two independent cohorts of HCC patients.We also used univariable and multivariable Cox proportional hazard regression models to explore the association of GCH1 expression level with overall and recurrence-free mortality of HCC patients.Results:(1)Western blotting and RT-qPCR assays showed that GCH1 protein and mRNA expression levels were markedly down-regulated in HCC tissues and hepatoma cell lines compared with peri-tumor tissues and MIHA cells.Methylation-specific PCR showed that promoter methylation was responsible for GCH1 down-regulation in HCC.(2)In Chongqing cohort,GCH1 expression was found to be significantly associated with sex(P=0.02),Barcelona Clinic Liver Cancer staging(P<0.01),tumor size(P<0.01),and tumor number(P=0.02);in Sichuan cohort,GCH1expression was found to be significantly associated with?-fetoprotein level(P=0.01),ALT level(P=0.02),Barcelona Clinic Liver Cancer staging(P<0.01),tumor size(P=0.02),and tumor number(P=0.02).Kaplan-Meier survival curves showed that HCC patients with low GCH1 expression had worse overall survival(P<0.01)and recurrence-free survival(P<0.01)in both Chongqing and Sichuan cohorts.Moreover,multivariable analyses in Chongqing and Sichuan cohorts showed that low GCH1 expression was an independent predictor of higher overall mortality and recurrence-free mortality(P<0.05).Conclusions:GCH1 is frequently down-regulated in HCC tissue and hepatoma cells due to promoter methylation;down-regulation of GCH1 is predictive of poor prognosis of HCC patients.Section 2 GCH1 silencing promotes the proliferation of hepatoma cellsBackground&Aims:Previous studies showed that GCH1 promoted the proliferation of mouse melanoma cells and human breast cancer cells as well as the growth of glioblastoma.In this section,we aimed to explore the potential impacts of GCH1 expression modulation on the proliferation,migration,and inducing angiogenesis of hepatoma cells.Methods:(1)We used CCK-8,plate colony formation,and EdU assays as well as HCC xenograft model to explore the influence of GCH1 on HCC proliferation.(2)We used wound healing assay to explore the influence of GCH1 on HCC migration.(3)We used human umbilical vein endothelial cell tube formation assay to explore the influence of GCH1 on HCC anagenesis.(4)We used flow cytometry to explore the influence of GCH1on cell cycle and cell apoptosis.(5)We used Western blotting to explore the influence of GCH1 on expression levels of cleaved caspase-3,Bax,and Bcl-2.Results:(1)CCK-8,plate colony formation,and EdU assays showed that GCH1 overexpression inhibited cell viabilities(P<0.05)and cell proliferation(P<0.05)of hepatoma cells,while GCH1 silencing had opposite effects.Meanwhile,both weight and volume of xenograft tumors were found to be significantly larger in GCH1 silencing group than in control group.(2)Flow cytometry showed that GCH1 overexpression enhanced(P<0.05)while GCH1 silencing inhibited(P<0.05)cell apoptosis;Western blotting showed that GCH1 overexpression promoted expression levels of cleaved caspae-3 and Bax but decreased the expression level of Bcl2,while GCH1 silencing had opposite effects.(3)Wound healing assay showed that GCH1 overexpression or silencing did not affect the migration of HCC cells;human umbilical vein endothelial cell tube formation assay showed that GCH1 overexpression or silencing did not affect the angiogenesis of HCC.Conclusions:Down-regulation of GCH1 promotes the proliferation of hepatoma cells,but has no impact on their migration and induction of angiogenesis.Section 3 The growth-promoting effect of GCH1 silencing is a direct consequence of BH4 biosynthesis reductionBackground&Aims:GCH1 is the first rate-limiting enzyme in BH4 de novo biosynthesis,which catalyzes the conversion of GTP into dihydroneopterin triphosphate.In this section,we aimed to clarify whether the role of GCH1 in HCC was dependent on BH4.Methods:(1)We used BH4 ELISA kit to detect BH4 levels after GCH1overexpression and silencing.(2)We used CCK-8 assay and nude mice xenograft model to demonstrate the influence of exogenous BH4 on HCC proliferation and whether BH4 biosynthesis reduction mediated the growth-promoting effect of GCH1 silencing.Results:(1)GCH1 overexpression significantly increased(P<0.05) while GCH1 silencing significantly decreased(P<0.05)intracellular BH4levels.(2)The metabolite BH4 inhibited HCC growth in vitro and in vivo.(3)Rescue experiments showed that GCH1 silencing exerted its growth-promoting effect through directly inhibiting BH4 de novo biosynthesis.Conclusions:BH4 inhibited HCC growth in vitro and in vivo;the reduction of BH4 de novo biosynthesis mediated the growth-promoting effect of GCH1 silencing in HCC.Section 4 GCH1 silencing activates ASK1/p38 signaling to promote the proliferation of hepatoma cellsBackground&Aims:The experiments in sections 1 to 3 showed that GCH1 silencing promoted the proliferation of hepatoma cells.In this section,we aimed to investigate molecular mechanisms underlying how GCH1silencing promoted HCC cell proliferation.Methods:(1)We used Western blotting to screen common signaling pathways in oncology field,and further used RT-qPCR to detect mRNA expression of down-stream genes.(2)We used SB203580 and si RNA to perform rescue experiments for clarifying whether the activation of p38signaling mediated the growth-promoting effect of GCH1 silencing.(3)We used Western blotting to detect the phosphorylation levels of ASK1,and used selonsertib to confirm whether ASK1 signaling was the upstream of p38 signaling.Results:(1)Western blotting showed that GCH1 silencing activated p38signaling;meanwhile,RT-qPCR showed that GCH1 silencing significantly increased the mRNA expression of down-stream genes positively regulated by p38 signaling.SB203580 inhibited the activation of p38 signaling,and reversed the growth-promoting effect of GCH1 silencing.(2)RNAi showed that si RNA-mediated knockdown of p38?or p38?significantly impaired cell proliferation;knockdown of both p38?and p38?completely reversed cell proliferation to the levels of control cells.(3)Western blotting showed that GCH1 silencing promoted the phosphorylation of ASK1;after using selonsertib to inhibit ASK1 signaling,p38 signaling was also inhibited.Conclusions:GCH1 silencing activates ASK1/p38 signaling to promote HCC cell proliferation.Section 5 The increase of O₂^·-mediates the ASK1/p38 activation by GCH1 silencingBackground&Aims:Above experiments showed that GCH1 silencing activates ASK1/p38 signaling to promote HCC cell proliferation.In this section,we aimed to investigate the exact mechanisms underlying how GCH1 silencing activated ASK1/p38 signaling.Methods:(1)We used Western blotting to detect the impacts of BH4 on ASK1/p38 signaling.(2)We used DHE fluorescence probe to detect the impacts of BH4 on intracellular levels of O₂^·-.(3)We used NAC,a reactive oxygen species scavenger,to perform rescue experiments for clarifying whether the increase of O₂^·-mediated the activation of ASK1/p38 signaling induced by GCH1 silencing.Results:(1)Western blotting showed that BH4 inhibited ASK1/p38 signaling in a dose-dependent manner.(2)GCH1 silencing increased intracellular levels of O₂^·-by inhibiting BH4 de novo biosynthesis.(3)Rescue experiment showed that the increase of O₂^·-mediated the activation of ASK1/p38 signaling induced by GCH1 silencing.Conclusions:GCH1 silencing activates ASK1/p38 signaling by increasing superoxide anion via inhibiting BH4 de novo biosynthesis.