C1QBP Regulates Hypoxanthine Catabolism And Apoptosis Of Renal Cell Carcinoma Via Xanthine Dehydrogenase (XDH)

Objectives: Renal cell carcinoma(RCC)is one of the most common malignant tumors in the urinary system.The early diagnosis rate of RCC is not high,patients are often accompanied with systemic metastasis.The postoperative recurrence rate of RCC patients is high and the survival rate is low.Renal cell carcinoma is insensitive to chemotherapy and radiotherapy,although immunotherapy and angiogenic targeted therapy have improved the survival rate of patients with renal cell carcinoma.However,the serious adverse reactions caused by immunotherapy and the side effects and drug resistance of targeted drugs pose challenges to the treatment of renal cell carcinoma.Therefore,in-depth study of the molecular basis of the progress of renal cell carcinoma and the development of new targeted anticancer drugs are of great significance to improve the survival rate of patients with renal cell carcinoma.In addition,RCC often undergoes extensive metabolic reprogramming,and abnormal tumor metabolites play an important role in cancer progression.Therefore,it is of great significance to explore new renal cancer treatment targets and biomarkers by identifying tumor metabolites and targeting key proteins or enzymes involved in the dysfunctional metabolic pathways of RCC,and applying them to diagnosis and prognosis.Complement C1 q binding protein(C1QBP)is a highly conserved multifunctional protein that plays an important role in inflammation,infection,energy metabolism and cancer.C1 QBP is highly expressed in breast cancer,colon cancer,gastric cancer and other cancers,and is associated to the poor prognosis of patients.In addition,as a mitochondrial matrix protein,C1 QBP plays an important role in maintaining the balance of oxidative phosphorylation(OXPHOS),reactive oxygen species(ROS)production and energy metabolism.Differently from other tumors,our previous studies have verified that the expression of C1 QBP is low in RCC and that knocking down C1 QBP promotes the adhesion and invasion of RCC cells.However,the mechanisms of C1 QBP in RCC purine metabolism,oxidative stress and apoptosis have not been elucidated.This study will explore that C1 QBP regulates hypoxanthine catabolism,ROS level and the apoptosis of renal cancer cells by regulating the expression of xanthine dehydrogenase(XDH)in vivo and in vitro.Meanwhile,this study will clarify the effect of C1 QBP on hypoxanthine metabolism of RCC and analyze the mechanism that C1 QBP inhibits the progression of RCC,so as to provide a new molecular theoretical basis for the therapeutic target of RCC.Methods: 1.Lentivirus-mediated RNA interference technique was used to construct C1 QBP differentially expressed stable ACHN and 786-O renal cell lines,and real-time PCR and western blot methods were used to verify the m RNA and protein expressions of C1 QBP.2.Metabonomic techniques were used to detect the differentially expressed metabolites between 786-O control group and C1 QBP overexpression group.3.The effects of down-expression and overexpression of C1 QBP on hypoxanthine content were detected in renal cancer cells ACHN and 786-O,respectively.4.Western blot and real-time PCR were used to detect the effects of downexpression and overexpression of C1 QBP on XDH expression in renal cancer cells ACHN and 786-O,respectively.5.Western blot and immunohistochemistry techniques were used to detect the expression and distribution of C1 QBP and XDH in renal cell carcinoma and para-carcinoma tissues.And statistical methods were used to analyze the correlation between C1 QBP and XDH,and correlations of XDH and C1 QBP with clinicopathological parameters.6.The effects of down-expression and overexpression of C1 QBP on ROS production and apoptosis were detected by enzyme labeling instrument and flow cytometry in renal cancer cells ACHN and 786-O,respectively.7.Western blot was used to detect the effects of down-expression and overexpression of C1 QBP on the expression of apoptosis-related proteins active caspase-3 and bax/bcl2 in renal cancer cells ACHN and 786-O,respectively.8.Real-time PCR and western blot were used to verify the efficiency of m RNA and protein expressions of silenced XDH after transfection of XDH si RNA,and flow cytometry was used to detect the effects of XDH knocked down on ROS production and apoptosis induced by C1 QBP in ACHN and 786-O cells.9.Western blot was used to detect the effects of low expression of XDH on the expression of apoptosis-related proteins active caspase-3 and bax/bcl2 induced by C1 QBP in ACHN and 786-O cells.10.The xenograft tumor model of renal orthotopic human ACHN cells in nude mice was established,and the bioluminescence intensity of C1 QBP overexpression on primary tumor and liver and lung metastasis was studied by live imaging system.The primary tumors were weighted.Detection of metastatic foci of liver and lung by H&E staining.The expressions of protein XDH and apoptosis-related proteins caspase-3 and bax/bcl2 were detected by immunohistochemical staining in primary tumor.Results: 1.The stable renal carcinoma cell lines with down-expression of C1 QBP were successfully constructed: ACHN-sh C1QBP/Scr and 786-O-sh C1QBP/Scr cells,the m RNA(***P<0.001)and protein levels of C1 QBP were downregulated,and the stable renal carcinoma cell lines with overexpression of C1QBP: ACHN-C1QBP/p CDH and 786-O-C1QBP/p CDH cells were successfully constructed,and the m RNA(***P<0.001)and protein levels of C1 QBP were increased.2.There were 109 metabolites were detected in 786-O cells by metabonomic techniques.Compared with the control group,17 kinds of metabolites were differentially expressed in 786-O C1 QBP overexpression cells.And the change of hypoxanthine content was the most significant,which decreased to 0.2-fold(*P=0.01).3.Consistent with the results of metabolomics,compared with the control group,C1 QBP knockdown decreased hypoxanthine content(***P<0.001),while C1 QBP increased hypoxanthine content(***P<0.001).4.C1 QBP positively regulates the expression of XDH at both m RNA(**P<0.01)and protein levels in renal cancer cells ACHN and 786-O.5.The results of western blot and immunohistochemistry showed that the expressions of C1 QBP and XDH in renal cell carcinoma were lower than adjacent normal tissues(*P<0.05,***P<0.001),and they were positively correlated(**P<0.01,***P<0.001).The expressions of C1 QBP and XDH were related to patients Fuhrman grade(***P<0.05),but not to gender,age and tumor size.In addition,C1 QBP expression was correlated with patients TNM stage,while XDH expression was not related to patients TNM stage.6.C1 QBP was positively regulating ROS production and apoptosis in renal cell carcinoma ACHN and 786-O cells(*P <0.05,***P<0.001).7.C1 QBP positively regulated the activation of pro-apoptotic protein caspase-3 and the expression of bax,and negatively regulated the expression of antiapoptotic protein bcl2 in renal cell carcinoma ACHN and 786-O cells.8.Small interference RNAs were transfected and successfully knocked down the m RNA(***P<0.001)and protein expression of XDH.XDH knockdown significantly inhibit C1QBP-induced ROS production(***P<0.001)and apoptosis(*P< 0.05,**P<0.01)in ACHN and 786-O cells.9.Knocking down XDH significantly inhibit the activation of caspase-3 and the increase of bax expression induced by C1 QBP in ACHN and 786-O cells,and restore the inhibition of bcl2 expression mediated by C1 QBP.10.Compared with the control group,the light signal intensity of renal orthotopic tumor,liver and lung were decreased and the weight of primary tumor decreased significantly after overexpression of C1QBP(***P<0.001).H&E staining showed that C1 QBP overexpression significantly reduced the metastatic foci of liver and lung(***P<0.001).The results of IHC showed that C1QBP could promote the expressions of XDH,bax and activated caspase-3 and inhibited the expression of bcl2 in renal tumor in nude mice.Conclusions: 1.C1 QBP inhibits the content of hypoxanthine in renal cancer cells.2.C1 QBP can positively regulate the expression of XDH in renal cell carcinoma,and both of them are low expressed in renal cell carcinoma,which are positively correlated.The low expressions of C1 QBP and XDH in renal cancer tissues are related to the high Fuhrman grade,in addition,the low expression of C1 QBP is still related to the high TNM stage.3.C1 QBP promotes ROS production,apoptosis and the expressions of bax and active caspase-3,and inhibits expression of bcl2 in RCC.4.C1 QBP promotes the production of ROS,apoptosis and the expressions of bax and active caspase-3,and inhibits expression of bcl2 by up-regulating the expression of XDH in RCC.5.In the nude mice model of transplanted renal cell carcinoma,C1 QBP promotes the expression of XDH and regulates apoptotic proteins expressions,and inhibits the size of the tumor and the metastasis of liver and lung.