Effect And Mechanism Of Oridonin Targeting HK1 On Bladder Tumor Cell Proliferation

Objective: The uncontrolled growth of bladder cancer cells is the main cause of cancer recurrence and high mortality.Exploring the molecular mechanism of the occurrence and development of bladder cancer,identifying the therapeutic targets of malignant proliferation,and finding effective small molecular drugs for the treatment of bladder tumors has become an important problem in the treatment of bladder tumor.Oridonin(oridonin)is a diterpenoid extracted from traditional Chinese herbal medicine Rabdosia rubescens,which can effectively inhibit the proliferation of a variety of tumor cells and induce cell apoptosis.In order to deeply understand the anti-tumor molecular mechanism of oridonin and provide guidance for its effective application in the treatment of bladder tumor,this study intends to find the direct targets of oridonin from the system level,and then deeply understand its anti-tumor mechanism from the source.Research methods:1.The potential target genes that may directly bind to oridonin were identified by human proteome chip detection and bioinformatics analysis.2.The binding of oridonin to hexokinase 1(HK1)was verified by small molecule pull-down assay in vivo.3.Western blotting(western blotting)was used to detect the expression of HK1 in bladder cancer tissues and corresponding normal tissues.HPA database was used to evaluate the relationship between HK1 expression and tumor grade,and TCGA database combined with gene enrichment analysis(gene set enrichment analysis,GSEA)was used to explore the potential biological function of HK1 in the malignant progression of bladder cancer.4.Using bladder cancer cell lines 5637 and UMUC3,the effects of HK1 knockdown on the proliferation and survive of bladder cancer cells were studied by CCK8 and 5-ethynyl-2'-deoxyuridine(EdU).5.Using t bladder cancer cell line 5637 and UMUC3,the effects of oridonin on the viability and HK1 expression of bladder cancer cells were studied by gene knockdown of HK1,CCK8 and western blotting.6.The binding between HK1 and oridonin was investigated by Octet K2 molecular interaction analyzer.Results:1.Using proteome chip technology,combined with bioinformatics analysis and binding verification,a large number of potential direct binding proteins of oridonin,including HK1,were preliminarily found.In this study,we found that the proteins interacting with oridonin are not only involved in glycolysis pathway,but also involved in a variety of important cellular signal pathways.2.The binding of oridonin to hexokinase 1(HK1)in vivo has been preliminarily confirmed.3.The HK1 protein's expression level in bladder cancer tissues was extremly higher than that in normal bladder tissues;the HK1's expression was highly up-regulated in highgrade bladder cancer tissues;in bladder cancer specimens,the HK1's high expression was closely related to metabolism,proliferation,apoptosis and other signal pathways.4.Knockdown of HK1 can inhibit the proliferation and survival of bladder cancer cells,which indicates that HK1 plays an oncogene role in bladder cancer.5.Oridonin has strong cytotoxicity to bladder cancer cells,and can effectively inhibit the proliferation of bladder cancer cells by inhibiting the expression of HK1.6.The results of intermolecular interaction suggest that there is binding and dissociation between HK1 and different concentrations of oridonin,and the binding is concentration-dependent.Conclusion: The anticancer drug oridonin can directly bind to HK1 and other functional proteins involved in signal regulation.The up-regulated expression of HK1 in bladder cancer tissue can inhibit the proliferation and survival ability of bladder cancer cells,which indicates that HK1 plays an oncogene role in bladder cancer.Oridonin directly binds to HK1,and effectively inhibits the proliferation of bladder cancer cells by inhibiting the expression of HK1,and then plays a cytotoxic role.