NDRG1 Promotes The Formation Of 5637 Drug-resistant Cells And Mediates The Molecular Mechanism Of Oxaliplatin Resistance In Bladder Cancer

Objective:Bladder cancer is a major health problem in the world.Bad lifestyle habits,such as smoking,can aggravate the damage to the bladder and induce cancer.Bladder cancer is one of the malignant tumors with poor prognosis.Platinum drugs are the first choice for the treatment of bladder cancer.Oxaliplatin,as a third-generation platinum drug,has the advantages of low side effects and fast DNA binding rate.However,oxaliplatin is easy to cause the body to develop drug resistance,leading to the failure of anti-tumor treatment.As a metastasis suppressor,NDRG1 has a complicated mechanism of action in different cancers.Through deep sequencing,qRT-PCR and Western Blot verification experiments,oxaliplatin resistant bladder cancer cell lines were screened for high expression of NDRG1.Inhibition of NDRG1 can reverse the resistance of bladder cancer,indicating that NDRG1 is an important factor in mediating the resistance of bladder cancer to oxaliplatin.NDRG1 can promote the formation of 5637 drug-resistant cells,thereby mediating bladder cancer resistance to oxaliplatin.The positive expression of Ki-67 of the drug-resistant strain increased,the expression of apoptotic protein decreased,and the EMT-related transfer protein was inhibited.Secondly,through the detection of mass spectrometry platform,it was found that DNA promoter methylation is related to the high expression of NDRG1 in oxaliplatin-resistant strains.The use of histone methyltransferase inhibitors can significantly reduce the expression of drug-resistant strains of NDRG1,and preliminarily clarify the important role of epigenetic modification in NDRG1 overexpression-mediated oxaliplatin resistance in bladder cancer.In order to further study the role of abnormal methylation-mediated NDRG1 in bladder cancer and the mechanism of drug resistance,this study further examined the downstream genes regulated by NDRG1.The study found that knocking down NDRG1 or using histone methyltransferase inhibitors both up-regulated the expression of senescent proteins p21 and p27,and down-regulated the expression of cyclin CDK6,revealing an important mechanism by which methylation regulates NDRG1 to affect drug-resistant cell proliferation through the p21 pathway.We did a preliminary study on the relationship between NDRG1 and tumor chemotherapy resistance and the molecular mechanism,the research results provide a solid experimental basis for reversing the drug resistance of bladder cancer cells and improving the sensitivity of chemotherapy drugs,as well as new ideas for clinical anti-bladder cancer treatment.Methods and results:1.The CCK8 method was used to detect the effects of different concentrations of oxaliplatin on cell proliferation of 5637P(blank group)and 5637R(drug resistance group).The nuclear antigen Ki-67 was used to detect cell proliferation by flow cytometry.Western Blot method was used to detect apoptosis protein and EMT(epithelial-mesenchymal-transition)related proteins to analyze tumor cell apoptosis and metastasis.The results showed that the long-term effect of oxaliplatin promoted the proliferation of 5637 R and inhibited the apoptosis and metastasis of bladder cancer cells.2.Detect NDRG1 mRNA expression by qRT-PCR method,and detect NDRG1 protein expression in 5637 P,5637P-OXI,and 5637 R by Western Blot.The CCK8 method was used to determine the changes in cell resistance of 5637 R after knocking down NDRG1 after treatment with different concentrations of oxaliplatin,and analyze the relationship between the expression of NDRG1 gene and the degree of resistance to oxaliplatin.Flow cytometry detected the changes in cell proliferation and cell cycle of 5637 R cells after knocking down NDRG1,and the results showed that NDRG1 was up-regulated in bladder cancer 5637R;knocking down NDRG1 resulted in a decrease in the resistance and proliferation ability of 5637 R to oxaliplatin.It also affects cell proliferation by inhibiting the S phase of the cell cycle,suggesting that NDRG1 can be used as a specific bladder cancer drug resistance target,and it can play a role in promoting the drug resistance process of bladder cancer.3.Using plasma nuclear separation technology,Western Blot method to detect the relative expression of NDRG1 protein in cytoplasm and nucleus.The laser confocal method was used to observe the intracellular localization of NDRG1 under oxaliplatin stimulation.The results showed that NDRG1 was highly expressed in the cytoplasm and nucleus during the formation of acquired drug resistance due to long-term drug stimulation of 5637 R.Western Blot method was used to detect the effect of adding histone methyltransferase inhibitor on NDRG1.The results showed that the degree of DNA methylation of 5637 R was significantly higher than that of 5637 P.It can be seen that the high level of DNA methylation mediates drug resistance in bladder cancer.4.Sequenom Mass ARRAY Methylation sequencing method detects the DNA methylation level of 5637 P and 5637 R.Western Blot method was used to detect the protein level of NDRG1 when cells challenge histone methyltransferase inhibitor.The results showed that the degree of histonemethylation of 5637 R was significantly higher than that of 5637 P.Histone methyltransferase inhibitor(CPI-169)inhibited the expression of NDRG1.Compared with 5637 P,5637R has a higher histone methylation level,and histone methyltransferase inhibitor(CPI-169)can simultaneously down-regulate the expression of Tri-Me-Histone H3 Lys27 and NDRG1,it shows that histone methylation can promote the expression of NDRG1.5.Western Blot method to detect the expression of cyclin p21,p27 and CDK6 after knocking down NDRG1 in bladder cancer cell lines 5637 R,T24,TCCSUP.The results showed that NDRG1 can promote the proliferation of drug-resistant cells by inhibiting p21,p27 and promoting the expression of CDK6,and participate in the occurrence of drug resistance.Conclusions:1.NDRG1 plays a role in promoting proliferation and inhibiting cell apoptosis in oxaliplatin-induced bladder cancer resistance.2.Oxaliplatin up-regulates histone methylation level and DNA methylation level to promote the expression of NDRG1,thereby regulating the drug resistance pathway of bladder cancer.3.NDRG1 inhibits the p21 pathway to regulate cell cycle and promote cell proliferation,which makes bladder cancer cells resistant to oxaliplatin.