Role Of The RNA Methyladenosine Methylase METTL3 In Cadmium Induced Malignant Transformation Model Of Cells

BackgroundBladder cancer,with the highest morbidity and mortality among the urinary system tumors,is one of the most common malignant neoplasms of the urinary system in China.Epigenetic regulatory mechanism plays an important role in the development of chemical carcinogenesis.N6-methyladenosine(m6A)is the most common and the most abundant modification of messenger RNA(mRNA),which occurs on the sixth N atom of adenine.The most important part of mRNA is methylation modification,which affects all basic aspects of mRNA metabolism.Recent studies have found that Methyltransferase-like 3(METTL3)gene encoding m6A RNA methylase,plays an important role in adipogenesis,spermatogenesis,development,carcinogenesis,stem cell renewal and other as yet uncharacterized processes.Cadmium is a widely exposed metal element in human occupation and environment,which has been listed as a carcinogen by the International Agency for Cancer Research(IARC).The carcinogenic mechanism of cadmium is very complicated.Due to its low mutagenicity and strong carcinogenicity,recent studies have focused on the epigenetic mechanism.So far,the role of m6A methyltransferase METTL3 in occurrence and development of bladder carcinoma is still poorly understood.ObjectiveTo investigate the expression and function of m6A Methyltransferase METTL3 in bladder cancer cells,and clarify the role of METTL3 in the malignant transformation induced by cadmium chloride,laying the foundation for RNA epigenetic regulation in bladder cancer,and METTL3 provide new strategies for the RNA epigenetic therapy in bladder cancer.Methods1.To examine the expression of METTL3 in human bladder cancer tissues and matched adjacent tissues of bladder cancer.The TCGA database was used to detect the expression of METTL3 in bladder cancer tissues and matched adjacent tissues of bladder cancer.2.To examine the expression of METTL3 in human bladder epithelium cell line and bladder cancer cell lines.Western blot was used to detect the expression of METTL3 in bladder epithelia cell line and bladder cancer cell lines.3.The sgRNA targeting METTL3 exons were designed and transfected into 293T cells,sgRNA has the maximum knockout efficiency by T7E1 assay,sgRNA was inserted in lentiCRISPRv2 vector to construct lentiCRISPRv2-sgRNA knockout expression vector.METTL3 gene was amplified by PCR with cDNA as template,The PCR amplified METTL3 gene was ligated into PLEX expression vector to construct PLEX-METTL3.4.The lentiCRISPRv2-sgRNA and PLEX-METTL3 were transfected into 293T cells.the supernatant was collected and filtered,then infected the SV-HUC-1 and T24 cells.The cells were then selected by Puromycin.The stable METTL3 overexpression SV-HUC-1 cell lines and METTL3 knockout T24 cells were constructed.The protein expression level of METTL3 in SV-HUC-1-METTL3 and T24-KO-METTL3 were detected by Western blot.5.MTS assay detected cell proliferation;Flow cytometry were used to determine cell cycle distribution and apoptosis;The migration potential was measured by wound healing assay;The invasive potential was measured by Transwell with matrigel.6.The model of malignant transformation of SV-HUC-1 cells induced by cadmium chloride were constructed.Then the METTL3 overxpression SV-HUC-1 cells were treated with CdCl2(10?mol/L)for 6 weeks to construct the model of malignant transformation of SV-HUC-1 cells induced by over expression of METTL3 byusing the same method,the phenotypic changes of cellular were detected.7.Statistical analysis.All experiments were repeated at three times.Statistics were assessed using SPSS 18.0 Statistical data are expressed as mean±SEM,the analysis between two groups of data by t test.In all cases,P<0.05 was considered statistically significant.Results1.METTL3 mRNA levels were obviously higher in tumor tissues than in the pairing adjacent non-tumor tissues by TCGA data base(P<0.05).2.Expression of METTL3 was examined at protein level in bladder epithelial cell line and bladder cancer cell lines using western blot.METTL3 protein is relative lower expression in bladder cell lines SV-HUC-1,compared to bladder cancer cell lines T24.3.Sequencing confirmed METTL3 sgRNA was correctly inserted into lentiCRISPRv2 vector,and METTL3 was correctly inserted into PLEX vector.4.Sequencing confirmed METTL3 was knockout in T24-KO-METTL3.Stable METTL3 knockout cell lines T24-KO-METTL3 and METTL3 overexpression cell lines SV-HUC-1-METTL3 were successfully constructed.5.The result of MTS showed that cells proliferation are increased in SV-HUC-1-METTL3 compared to SV-HUC-1-control,cells proliferation are decreased in T24-KO-METTL3,compared to T24-V2.The result of the cell cycle showed that the number of SV-HUC-1-METTL3 cells stop in the period of G0/G1 are less than SV-HUC-1-control,and the number of T24-KO-METTL3 cells stop in the period of G0/G1 are more than that of T24-V2.The apoptosis rate of T24-KO-METTL3 was significant higher than that of T24-V2.Cells migration showed SV-HUC-1-METTL3 moved faster than SV-HUC-1-control,and T24-KO-METTL3 moved more slowly than T24-V2.The result of Transwell Migration and Invasion assay showed that the migrated cells were significantly lower in T24-KO-METTL3 cells than T24-V2.6.The cellular models of chemical carcinogen cadmium chloride-induced malignant transformation of METTL3 overexpression bladder epithelial cells was successfully constructed.Compared with the control group,the cellular proliferation,migration and invasion were enhanced in SV-HUC-1-METTL3-6W-Cd.Conclusions1.METTL3 mRNA levels were obviously higher in human tumor tissues and human bladder cancer cell lines.2.METTL3 can promote bladder cancer cell growth,proliferation,migration and invasion,inhibit cell apoptosis both in vivo and in vitro.3.METTL3 can promote malignant transformation in cadmium chloride-induced malignant transformation of human bladder epithelial cell.