The Effect Of Knocking Down TRMT61A On Gene Expression Profiles And FABP4 In Bladder Cancer 5637 Cells

Background and objectiveBladder cancer is the most common malignancy of the genitourinary system. In recent years, the incidence of bladder cancer in Western countries decreased slowly while the incidence and mortality in china was an upward trend over the past decade. On the other hand, because of long term survival and the need of lifelong routine monitoring, the cost per patient of diagnosis, treatment, monitoring and other medical treatment was the highest in all tumors which bring a heavy economic burden to patient and society.Cystoscopy and voided urine cytology is the most common means of detection and monitoring of bladder cancer. But cystoscopy was invasive and expensive while voided urine cytology has a low sensitivity although high specificity. Therefore this highlights the need for the new methods of screening and monitoring of bladder cancer.Urine modified nucleosides were very promising tumor markers for the diagnosis, detection, monitoring and prognosis of many kinds of tumors. Our previous study found that the combined detection of 1-methyl-adenosine(m1A) and 1-methylinosine(1-mel) has a high sensitivity(92.45%) and specificity(87.50%) for bladder cancer. Thus, the urine modified nucleosides may be the potential and promising biomarkers for monitoring and detection of bladder cancer.m1A58 methyltransferase(hTrm6P/hTrm61P) catalyzed t RNA58 adenosine(A) which generated m1A58. The catalytic subunit of m1A58 methyltransferase is h Trm61 P encoded by TRMT61 A. In subsequent study, we found the elevated expression of TRMT61 A in bladder cancer tissue and 5637 cell line which has a high level of TRMT61 A mRNA among the bladder cancer cell lines T24, 5637, EJ. And we found the significant reduction of proliferation and invasion and increased apoptosis when knocking down of TRMT61 A in 5637 cell line. These indicate that TRMT61 A plays an important role in development of bladder cancer.In this study we test the impact of knocking down TRMT61 A in 5637 cell line on gene expression profiles by gene chip and analyze the different expressed genes by panther data base and explore the possible pathway of TRMT61 A involved in bladder cancer. The fold change of four different expressed genes is more than 5, and as one of them, FABP4 shows a higher expression in the 5637 cells that TRMT61 A was knocked down. Numerous studies demonstrate that FABP4 show reduced expression in bladder cancer tissue compared with bladder cancer adjacent tissue which related to pathological grade, stage and poor prognosis. Therefor the RT-q PCR technology was used to validate the effect of knocking down TRMT61 A on FABP4 mRNA expression in 5637 cells.Methods 1 Gene chip : Grouped in two: Lip2000 group( 5637 cells dealed with lipofectamineR2000),siRNA group(5637 cells transfected by TRMT61A-siRNA). Knock down TRMT61 A in 5637 cells by siRNA technology and detect the efficiency. Submit the samples for gene chip when the efficiency was more than 70%. Analyze the different expressed genes by panther data base.2 The validation of the effect of knocking down TRMT61 A on FABP4 mRNA in 5637 cells by RT-q PCR technology.:Grouped in four: blank group(5637 cells without any processing), Lip2000 group(5637 cells dealed with lipofectamineR2000),NC group(5637cells transfected by negative control siRNA), siRNA group(5637 cells transfected by TRMT61A-siRNA).3 The relative expression of TRMT61 A and FABP4 mRNA in bladder cancer tissues:The bladder cancer tissues confirmed by pathology were collected in the first affiliated hospital of Zhengzhou university between January 2014 and January 2016. The relative mRNA level of TRMT61 A and FABP4 in bladder cancer was determined by RT-q PCR technology.Result 1 The result of gene chip: the effect expressed genes were screened by the threshold that the probe signalsdiffer from background signals and the standardized signals value was more than 8. The standard of significant difference was FC(fold change)>2 or FC<0.5. The number of genes which were obviously up-regulated(FC>2)after knocking down TRMT61 A was 99 while the number of genes which were obviously up-regulated(FC<0.5)after knocking down TRMT61 A was 107. The fold change of STC2, FABP4, KRT13, ERMP1 genes was more than 5 or 0.2 among them. The result of Go Term analysis indicates that the different expressed genes involved in multiple molecular function, biological process, cell component and pathway.2The TRMT61 A mRNA in siRNA group(0.563±0.050)was significantly down regulated compared with NCgroup(0.999±0.041),and the difference between two groups was statistically significant(P <0.05). While The FABP4 mRNA in siRNA group(2.260±1.167)was significantly up regulated compared with NCgroup(1.000±0.019), and the difference between two groups was statistically significant(P <0.05).3 The TRMT61 A mRNA in 30 bladder cancer tissues(2.076±1.349) was obviously overexpression compared with the bladder cancer adjacent tissue(1.000±0.000), and the difference between them was statistically significant(P<0.05). On the contrary,the FABP4 mRNA in 28 bladder cancer tissues(0.568±1.420) was obviously lower-expression compared with the bladder cancer adjacent tissues(1.000±0.000), and the difference between them was statistically significant(P <0.05)Conclusion 1 Knocking down of TRMT61 A has a significant influence on the gene expression profiles in 5637 cells.2 Knocking down of TRMT61 A up regulates FABP4 mRNA expression significantly in 5637 cells. This indicates that the anticancer activity of knocking down TRMT61 A is mediated by the up regulation of FABP4. Therefore, TRMT61 A may be a promising target for therapy of bladder cancer.