| Background and objectiveIn urinary system, bladder cancer is the most commom malignant rumor,with the rising morbidity year by year.Only in 2010,in the USA bladder cancer account for an estimated 70530 new cases and 14680 deaths.The most commom presenting pathology of bladder rurmors is transitional cell carcinoma(90% of cases),while squamous cell carcinomas, adenocarcinomas and other rare subtypes comprise a minority of cases.Bladder cancer have seriously influenced people,work and life. On one side, it needs expensive cost on the diagnosis,trewtment,monitoring of relapse and treatment with related complications in bladder cancer,which can bring a serious economic burden to patients. On the other side,bladder cancer has very high replase rate, and the stage of a rather part of non-invasive patients will rise when bladder cancer relapsed. However, 10%-20% of non-invasive bladder cancer will become invasive bladder, which can only be treated by a radical cystectomy and urinary diversion surgery. So clear diagnosis of bladder cancer and good control of bladder cancer,development are the key factors in treatments of bladder cancer in clinical.As a trustworthy tumor mark, urine modified nucleosides play a significant role in early diagnosis, monitoring and prognosis of a lot of tumors. Our earlier study makes clearly that the combined detection of m1 A and 1-mel has a rather high sensitivity(92.45%) and specificity(87.50%). The m1A58 methyltransferase(h Trm6p/h Trm61p)is a kind of proteins produced by the body which is made of two subunits. It can catalyze the fifty-eighth adenosine A of t RNA into m1 A.Our early experimentsshow that compared with normal people, the level of m1 A in bladder cancer tissue and bladder cancer patients,urine are both rised significantly.HTrm61 is one of integrant genes in producing m1 A methyltransferase.When h Trm61 is knocked down, the expression of m1 A is also reduced. Throughout screening,5637 cell is the cell which highly expresses h Trm61. When h Trm61 in 5637 cell is knocked down,the proliferation of 5637 cell is inhibited, and the apoptosis of 5637 cell is enhanced,which illustrated that h Trm61 has an importantsignificance in the pathogenesis of bladder cancer.On the basis of our early experiments, the changes of the invasion ability of 5637 cell is further explored when h Trm61 was knock down in 5637 cell. What is more, the 5637 cells transfected by h Trm61-si RNA and 5637 cell transfected only by lip2000 are combined to sent to do Affymetrix U133 plus 2.0 array genechip, by which we discover that the differentially expressed genes in expression profiling when h Trm61 was knock down.Through the expression differences of genes,we can research which signal pathway can be influenced.Methods1.We got the chemical synthesized h Trm61-si RNA and negative control si RNA from shanghai Gene Pharma co.,Ltd. The experiment was made up of three groups, which comprise experimental group,negative group and blank group. Bladder cancer 5637 cells were transfected with h Trm61-si RNA(as experimental group) and negative control si RNA(as negative group) and nothing(as blank control),respectively. When they were transfected after twenty-four hours, we detected the knocked-down rate of h Trm61.Finally, we checked the changes of invasion ability of 5637 cell when h Trm61 was knock down by Transwell. 2. Through Affymetrix U133 plus 2.0 array genechip to Study the different expression profiling in 5637 cell transfected by h Trm61-si RNA compared with 5637 cell transfected only by lip2000 through genechip. Screen the differentially expressed genes. Do the GO-Analysis on the differentially expressed genes and study which signaling pathway can be influced by the differentially expressed genes.Results1. In the 5637 transwell experiment,the transmembranel cell numbers in blank group, negative control group and experimental group are 76±4.6, 74±5.2,and 32±2.2, respectively. Compared with blank group, the transmembranel cell number of the negative group has no statistical significance(P>0.05). However, compared with blank group, the transmembranel cell number of the experimental group has significant statistical significance(P<0.05). 2 The results of Affymetrix U133 plus 2.0 array genechip reveal that according to the significantly differential standard of Foldchange ≥ 2.0 or ≤ 0.5, the number of differentially expressed genes is 2423,1337 of which are up-regulated. And other 1086 differentially expressed genes are down-regulated. When the significantly differential standard of Foldchange ≥5.0 or ≤0.2, the number of differentially expressed genes is 419, 221 of which are up-regulated. And other 198 differentially expressed genes are down-regulated.what is more, a lot of signaling pathway can be influenced by the differentially expressed genes.Conclusion1. When knocking down h Trm61 in 5637 cell, we found that the invasive ability of 5637 cell was reduced, which show that h Trm61 can increase the invasive ability of 5637 cell. It indicate that h Trm61 may play a important role in the mechanism of invision in bladder cancer. 2. Preliminarily build the different expression profiling between the knocked-down 5637 cell and 5637 cell transfected with only by lip2000. 3. Through Affymetrix U133 plus 2.0 array genechip, we found thousands of differentially expressed genes, which involved in lots of signaling pathways. It may lay the first stone for the further research of related pathogenesis of bladder cancer. |
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