| Background and objectiveBladder cancer is the most common cancer of the urinary system, and showing arising trend. During the year of2013, the incidence and mortality of bladder cancer inmost western countries shows a decreasing trend overall, but it still shows a risingtrend in developing countries. One side, the cost of diagnosis, treatment, monitoringof relapse and treatment of related complications spent in baldder cancer rearchseventh place in all cancers, which give patients a heavy economic burden. The otherside, the replase rate of bladder cancer in5years can get to50%-90%, when replaseaboult35%non-invasive patient will increase in stage classification, in which aboult10%-20%will change into invasivation and need a radical cystectomy and urinarydiversion surgery. At the same time, incontinence, dysuria and sexual dysfunction andother complications may appear, which affect the social activities and quality ofpatients. So, effective treatment of bladder cancer, reduction of the risk of recurrenceis urgent problems need to be sloved in clinical. Urine modified nucleosides have been found an significant role in the diagnosis,monitoring and prognosis of a variety tumors as tumor markers. Prior to this study themodified nuclesides level in urine of bladder cancer patients were tested, and wefound that m1A (1-methyl adenosine) and1-meI (1-methyl inosine) joint detectionshowed high sensitivity (92.45%) and specificity (87.50%). So, Detection of modifiednucleosides in urine is expected to become a higher value and broad applicationprospects of bladder cancer tumor markers in bladder cancer recurrence andprognosis for effective monitoring.The main source of modified nucleosides in urine are tRNAs, the increase ofactivity and expression of tRNA modification enzymes is the main cause of theincreasement of modified nucleosides in urine. Decades of research proves theexistence of a large number of rRNA and tRNA modified eyzemes ofpost-transcription, at least100kinds, but few large functional studies were conductedon them. It is reported that high activity tRNA modification enzymes exist inmalignant cells. m1A58methyl transferase (hTrm6p/hTrm61p) is an importantenzyme which can produce m1A in the58site of tRNA. It is heterologoustetramercomposed of two subunits.The post-transcriptional modification of tRNAs play an essential role in keepingstable of secondary structure. These modifications can also influence the transcriptionaccuracy of translation, maintain the open coding frame and identification ofaminoacyl tRNA. Methylation is very common in these modifications. A is aconserved nucleoside in the58site of T loop of tRNA, the methylation of A58N1isvery common, it is found in eukaryotes, bacteria and archaea. The methylation of A58of tRNAiMet is essential for the growth of yeast and the stability itself. The hTrm61psubunit can bind SAM, and both two subunits take part in the combination with tRNAby N-terminal amino acid sequences and methylase activity area. hTrm6p/hTrm61plocated in the nucleus plays a crucial role in the initial stages of translation, becausemethylation modification of tRNAiMet58-bit A is essential for its stability.hTrm6p/hTrm61p has little effect on the extension phase. Prio to this study we testedthe level of hTrm6p/hTrm61p in bladder tissue and adjacent normal tissues and found that hTrm6p/hTrm61p was significantly higher expressed in cancer tissues. It remindsthat hTrm6/hTrm61may play an important role in the pathogenesis of bladder cancer,hTrm6/hTrm61may be involved in and contributed to the pathogenesis of bladdercancer.In this study we test the hTrm61mRNA level in bladder cancer tussiues andadjacent normal tussiues and cell lines of EJ, T24,5637and HEK-293; then we knockdown the hTrm61in5637cell line; and test the proliferation and apoptosis of5637.MethodsThe samples of23bladder cancer patients conformed by histology were collected:gender: male:18cases, female:5cases; Incipient:15cases; Replase:8cases;Invasive:12cases; non-innasive:11cases; Histological grade: grade Ⅰ:9cases,gradeⅡ:8cases, grade Ⅲ:6cases. Surgical approach were cystectomy or partialresection; urine specimens were collected before surgery, the tissue specimens wererapidly frozen in liquid nitrogen after resection during the surgery. hTrm61mRNAexpression level was tested in bladder cancer tussiues and adjacent normal tussiuesbesides the cell lines of EJ, T24,5637and HEK-293by qPCR, then the highestexpressed cell line was selected; the hTrm61gene was konckdowned by siRNAtechnology; the proliferation and apoptosis of5637was tested by CCK-8and Flowcytometry(FCM).Results1. Compared with normal tissues, hTrm61mRNA expression in bladder cancer tissueswas significantly increased. hTrm61mRNA expression in bladder cancer tissue was(2.02±1.01), the expression in adjacent normal tissue was (1.00±0.00), astatistically significant difference between the two (p <0.05).2. Incipent patients hTrm61mRNA expression level was2.050±1.090, relapsepatients was1.954±0.856, P=0.833>0.05, the difference was not statisticallysignificant; hTrm61mRNA expression levels of male patients was1.952±0.801, and2.250±1.105for the female patients, P=0.570>0.05, the difference was notstatistically significant; hTrm61mRNA expression levels in patients with invasive cancer was2.116±1.254, non-invasive cancer patients was1.908±0.686, P=0.632>0.05; histological grading F=0.436, P=0.653>0.05, no statisticallysignificant difference among the groups.3.The m1A level in urine increases with the rise of hTrm61in cancer tissue. ThePearson correlation of two variables are as follows: r=0.644, p=0.000<0.05. Thereis a positive correlation between the hTrm61mRNA expression levels in cancer tissueand the m1A level in urine.4.The hTrm61mRNA expressions in human bladder cancer cell lines T24,5637, EJand human embryonic kidney cell line HEK-293had significant differencestatistically (F=58.081P=0.000). Compared with HEK293(1.00±0.00) cell line,5637was5.46±1.38, T24was1.03±0.24, EJ was1.17±0.19. Conducted LSDpairwise comparison:5637compared with T24,p=0.000;5637compared with EJ p=0.000;5637compared with the statistical HEK-293, p=0.000; T24compared withHEK293p=0.937, the difference was not statistically significant; EJ comparedwith HEK293p=0.679, the difference was not statistically significant.5. The knockdown of hTrm61in human bladder cancer cell line5637. Settingblank(BLANK), negative control group (NC) and siRNA1, siRNA2(siRNA1andsiRNA2are knockdown group) groups. Extracted total RNA at24h after transfectionand detected the knockdown effect. Among blank control (1.00±0.00), negativecontrol (0.96±0.09), siRNA1(0.30±0.01) and siRNA2(0.48±0.01) F=120.559, P=0.000. siRNA1group which can reach more than70%in knockdown effect wasselected for the subsequent experiments.6. When hTrm61gene was knocked down in the cell line of5637, its proliferationwas affected significantly.24h after the knockdown, the OD value of BLANK group,NC group and sihTrm61group was0.87±0.03、0.81±0.02'0.72±0.09, differencein the groups was statistically significant.48h after the knockdown, the OD value ofBLANK group, NC group and sihTrm61group was1.03±0.09,0.95±0.06and0.83±0.02, difference in the groups was statistically significant.72h: the OD value ofBLANK group, NC group and sihTrm61group was1.04±0.04,0.99±0.03and0.92±0.04, difference in the groups was statistically significant.7.48h after the knockdown of hTrm61gene in5637cell line, we detected the apoptosis of the cell line in FCM. The percentage of apoptotic cells were6.06±0.67in BLANK group;6.01±1.41in NC group;9.81±0.14in sihTrm61group,difference in the groups was statistically significant(F=11.490, p=0.039). LSDpairwise comparison: between BLANK and NC, p=0.96, the difference was notstatistically significant; between BLANK and sihTrm61, p=0.026, statisticallysignificant in difference; between NC and sihTrm61, p=0.025, a statisticallysignificant difference.Conclusion1. The expression level of hTrm61mRNA in bladder cancerous tissues wassignificant higher than that of normal tissues in the same patient, and the level ofm1A in urine has an positive correlation with the expression level of hTrm61mRNAin cancerous tissues, which suggests that hTrm61play a vital role in the appearanceof bladder cancer, the high expression of hTrm61is a vital cause of the high level ofm1A in urine of the bladder cancer patients.2. The expression of hTrm61mRNA in bladder cancer tissues were not relevant toclinical pathological features.3. When hTrm61was knockdowned in the bladder cancer cell line of5637, itsproliferation and apoptosis were affected significantly, which suggests that hTrm61played an important role in the pathogenesis of bladder cancer, hTrm61mayparticipate in and promote the occurrence of bladder cancer, and also strongly suggesthTrm6p/hTrm61p was a potential therapeutic target of bladder cancer. |
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