Significance And Expression Of M1A Methyltransferase HTrm6p/HTrm61p In Bladder Urothelial Carcinoma

Background and objectiveBladder tumor is the most common malignant tumor in all the tumors of urinary system, the latest statistics show that its incidence increased year by year. The recurrence rate of bladder cancer is high, the recurrence rate as high as50%～90%in5years, about35%of the muscular layer noninvasive carcinoma will increase its grade, stage when recurrence, accordingly, if not treatment timely,10%～20%of them can be further progress to muscle invasive carcinoma, and needs radical bladder cut and urinary diersion, and the complications such as bowel atresia, dysuria and sexual dysfunction may come, this bring great difficulties to social activities of the patients, decline their life quality seriously, bring huge economic,mental stress. Therefore, early diagnosis of bladder cancer and effectively predict the risk of recurrence is the present clinical problems to be solved。Urinary modified nucleoside as tumor molecular markers are studied greatly in recent years, it has a stable molecular structure, it react and change shape not easily, and the molecular mass are small relatively, the collection and storage urine sample is easy, and it is noninvasive, can be directly determined after urine were clllected, and the detection range is not affected by tumor pathology classification and tumor types, patient could accept it easily. Research shows that the level of modified nucleosides in urine can be used for diagnosis of a variety of tumor, therapeutic evaluation and predicting prognosis. Our previous studies have shown that the levels of4kinds of modified nucleosides in urine of patients with bladder cancer is significantly higher than normal, they are N4interchange-acetyl cytidine (ac4C),06-methyl guanosine (06-MeG),1-MeI and1-methyl adenosine; and the joint detection of m1A and1-MeI2kinds of urinary modified nucleosides is of great significance to the diagnosis and predicting prognosis of bladder cancer, so they can be used as the first chosen tumor markers of bladder cancer.However it is not clear why urinary modified nucleoside increased significantly in patients with bladder cancer, research suggested that there are highly active tRNA modification enzyme in malignant cells, it can process height modification to the normal structure of tRNA, and abnormal tRNA are produced, then a large number of modified nucleosides are produced in the metabolic process. M1A are produced from A by catalytic transformation of tRNA methyltransferase. Studies have shown that the tRNA methyltransferase which catalytic the formation of m1A is m1A58methyl transferase in human body, namely hTrm6p/hTrm61p.This study through the detection of levels of m1A in the urine in bladder cancer patients and normal healthy people, different expression of hTrm6p/hTrm61p in carcinoma tissues and adjacent cancerous tissue removed in surgery of bladder cancer patients, the relationship of expression levels of hTrm6p/hTrm61p in cancerous tissues and the lecel of m1A in urine of the same patients, and the relationship between expression levels of hTrm6p/hTrm61p in cancerous tissues and the clinical pathologic characteristics.and to explore the role of hTrm6p/hTrm61p in the high level expression of m1A in bladder cancer patients, and then provide theoretical and experimental basis for the clinical application of m1A as the tumor markers of bladder cancer. Methods32cases bladder urothelial cell carcinoma (bladder urothelial cell carcinoma, BUCC) patients confirmed by pathology were chosen;25cases of men,7cases women;22cases of incipient, recurrence; histologic classification:14cases I level,10cases of Π level, magnitude8cases Ⅲ lecel;18cases of invasive carcinoma,14cases of noninvasive carcinoma; the surgery for bladder are full or partial resection; urine are collected before surgery, carcinoma tissues and adjacent tissues are collected intraoperative.16normal healthy people are selected as control group. High performance liquid chromatography/electrospray-quadrupole time-of-flight mass spectrometry (HPLC/ESI-Q-TOF-MS) are adopted to detect the levels of m1A in urine of bladder cancer patients and normal healthy people; Western blot method are used to detect the expression level of hTrm6p/hTrm61p in carcinoma tissues and adjacent tissues of patients with bladder.Results1. The levels of mlA in urine in the bladder cancer group (5.76±0.90) are significantly higher than those of healthy volunteers (2.79±0.54), the difference was statistically significant (P<0.05), and each patients in bladder cancer group were higher than the control group.2. The expression levels of hTrm6p/hTrm61p of31cancer tissue (97%) of the all32patients were obviously higher than those of tissues adjacent to carcinoma, the expression level of hTrm6p/hTrm61p in bladder cancer tissues was0.701±0.259, and its expression level in the tissue adjacent to carcinoma was0.443±0.239, the difference was statistically significant.3. The expression level of hTrm6p/hTrm61p in incipient patients was0.679±0.250, and recrudescent patients was0.743±0.283, t=0.641, P=0.526>0.05, the difference was not statistically significant; The expression level of hTrm6p/hTrm61p in male patients was0.703±0.273, female patients was0.696±0.221, t=0.057, P=0.955>0.05, the difference was not statistically significant; The expression level of hTrm6p/hTrm61p in invasive carcinoma was0.689±0.272, noninvasive carcinoma patients was0.716±0.251, t=0.289, P=0.775>0.05, the difference was not statistically significant; Among the histologic classification, F=1.176, P=0.323>0.05, the difference was not statistically significant, and further S-N-K method comparing between two, P=0.412>0.05, the difference was not statistically significant between two groups.4. m1A levels in urine has a upward trend with the high expression level of hTrm6p/hTrm61p protein in cancerous tissue, Pearson correlation analysis was used for two variables, r=0.799, p=0.000<0.05, illustrate that m1A levels in urine were correlate to the expression levels in cancerous tissue. Then linear regression analysis was used for the two variables, coefficient of determination R2=0.639, reflecting that the63.9%reasons of high expression levels of m1A in urine was the high expression level of hTrm6p/hTrm61p protein in cancerous tissue; Inspection result F=53.018, P=0.000<0.05, regression model was statistically significant, there is a linear relationship between the high expression levels of m1A in urine of bladder cancer patients and the high expression levels of hTrm6p/hTrm61p protein in cancerous tissue, constant and hTrm6p/hTrm61p expression levels in bladder cancer tissue have statistical significance. Constants a=3.816, the regression coefficient b=2.782, the regression equation Y=3.816+2.782X.Conclusion1. The expression level of m1A in urine of bladder cancer patients was obviously higher than that of normal healthy people, so the detection of m1A in urine can be used as tumor markers for the screening and early diagnosis of bladder cancer.2. The expression level of hTrm6p/hTrm61p in cancerous tissues was significantly higher than that of tissues adjacent to carcinoma of the same patients, and the levels of m1A in urine exist positive correlation to the expression level of hTrm6p/hTrm61p in cancerous tissues, this suggests that hTrm6p/hTrm61p play an important role in the occurrence of bladder cancer, the high express levels of hTrm6p/hTrm61p is an important cause of the high levels of mlA in urine of the bladder patients.3. The expression levels of hTrm6p/hTrm61p in bladder cancer tissues were irrelevant to clinical pathological features.