The Impact Of TRM61 Connecting With PKC? On The Expression Of TRNAi^Met In Bladder Cancer And The Study Of Mechanism

Background and objectiveBladder cancer is the seventh most common cancer in the world.This cancer is more common in males than females and is one of the most expensive to manage.The 5-year survival rate in the patients with invasive bladder cancer who have implemented the preserve bladder surgery is from 59 to 70 percent.While 50 percent of the patients who have radical cystectomy will occur metastasis.Bladder cancer seriously threatens the life of the patients.At the same time,it brings huge economic and social pressure to the patients.Cystoscopy is an important examination which is used to diagnosis and monitor the recurrence,however it is invasive and expensive.Therefore looking for the new bio-markers which used to diagnosis and monitor the recurrence has a crucial clinical significance.We have found that the levels of m1 A and 1-mel in the urine of bladder cancer patients were higher than that in the urine of the healthy volunteers in our early study.Meanwhile,connecting m1 A with 1-mel to detect the bladder cancer,the sensitivity and specificity can be as high as 92.45 % and 87.50 % respectively.Therefore urine modified nucleosides as molecular markers have the function of diagnosing and monitoring the bladder cancer.m1A58 methyltransferase?hTrm6p/hTrm61p?can create m1 A because it make the adenosine in the position 58 methylation.hTrm61 p is the catalytic subunit of m1A58 methyltransferase and is encoded by TRM61.TRM61 plays an important role in maintaining the stabilization of tRNAi^Met.However inactivation of TRM61 results in growth arrest and cell death due to the rapid degradation of tRNAi^Met lacking m1A58 modification.tRNAi^Met mainly recognize the iniation codon and plays an important role in translation initiation.In our previous studies,we have found that the expression level of TRM61 in bladder cancer tissues were higher than that in adjacent normal tissues.Knocking down the expression of TRM61 in 5637 cell line,the apoptosis of 5637 cell is increased and the proliferation is slowed and the invasion is weakened.The above results suggest that TRM61 plays a role in promoting the development and progression of bladder cancer.However,the mechanism of cancer promoting effects of TRM61 and the mechanism of its elevated expression are not clear.Protein kinase C?PKC?,a prototypical class of serine/threonine kinases,involves in gene expression,cell proliferation,apoptosis,metastasis,signal transduction and other functions.Protein kinase C-alpha?PKC??,a classic subtype of Protein kinase C family,participates in the tumor occurrence,development and resistance.The expression of PKC? is different in different tumors,while the level of PKC? differentially alters as a function of tumor differentiation.The role of PKC? in the tumor makes it as an important drug target in clinic.Based on the previous experiments,we detect the mRNA expression level of TRM61 and PKC? in bladder cancer tissues and adjacent tissues and cell lines of 5637,T24 and HEK-293,at the same time we test the expression level of tRNAi^Met in this study.Knocking down the level of TRM61 in 5637 cells by the siRNA interference technique,we test the mRNA expression level of TRM61 and the expression level of tRNAi^Met.Using the cDNA Gene Sequencing technology,we analyse the cDNA Gene Sequence of TRM61.Then we detect the TRM61 mRNA expression level of 5637 and T24 cells after treatment with 5-Aza-2dc of different concentrations.By immunofluorescence,we observe the localization of TRM61 and PKC? in 5637 cells.Methods1 The relative expression levels of TRM61,PKC? and tRNAi^Met in bladder cancer tissues.23 cases of bladder cancer diagnosed by pathological examination were collected in The First Affiliated Hospital Of Zheng Zhou University during February 2015 to February 2017.The relative expression levels of TRM61,PKC? and tRNAi^Met in bladder cancer tissues and adjacent tissues were detected by qPCR technique.2 The relative expression levels of TRM61,PKC? and tRNAi^Met in cell lines of 5637,T24 and HEK-293.The relative expression levels of TRM61,PKC? and tRNAi^Met in cell lines of 5637,T24 and HEK-293 were tested by qPCR technique.3 Effect of knocking down TRM61 on expression of tRNAi^Met in bladder cancer 5637 cells.The expression levels of TRM61 and tRNAi^Met in the NC group?5637 cells transfected with NC-si RNA?and siRNA group?5637 cells treated with lipofectamine R3000 TRM61-siRNA?were detected by qPCR technique.NC group and siRNA group were transient transfection for 48 h.4 cDNA Sequence of TRM61 in Bladder Cancer Tissue and adjacent Tissues.The TRM61 cDNA of 23 cases of bladder cancer tissues and adjacent tissues were extracted and sequenced.The gene sequence of TRM61 gene coding region was detected.5 Effects of DNA methyltransferase inhibitor?5-Aza-2dc?on the expression level of TRM61 in bladder cancer cells lines of 5637.Different concentrations of DNA methyltransferase inhibitor?5-Aza-2dc?were dealed with 5637 cells.Then TRM61 mRNA levels in cell lines were measured after 72 h.6 Double immunofluorescence was used to detect the localization of TRM61 and PKC? protein in 5637 cells.Result1 In 23 cases of bladder cancer tissues,the TRM61 mRNA expression level?2.195±1.100?and PKC? mRNA expression level?1.772±1.439?and tRNAi^Met expression level?2.200±2.446?were significantly increased compared with the adjacent tissues?1.000 ± 0.000?.The differences were statistically significant?P<0.05?.Two-variable Pearson correlation analysis of the mRNA levels of TRM61 and PKC? in cancer tissues showed that there was a obviously positive correlation between them?R2= 0.270,P<0.05?.PKC? mRNA expression level in high-grade bladder cancer tissues?2.146±2.264?was higher than it in low-grade?0.976±0.805?.The differences were statistically significant?P<0.05?.2 Compared with HEK-293 cells?1.000 ± 0.000?,the mRNA expression level of TRM61 in 5637 cells was 3.846±0.498,the mRNA expression level of PKC? was 0.492±0.131,the expression level of tRNAi^Met was 1.423±0.114,The differences were statistically significant?P<0.05?;the mRNA expression level of TRM61 in T24 cells was 0.700±0.030,the mRNA expression level of PKC? was 1.797±0.255,the expression level of tRNAi^Met was 0.758±0.198,The differences were statistically significant?P<0.05?.3 Changes of expression level of tRNAi^Met in knockdown of TRM61 in bladder cancer 5637 cells.Compared with NC group?1.000±0.000?,The TRM61 mRNA expression in siRNA group?0.450±0.088?was significantly down regulated.Silence efficiency can reach 55%.The differences were statistically significant?P<0.01?.Compared with NC group?1.000±0.000?,The tRNAi^Met expression in siRNA group?0.583±0.032?was significantly down regulated.4 The results of TRM61 cDNA sequences in 23 cases of bladder cancer tissues and adjacent tissues showed that the TRM61 cDNA sequences in cancer tissues were not mutated compared with that in adjacent tissues.5 As the DNA methyltransferase inhibitor?5-Aza-2dc?concentration increased,the TRM61 mRNA expression in 5637 cell lines were elevated and concentration-dependent.Compared with control group?1.000±0.000?,TRM61 mRNA expression in 5637 cells treated with 5-Aza-2dc were 1.088±0.036?drug concentration was 1 uM?,1.336±0.150?drug concentration was 5 uM?,1.505±0.117?drug concentration was 10 uM?,1.862±0.273?drug concentration was 15 uM?.The differences were statistically significant?P<0.05?.6 By double immunofluorescence,we observed that TRM61 was in cytoplasm and nucleus,while the PKC? was mainly in cytoplasm.Conclusion1 The mRNA expressions of TRM61 and PKC? and the tRNAi^Met expression in bladder cancer tissues were significantly higher than those in adjacent tissues.The TRM61 mRNA expression was positively correlated with the PKC? mRNA expression.Knockdown of TRM61 in bladder cancer 5637 cell line,the tRNAi^Met expression was significantly decreased.This suggests that TRM61 combined with PKC? affects t RNAiMet expression,which provides a new theoretical basis for the carcinogenesis of bladder cancer.2 There was no gene mutation in TRM61 cDNA sequences of bladder cancer tissues,which indicated that TRM61 gene genetics could not be explained the high expression of TRM61 in bladder cancer and the carcinogenesis of bladder cancer.3 As the DNA methyltransferase inhibitor?5-Aza-2dc?concentration increased,the TRM61 mRNA expression in 5637 cell lines were elevated and concentration-dependent.This suggests that Epigenetic regulation may be a new mechanism for TRM61 high expression in bladder cancer and provides new ideas for the clinical treatment of bladder cancer.4 In 5637 cells,TRM61 was in cytoplasm and nucleus,while the PKC? was mainly in cytoplasm.This lays the theoretical foundation for further studying the mechanism that the both affect translation initiation of tRNAi^Met in bladder cancer.