| Background and purposeBladder cancer is the most common malignant tumor of the urinary system.In recent years,the incidence in our country has increased year by year.Among them,70~80% of patients with bladder cancer are non-muscle invasive bladder cancer(NMIBC)at the initial visit.Currently,transurethral electrotomy(TUR)combined with intravesical instillation is commonly used in clinical treatment.However,the recurrence rate after treatment is high,and about 30% NMIBC patients develop into invasive bladder cancer.Therefore,exploring the pathogenesis and finding effective therapeutic targets have been important issues for urologists to study bladder cancer.Our previous study found that the urinary levels of 1-Methyladenosine(m1A),1-Methyl Inosine(1-mel),O6-methylguanosine(O6-meG),N4 Acetylcytidine(ac4c)in urinary bladder cancer patients were significantly higher than those in normal subjects.Among them,the combination of m1 A and 1-meI had extremely high sensitivity(92.45%)and specificity(87.50%)to detect bladder cancer,which was of great significance for the early diagnosis and monitoring recurrence of bladder cancer.M1 A is formed by the m1A58 methyltransferase which catalyzes the adenosine(A)atits tRNA position 58.M1A58 methyltransferase consists of two subunits of hTrm6 p and hTrm61 p,in which hTrm61 P is encoded by the TRMT61 A gene and is the catalytic subunit.Our follow-up study found that the expression of hTrm61 p mRNA and protein in bladder cancer tissue was significantly higher than that in paracancerous tissue.The cell proliferation and invasion of bladder cancer cell line5637 significantly decreased and the apoptosis rate increased significantly with the down-regulation of hTrm61 p expression.These studies suggest that hTrm61 p may promote the occurrence and development of bladder cancer,but the mechanism of its elevated expression is not yet clear.If the relevant mechanisms of hTrm61 p regulation can be clarified,its value in the prevention and treatment of malignant tumors must be further clarified.Nuclear factor-кB(NF-кB),a nuclear factor-activated B-cell K-chain light enhancer(nuclear factor kappa-light-chain-enhancer of activated B cells),belongs to a family of transcription factor proteins.It plays a central role in many cell-mediated transcriptional regulation and involves in the expression and regulation of a variety of genes.In addition,it plays an important role in a variety of cellular activities such as apoptosis and proliferation,immune responses,and inflammatory responses.Abnormal activation of NF-кB has been found in a variety of malignancies.By promoting the nuclear translocation of activated NF-кB,it is involved in the regulation of downstream target gene sequences,thereby promoting the development of tumors.The important role of NF-кB in tumors has made it an important part of revealing the development and regulation of tumor cells.After repeated comparisons and analysis of gene banks,it was found that the NF-кB binding site was present in the hTrm61 p mRNA promoter region.It was speculated that NF-кB may have an important influence on the regulation of hTrm61 p expression.However,the relationship between them and its specific mechanism have not been reported.Based on previous work,this study was designed to further examine the mRNA expression levels of hTrm61 p and NF-кB in bladder cancer tissues and paracancerous tissues.At the same time,a correlation analysis between then was analyzed.To observe the cell proliferation and detect the m RNA and protein expression levels of hTrm61 p,the 5637 cell line was treated with different concentration of NF-кB inhibitor PDTC.Immunofluorescence method was used to observe the localization of hTrm61 p and NF-кB protein in 5637 cells.Finally,the co-immunoprecipitation technique was used to detect the binding of hTrm61 p and NF-кB protein in 5637 cells.Through this study,we can obtain direct evidence of the regulation of hTrm61 p expression in human bladder cancer cell line 5637 by NF-кB.And this research can provide a new theoretical basis for further exploring the pathogenesis of bladder cancer and finding effective therapeutic targets.Method:1.Detection of hTrm61 p and NF-кB mRNA in bladder cancer and paraneoplastic tissues: 34 cases of complete bladder or subtotal cystectomy were selected in the First Affiliated Hospital of Zhengzhou University from February 2016 to February 2017(27 males and 7 females,aged 33 to 81 years,median age 62 years,14 low-grade bladder cancers,and 20 high-grade bladder cancers).Urinary urothelial carcinoma of the bladder was diagnosed by postoperative biopsy.Cancer tissues and normal tissues 5 cm away from the tumor were taken respectively.The expression of hTrm61 p and NF-кB m RNA in bladder cancer and paracancerous tissues were detected by Fluorescence quantitative polymerase chain reaction(RT-qPCR),and the correlation between them was analyzed.2.Detection of hTrm61 p and NF-кB mRNA expression in bladder cancer 5637 cells and normal human urothelial sc-huv-1 cells: Bladder cancer line 5637 cells and normal human urothelial SV-HUV-1 Cells were from the Chinese Academy of Sciences cell bank.And then,they were resuscitated,passaged and cultured.RNA was extracted from well-grown cells and the expression levels of hTrm61 p and NF-кB mRNA were detected by RT-qPCR.3.Observation of the effect of NF-кB inhibitor PDTC on the proliferation of bladder cancer cell line 5637 in vitro: the experiment was divided into 4 groups,blank control group(without PDTC treatment),low concentration PDTC group(25μmol/L),medium concentration PDTC group(50μmol/L),high concentration PDTC group(100μmol/L).After cultured for 24 h,48h,and 72 h,CCK-8 method was used to detect proliferation of 5637 cells in different concentrations of PDTC.4.Detection of the effect of NF-кB inhibitor on the expression of hTrm61 p mRNA and hTrm61 p protein in bladder cancer cell line 5637: RT-qPCR and Western Blot were used to detect hTrm61 p mRNA and hTrm61 p protein in 5637 cells treated with different doses of PDTC.5.Observation of the localization of hTrm61 p and NF-кB proteins in 5637 cells:the localization of hTrm61 p and NF-кB proteins in bladder cancer cells 5637 was observed by double immunofluorescence.6.Detection of hTrm61 p and NF-кB protein interactions in bladder cancer 5637 cells by co-immunoprecipitation: 5637 cell lysates were precipitated using anti-hTrm61 p monoclonal antibody and anti-NF-кB monoclonal antibody,respectively.The whole protein group and IGg group were used as control groups to detect the binding of hTrm61 p and NF-кB protein in 5637 cells.Result:1.Detection of hTrm61 p and NF-кB mRNA in bladder cancer and paraneoplastic tissues: Compared with the paracancerous tissues,the relative expression level of hTrm61 p mRNA(2.218±0.630)and NF-кB mRNA(2.291 ±0.729)in bladder cancer tissues was increased significantly and there are statisticaldifferences(P<0.01).SPSS 21 statistical software was used to Pearson correlation analysis between the relative expression level of hTrm61 p mRNA and NF-кB mRNA(r=0.834),which suggested that the expression of hTrm61 p m RNA and NF-кB mRNA are positively correlated in bladder cancer tissues.2.Detection of hTrm61 p mRNA and NF-кB mRNA expression in normal urothelial SV-HUV-1 cells and bladder cancer 5637 cells: Compared with normal urothelial SV-HUV-1 cells,the level of hTrm61 p mRNA(1.955±0.153)and NF-кB mRNA(2.802 ± 0.048)in bladder cancer 5637 cells were significantly increased,there are statistical differences(P<0.01).3.Effect of NF-кB specific inhibitor PDTC on proliferation of bladder cancer5637 Cells: After 24 h incubation with different concentration of PDTC,the OD values of the blank control group and low,medium,and high PDTC groups were0.716±0.036,0.586±0.042,0.475±0.029,and 0.396±0.033,respectively.Compared with the blank control group,the OD value of the PDTC group decreased,showing a concentration-dependent,and there are statistical differences(P <0.05).After 48 h of culture,the OD values of the blank control group and low,medium,and high PDTC groups were 0.988±0.065,0.701±0.039,0.570±0.035,and 0.449±0.025,respectively.Compared with the control group,the OD value of the PDTC group decreased,showing a concentration-dependent,and there are statistical differences(P <0.05).After 72 h of culture,the OD values of the blank control group and low,medium,and high PDTC groups were 1.127±0.052、0.827±0.012、0.647±0.029、0.499±0.036,respectively.Compared with the blank control group,the OD value of the PDTC group decreased,showing a concentration-dependent,and there are statistical differences(P <0.05).4.Effect of NF-кB inhibitors on the expression of hTrm61 p in bladder cancer cell line 5637: Compared with the blank control group,the relative expression level of hTrm61 p m RNA in the 25μmol/L PDTC group,50μmol/L PDTC group and100μmol/L PDTCgroup was 0.555±0.044,0.457±0.011 and 0.352±0.034,and there are statistical differences(P <0.05).Consistent with the results of gene levels,as the concentration of PDTC increased,the expression of hTrm61 p protein gradually decreased.Compared with the blank control group,the relative expression level ofhTrm61 p protein in the 25μmol/L PDTC group,50μmol/L PDTC group and100μmol/L group was 0.814 ± 0.022,0.641 ± 0.055 and 0.469 ± 0.036,With increasing PDTC concentration,hTrm61 p protein expression gradually decreased,and there are statistical differences(P <0.05),respectively.5.The localization of hTrm61 p and NF-кB proteins in bladder cancer 5637 cells:hTrm61p protein localizes in the cytoplasm and nucleus of 5637,and is mainly located in the cytoplasm.NF-кB protein is also localized in the cytoplasm and nucleus of 5637 cells.It is mainly located in the cytoplasm.Both colocalization signals exist in the cytoplasm and the nucleus,and are mainly found in the cytoplasm.6.Co-immunoprecipitation of h Trm61 p and NF-кB protein in bladder cancer 5637 cells:Simultaneous exist of NF-кB p65,hTrm61 p protein were detected in whole protein extraction group.In the anti-NF-kappa B p65-treated supernatant with co-immunoprecipitation,only hTrm61 p protein was detected,but no NF-кB p65 was detected.In the anti-NF-kappa B p65-treated precipitation complex with co-immunoprecipitation,both of hTrm61 p protein and NF-кB p65 protein could be detected.In the anti-hTrm61p-treated supernatant with co-immunoprecipitation,only NF-кB p65 protein was detected,but no hTrm61 p was detected.In the anti-hTrm61p-treated precipitation complex with co-immunoprecipitation,both of hTrm61 p protein and NF-кB p65 protein could be detected.Conclusion:1.Both hTrm61 p mRNA and NF-кB m RNA are significantly over-expressed in cancer tissues and cells of bladder cancer.In addition,there is a positive correlation between hTrm61 p mRNA and NF-кB mRNA expression,which indicates that the role of hTrm61 p in the development of bladder cancer is closely related to NF-кB.2.NF-кB plays an important role in regulating the expression of hTrm61 p in bladder cancer cells.Inhibiting the activity of NF-кB can inhibit the expression of hTrm61 p and further inhibit the proliferation of tumor cells.3.NF-кB and hTrm61 p have interaction in bladder cancer 5637 cells,which provides new ideas for further exploring the pathogenesis of bladder cancer and finding effective therapeutic targets. |
PDF Full Text Request