Mechanism Of Cisplatin Promoting Apoptosis Of Bladder Cancer Cells By Affecting MiR-141 Expression

Objective:Part one:To investigate the difference of apoptosis and expression of miR-141in urinary epithelial cancer cells cultured with different concentration gradients of cisplatin and chloramine,and to provide some clinical enlightenment.Part two:To explore the effect mechanism of cisplatin on the apoptosis of bladder cancer cells by affecting miR-141,and to expatiate from the perspective of mir-141 and the silencing information regulator 1(Sirt1)pathway,in order to provide help for the clinical treatment of drug resistance of bladder cancer.Materials and Methods:Part one:T24 cell line of bladder cancer was selected for culture,and the third-generation growth cells were selected as the research object.They were divided into the following groups:Control group(equal amount of normal saline);H-DDP group(DDP culture at 50μmol/L for 24 h);M-DDP group(DDP culture at 20μmol/L for 24 h);L-DDP group(DDP culture at 10μmol/L for 24 h);The specific detection items were as follows:real-time fluorescence quantitative nucleic acid amplification detection system(RT-PCR)was used to detect the mRNA expression level of miR-141 in bladder cancer cell lines and normal bladder epithelial tissues.MTT assay was used to detect the proliferation inhibition of cancer cells in each group.Transwell assay was performed to detect cell metastasis and invasion.Cell apoptosis was detected by flow cytometry.Detection of cell cycle by DNA ploidy;Mitochondrial transmembrane potential was detected.Changes of organelles were detected by transmission electron microscopy.The expressions of miR-141 in cells of each group were detected by western-blot,and finally statistical analysis was performed.Part two:On the basis of the first group,the Control group(equivalent normal saline);H-DDP group(50μmol/LDDP for 24 h);M-DDP group(20μmol/L DDP for24 h);L-DDP group(10μmol/L DDP for 24 h);The following studies were carried out as:RT-PCR was used to detect the expression differences of Sirt1,pro-apoptotic protein Bax and Bcl-2 in cells of each group.The regulation relationship between Sirt1 and miR-141 was detected by dual-luciferase assay.Western blot was used to detect the apoptosis-related proteins Bax,Caspase-3,Bcl-2,Sirt1 as well as p53,FOXO1 andβ-catenin.Finally,statistical analysis was performed to determine the specific regulatory mechanism of cisplatin on the apoptosis of bladder cancer cells.Results:Part one:RT-PCR analysis:there were three obvious bands in the figure,rRNA28S,rRNA 18S and rRNA 5S,respectively,from top to bottom,indicating that the total RNA structure of the extracted samples was intact without degradation.The data indicated that the concentration of miR-141 in the control group was the highest and the brightest.With the intervention of cisplatin chloramine,the color of the bands gradually darkened,and with the increase of the concentration,the darker the bands became,indicating that the content of miR-141 gradually decreased.The OD value of total RNA of the extracted samples was detected by microscale nucleic acid detector,and a 260/280 ratio of each sample was within the range of 1.8~2.0.The dissolution curve between miR-141 and the internal reference gene U6 in each sample showed a specific single peak,fully indicating that the amplification primer used had no other foreign body dimer or other non-specific amplification process.In groups T24 bladder cancer cell expression level of statistical analysis,in groups of2-~ΔΔctΔct numerical size comparison for H-DDP<M-DDP<L-DDP<Control,data comparison between groups found P<0.05,differences were statistically significant.HE staining analysis:the control group had more cells on the whole,more blood vessels and villi around the cells with higher density,and weaker apoptosis trend of bladder cancer cells.With the full intervention of cisplatin chloramine,the shape of cells gradually became irregular,the overall density was low,the cell gap was large and filled with more tissue fluid,and the cells were mostly irregular polygons.With the increase of the concentration of cisplatin chloramine solution,the cell density gradually decreased,and the space around the cells gradually increased,and the regular cube gradually turned into an irregular polygon,indicating a serious trend of cell apoptosis.MTT assay analysis:with the extension of time,the cell proliferation trend of each group gradually decreased,the overall change of the control group was small,and the growth of tumor cells was stable.With the full intervention of cisplatin and chloramine,the T24 cell growth of bladder cancer in each group was gradually inhibited and the trend of cell proliferation was gradually reduced.With the increase of concentration of cisplatin chloramine solution,the trend of cell proliferation gradually slowed down,showing a certain degree of drug resistance.Transwell assay of cell invasion:H-DDP(18.68±6.46)<M-DDP(23.56±7.21)<L-DDP(34.70±7.88)<Control(53.44±12.09),the difference was statistically significant(P<0.05).Mitochondrial membrane potential analysis:the abnormal mitochondrial membrane potential of the control group cells was the most obvious,the overall green fluorescence,mitochondrial membrane potential changes were large.The T24 cells after fully interference by cisplatin chloramine,its internal miR-141 expression gradually reduces,T24 cells overall affect sexual,mitochondrial membrane potential sex gradually obvious,gradually from the pure green to yellow-green,highest when the concentration of cisplatin chloramine,cell mitochondria membrane potential yellow-green color monomer model,with the increase of its concentration,this phenomenon is increasingly obvious.TUNEL cell apoptosis:compared with the Control group(0.78%)<L-DDP(5.25%)<M-DDP(13.14%)<H-DDP(21.25%),the apoptosis trend gradually increased with the increase of cisplatin chloride concentration,the difference was statistically significant(P<0.05).Comparison analysis of T24 cells of bladder cancer in S phase in each group showed that H-DDP(25.38%)<M-DDP(30.46%)<L-DDP(39.80%)<Control group(43.45%),and the difference was statistically significant(P<0.05).The proportion of G2/M phase cells was analyzed as H-DDP(6.38%)<M-DDP(12.46%)<L-DDP(18.80%)<Control group(23.45%),and the difference was statistically significant(P<0.05).Transmission electron microscopy analysis:lipid deposition was observed to a certain extent in the cells of the control group,many vacuoles were observed in the cells,prominent processes were observed in the cells,and the mitochondria and golgi bodies in the cells were enlarged.With the full intervention of cisplatin and chloramine solution,the degree of internal lesions in T24 cells of bladder cancer decreased,and showed a dose-dependent feature.The vacuoles in the cells gradually decreased,the number of primary lysosomes,secondary lysosomes and even phagosomes also gradually decreased,and the mitochondrial volume tended to be normal.Western-blot analysis:with the increase of cisplatin chloride concentration,the expression level of miR-141 in bladder cancer T24 cells gradually decreased,and the expression rate between the groups was H-DDP(0.38±0.04)<M-DDP(0.43±0.10)<L-DDP(0.69±0.18)<Control group(0.83±0.15),and the difference between the groups was statistically significant(P<0.05).Part two:Detection and analysis of dual-luciferase assay showed that miR-141was more abundant in the region of Sirt1 3’UTR.It can be seen that there is a certain correlation between them and a certain regulatory relationship.miR-141inhibitor co-transfected with its recombinant plasmid in bladder cancer T24 cells significantly increased the activity of wild-type Sirt1 3’UTR,while the activity of mutant Sirt1 3’UTR was stable,with statistically significant difference(P<0.01).In bladder cancer T24 cells co-transfected with miR-141 promoter and its recombinant plasmid,the activity of wild-type Sirt1 3’UTR was significantly reduced,but the activity of mutant Sirt1 3’UTR was increased,and the difference was statistically significant(P<0.01).RT-PCR analysis:he expression trend of Bax and Caspase-3 was Control<L-DDP<M-DDP<H-DDP,and the difference between the groups was statistically significant(P<0.01).The Sirt1 concentration expression relationship was Control<L-DDP<M-DDP<H-DDP,and the difference between the groups was statistically significant(P<0.01).Sirt1 protein concentration expression:Control(0.19±0.04)<L-DDP(0.43±0.12)<M-DDP(0.93±0.34)<H-DDP(1.26±0.56).There were statistically significant differences in data between the groups(P<0.05).Bax protein and Caspase-3 protein concentration expression:Control<L-DDP<M-DDP<H-DDP,the data between the groups were statistically significant(P<0.05).Bcl-2concentration expression:H-DDP(0.46±0.18)<M-DDP(0.73±0.25)<L-DDP(0.94±0.35)<Control(1.21±0.34),the data between the groups were statistically significant(P<0.05).p53 concentration expression:H-DDP(0.58±0.14)<M-DDP(0.83±0.25)<L-DDP(1.34±0.40)<Control(1.66±0.56),the data between the groups were statistically significant(P<0.05).FOXO1 concentration expression:H-DDP(0.38±0.09)<M-DDP(0.47±0.20)<L-DDP(1.15±0.23)<Control(1.17±0.36),the data between the groups were statistically significant(P<0.05).β-catenin concentration expression:H-DDP(0.32±0.10)<M-DDP(0.43±0.22)<L-DDP(1.13±0.23)<Control(1.20±0.28),the data between the groups were statistically significant(P<0.05).Conclusions:Part one:Cisplatin chloramine can fully inhibit the expression effect of miR-141 in T24 bladder cancer cells and the apoptosis effect of bladder cancer cells,providing some reference for the following studies.Part two:Sirt1 is one of the target genes directly regulated by miR-141,both of which present negative feedback regulation and play an important role in the development of bladder cancer cells.miR-141 can fully promote the proliferation and inhibit the apoptosis of bladder cancer cells,and Sirt1 can fully promote the apoptosis of bladder cancer cells.Cisplatin chloride solution and the target gene Sirt1 showed a positive feedback trend,both of which jointly promoted the apoptosis process of bladder cancer cells,and had certain regulation on the key factor FOXO1in DNA transcription in the nucleus,the inflammatory circuit protein p53,and the key protein-catenin in the apoptosis pathway,etc.,which had certain clinical significance.