前信使RNA加工因子19在膀胱癌组织的表达及其对细胞增殖、迁移、侵袭的影响

The expression of pre-messenger RNA processing factor 19 in bladder cancer and its effect on cell proliferation,migration and invasion

目的:检测前信使RNA(mRNA)加工因子19(Prp19)在膀胱癌组织及细胞中的表达水平,探讨Prp19对膀胱癌细胞生物学功能的影响。方法:运用UALCAN数据库分析Prp19基因在膀胱癌组织中的表达水平,利用GEPIA数据库分析Prp19与膀胱癌患者预后的关系,收集膀胱癌及癌旁标本,采用实时定量反转录聚合酶链反应(RT-qPCR)和蛋白质印迹法(Western blot)技术检测膀胱癌及癌旁标本、正常膀胱上皮细胞及膀胱癌细胞中Prp19 mRNA和蛋白质的相对表达水平。利用免疫组织化学染色检测膀胱癌患者及其癌旁组织中Prp19蛋白的表达。在膀胱癌T24细胞中转染小干扰RNA(siRNA)-Prp19,将细胞分为转染组(si-Prp19)和对照组(NC),采用RT-qPCR和Western blot验证转染效率。利用细胞克隆形成实验和细胞计数盒(CCK-8)实验检测细胞增殖能力;利用Transwell实验检测细胞迁移和侵袭能力;利用Western blot检测磷脂酰肌醇3激酶(PI3K)、磷酸化PI3K(p-PI3K)、蛋白激酶B(Akt)、磷酸化Akt(p-Akt)及上皮-间充质转化(EMT)标志蛋白的表达水平;两组间比较采用t检验。结果:UALCAN数据库显示Prp19在膀胱癌组织中表达水平升高(P<0.001),GEPIA数据库显示Prp19表达与患者总生存期(OS)呈负相关(P<0.05);膀胱癌组织中Prp19 mRNA和蛋白相对表达水平高于癌旁组织(mRNA:1.36±0.05比1.00±0.05,t=7.763,P<0.05;蛋白:149.33±6.51比99.33±4.04,t=11.307,P<0.05),T24和5637细胞中Prp19相对表达水平高于正常膀胱上皮细胞(mRNA:3.11±0.15比1.01±0.12、1.92±0.12比1.01±0.12,t=18.562、9.192,P<0.05;蛋白:224.00±5.00比99.67±4.51、183.67±6.03比99.67±4.51,t=31.984、9.328,P<0.05);另外转染组细胞Prp19 mRNA和蛋白相对表达水平低于对照组(mRNA:0.49±0.09比1.00±0.09,t=6.661,P<0.05;蛋白:56.33±6.03比99.67±6.51,t=8.462,P<0.05);转染组细胞克隆形成数目低于对照组[(100.33±10.69)个比(200.33±13.58)个,t=10.022,P<0.01];转染组细胞在48、72、96 h的增殖活性均低于对照组(48 h:0.32±0.05比0.50±0.07、72 h:0.48±0.05比0.72±0.06、96 h:0.76±0.05比1.15±0.09,t=3.719、5.144、6.331,P<0.05);转染组细胞迁移数目低于对照组[(74.33±7.02)个比(152.67±11.68)个,t=9.957,P<0.01],转染组细胞侵袭数目低于对照组[(96.33±7.51)个比(161.33±8.02)个,t=10.249,P<0.01],差异均有统计学意义。结论:Prp19在膀胱癌组织和细胞中异常高表达,可作为促癌基因促进膀胱癌的发生及发展。

Objective To detect the expression level of pre-messenger RNA(mRNA)processing factor 19(Prp19)in bladder cancer tissues and cells,and to investigate the effect of Prp19 on the biological function of bladder cancer cells.Methods UALCAN database was used to analyze the expression level of Prp19 gene in bladder cancer tissues,and GEPIA database was used to analyze the relationship between Prp19 and the prognosis of patients with bladder cancer.real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)and Western blotting were used to detect the relative expression levels of Prp19 mRNA and protein in bladder cancer and adjacent tissues,normal bladder epithelial cells and bladder cancer cells.Immunohistochemical straining was used to detect the expression of Prp19 protein in bladder cancer and adjacent tissues.Bladder cancer T24 cells were transfected with small interfering RNA(siRNA)-Prp19,and the cells were divided into transfection group(si-Prp19)and negative control(NC).RT-qPCR and Western blotting were used to verify the transfection efficiency.Cell clone formation assay and cell counting kit-8(CCK-8)assay were used to detect cell proliferation.Transwell assay was used to detect cell migration and invasion ability.Western blotting was used to detect the expression levels of phosphatidylinositol 3 kinase(PI3K),phosphorylated PI3K(p-PI3K),protein kinase B(Akt),phosphorylated Akt(p-Akt)and epithelial-mesenchymal transition(EMT)marker proteins.The results were analyzed by SPSS 26.0 statistical software.Results UALCAN database showed that the expression level of Prp19 was increased in bladder cancer tissues(P<0.001).GEPIA database showed that Prp19 expression was negatively correlated with overall survival(OS)of patients(P<0.05).RT-qPCR and Western blotting results showed that the relative expression level of Prp19 mRNA and protein in bladder cancer tissues was higher than that in adjacent tissues(mRNA:1.36±0.05 vs.1.00±0.05,t=7.763,P<0.05;protein:149.33±6.51 vs.99.33±4.04,t=11.307,P<0.05),and the relative expression level of Prp19 in T24 and 5637 cells was higher than that in normal bladder epithelial cells(mRNA:3.11±0.15 vs.1.01±0.12,1.92±0.12 vs.1.01±0.12,t=18.562,9.192,P<0.05;protein:224.00±5.00 vs.99.67±4.51,183.67±6.03 vs.99.67±4.51,t=31.984,9.328,P<0.05).In addition,the relative expression levels of Prp19 mRNA and protein in transfected group were lower than those in control group(mRNA:0.49±0.09 vs.1.00±0.09,t=6.661,P<0.05;protein:56.33±6.03 vs.99.67±6.51,t=8.462,P<0.05).The results of cell clone formation assay showed that the number of cell clone formation in the transfection group was lower than that in the control group[(100.33±10.69)colonies vs.(200.33±13.58)colonies,t=10.022,P<0.01].In CCK-8 experiment,the proliferation activity of T24 transfection group was lower than control group at 48,72,96 h(48 h:0.32±0.05 vs.0.50±0.07,72 h:0.48±0.05 vs.0.72±0.06,96 h:0.76±0.05 vs.1.15±0.09,t=3.719,5.144,6.331,P<0.05).In Transwell migration experiment,the number of cell migration in the transfection group was lower than that in control group[(74.33±7.02)cells vs.(152.67±11.68)cells,t=9.957,P<0.01],in invasion experiment,the number of cell invasion in the transfection group was lower than that in control group[(96.33±7.51)cells vs.(161.33±8.02)cells,t=10.249,P<0.01].All the above differences were statistically significant.Conclusion Prp19 is highly expressed in bladder cancer tissues and cells,and it can be used as an oncogene to promote the formation and development of bladder cancer.