二肽基肽酶-4基因表达促进膀胱癌进展并抑制顺铂化疗敏感性

Dipeptidyl peptidase-4 gene expression promoted the progression of bladder cancer and inhibited the sensitivity of cisplatin to chemotherapy

目的探讨膀胱癌组织中二肽基肽酶-4(DPP4)的表达,以及其对膀胱癌增殖,迁移和侵袭的以及顺铂化疗敏感性调节。方法收集江苏省人民医院32例膀胱癌患者的根治性膀胱切除术后的病理标本,包括膀胱癌组织及癌旁组织。使用实时定量反转录聚合酶链反应(RT-qPCR)技术检测DPP4在膀胱癌组织和细胞株中的表达。在BIU87细胞系中转染DPP4小干扰RNA(siRNA)及其阴性对照,分组为DPP4敲低组(si-DPP4)和对照组(si-NC),通过细胞计数试剂盒(CCK-8)实验,细胞克隆,划痕实验和Transwell实验分别观察DPP4对膀胱癌增殖,迁移和侵袭的影响。通过蛋白质印迹法(Western blot)法检测DPP4蛋白表达情况和顺铂耐药实验验证DPP4对膀胱癌化疗敏感性的影响。结果 RT-qPCR结果表明DPP4在膀胱癌组织中表达水平高于正常组织表达水平,差异有统计学意义(t=3.90,P<0.01);膀胱癌细胞系中表达水平(2.55±0.29、1.54±0.23)高于人正常膀胱上皮细胞系(1.00±0.03),差异有统计学意义(t=9.30、3.92,P<0.01),生存分析显示DPP4高表达与预后不良明显相关(P<0.05)。与对照组比较,DPP4敲低组的细胞增殖能力(0.84±0.02,0.87±0.02)低于敲低对照组(0.92±0.02),差异有统计学意义(t=5.08、3.65,P<0.05)、DPP4敲低组的克隆数[(203.98±6.73)、(252.77±5.83)个]低于敲低对照组[(351.64±8.53)个],差异有统计学意义(t=23.53、16.57,P<0.01)、DPP4敲低组的迁移能力(2.24±0.43,3.12±0.47)低于敲低对照组的迁移能力(7.59±0.44),差异有统计学意义(t=15.11、12.05,P<0.01);DPP4敲低组的侵袭数目[(147.23±8.71)、(179.19±9.83)个]低于敲低对照组[(372.21±18.52)个],差异有统计学意义(t=19.02、15.94,P<0.01)能力下降。顺铂药物敏感性实验表明,DPP4敲低组(18.63±1.40、19.56±1.47)膀胱癌细胞顺铂半数抑制浓度(IC50)值低于敲低对照组(26.21±1.78),差异有统计学意义(t=5.78、4.98,P<0.01),并且在同时应用顺铂和DPP4抑制剂西他列汀时IC50值(16.96±1.10)低于顺铂单药对照组IC50值(23.62±1.88),差异有统计学意义(t=5.284,P<0.01)。结论敲低DPP4基因可以通过抑制膀胱癌细胞的增殖,迁移和侵袭能力,同时增加顺铂化疗敏感性,最终限制膀胱癌进展。

Objective To investigate the expression of dipeptidyl peptidase-4(DPP4)in bladder cancer and its regulation on the proliferation,migration and invasion of bladder cancer(BCa)and the sensitivity to cisplatin chemotherapy.Methods The pathological specimens of 32 patients with BCa who underwent radical cystectomy in Jiangsu Provincial Hospital from 2014 to 2017 were collected,including tumor tissues and paracancerous tissues.Real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)was used to detect the expression of DPP4 in BCa tissues and cell lines.DPP4 small interfering RNA(siRNA)and its negative control were transfected into BIU87 cell lines,we divided them into si-DPP4 group and si-NC group.The effects of DPP4 on the proliferation,migration and invasion of BCa were investigated by cell counting kit-8(CCK-8)assay,cell cloning assay,scratch assay and transwell assay.The influence of DPP4 on the sensitivity to cisplatin of BCa was verified by CCK-8 assay and half maximal inhibitory concentration(IC50)value.Results qRT-PCR results confirmed the expression of DPP4 was significantly upregulated in BCa tissue and in BCa cells(t=3.90,P<0.01).The expression level of BCa cell lines(2.55±0.29,1.54±0.23)was significantly higher than that of SV-HUC(1.00±0.03,t=9.30,3.92,P<0.01).Survival analysis showed that the high expression of DPP4 was associated with poor prognosis in BCa patient(P<0.05).Compared with the control group,knockdown of DPP4 decreased the cell proliferation(0.84±0.02,0.87±0.02;0.92±0.02,t=5.08,3.65,P<0.05),cloning formation[(203.98±6.73)cells,(252.77±5.83)cells;(351.64±8.53)cells,t=23.53,16.57,P<0.01],migration(2.24±0.43,3.12±0.47),(7.59±0.44),(t=15.11,12.05,P<0.01)and invasion abilities[(147.23±8.71)cells,(179.19±9.83)cells;(372.21±18.52)cells,t=19.02,15.94,P<0.01]in BCa cells,suggesting that DPP4 may promote the progression of BCa.Compared with the control group,the IC50 value(26.21±1.78)of BCa cells was decreased after DPP4 knockdown[(18.63±1.40,19.56±1.47),t=5.78,4.98,P<0.01]and the IC50 value was even lower than cisplatin group(23.62±1.88)when cisplatin and the DPP4 inhibitor sitagliptin(16.96±1.10)were used together(t=5.284,P<0.01).It is suggested that DPP4 knockdown can mediate the sensitivity of BCa to cisplatin chemotherapy.Conclusion DPP4 was highly expressed in BCa tissues and cell lines.Knockdown of DPP4 expression and inhibited the proliferation,migration and invasion ability of BCa cells and increased the sensitivity to cisplatin chemotherapy.Ultimately limiting the progression of bladder cancer.