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敲低卷曲螺旋结构域蛋白34表达对膀胱癌T24细胞增殖、迁移和侵袭能力的影响

巩艳青 宁向辉 李学松 周利群 郭应禄

100034 北京大学第一医院泌尿外科 北京大学泌尿外科研究所 国家泌尿男生殖系肿瘤研究中心

巩艳青,Email: yqgong@bjmu.edu.cn

北京市自然科学基金面上项目(7172219)

摘 要:目的 研究应用慢病毒介导的RNA干扰(RNA interference)技术敲低卷曲螺旋结构域蛋白34(CCDC34)的表达,分析其对膀胱癌T24细胞生物学行为的影响。方法 选取膀胱癌T24细胞分别同时感染阴性对照siRNA(阴性对照组)和CCDC34-siRNA(CCDC34干扰组)慢病毒载体,荧光显微镜下观察慢病毒感染效率;实时定量PCR和Western blot验证CCDC34的干扰效率;BrdU掺入实验、流式细胞术、划痕实验和Transwell小室侵袭实验检测干扰CCDC34表达对T24细胞增殖、凋亡、迁移及侵袭能力的影响。结果 与阴性对照组相比,感染CCDC34-siRNA的T24细胞中CCDC34 mRNA及蛋白表达水平均显著下降。BrdU实验显示CCDC34干扰组的细胞增殖能力较阴性对照组受到显著抑制(P<0.05),且细胞凋亡显著增加[(8.77±0.38)% vs.(3.91±0.08)%,P<0.01]。划痕实验显示,慢病毒感染24 h后CCDC34干扰组的细胞迁移率显著低于阴性对照组(0.40±0.04 vs. 0.79±0.01,P<0.01)。Transwell侵袭实验同时显示CCDC34干扰组膀胱癌T24细胞的侵袭率也显著低于阴性对照组(1.44±0.03 vs. 1.63±0.05,P<0.05)。结论 慢病毒介导的RNA干扰技术可下调CCDC34蛋白在T24细胞中的表达,显著抑制了膀胱癌T24细胞的增殖、迁移和侵袭能力,并促进了肿瘤细胞的凋亡。CCDC34有望成为膀胱癌生物标记物和治疗的潜在新靶点。

关键词:膀胱肿瘤; 卷曲螺旋结构域蛋白34; 细胞增殖; 迁移; 侵袭

Impact of CCDC34 down-regulation on the proliferation, migration and invasion of bladder cancer cell

Gong Yanqing, Ning Xianghui, Li Xuesong, Zhou Liqun, Guo Yinglu.

National Urological Cancer Center; Institute of Urology, Peking University; Department of Urology, Peking University First Hospital, Beijing 100034, China

Gong Yanqing, Email: yqgong@bjmu.edu.cn

Abstract:  Objective To evaluate the impact of CCDC34 down-regulation on the proliferation, apoptosis, migration and invasion of human bladder cancer cell lines. Methods T24 cells were infected with lentivirus-mediated CCDC34 negative control siRNA (negative control group) and CCDC34 siRNA (CCDC34-siRNA group) respectively. Fluorescence microscopy was used to observe the infection efficiency. qPCR and Western blot was used to confirm the interfering efficiency of CCDC34. The cell proliferation, apoptosis, migration and invasion ability were analyzed by BrdU incorporation assay, flow cytometry, wound healing assay and transwell experiments. Results The lentivirus infection efficiency was above 85% for both CCDC34-siRNA group and negative control group. The mRNA level and protein expression of CCDC34 in the CCDC34-siRNA group were greatly reduced. Compared with negative control group, cell proliferation was significantly inhibited in the CCDC34-siRNA group (P<0.05); the percentage of T24 cells in apoptosis phase was also significantly increased in the CCDC34-siRNA group [(8.77±0.38)% vs. (3.91±0.08)%, P<0.01]. The migration rate of CCDC34-siRNA group was significantly lower than the negative control group (0.40±0.04 vs. 0.79±0.01, P<0.01). The cell invasion rate of CCDC34-siRNA group was also significantly lower than the negative control group (1.44±0.03 vs. 1.63±0.05, P<0.05). Conclusions Down-regulation of CCDC34 in bladder cancer cell line T24 via lentivirus-mediated siRNA can inhibit the cell proliferation, migration and invasion; and induce cell apoptosis. CCDC34 may serve as a biomarker or even a therapeutic target for bladder cancer.

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文献标引:巩艳青 宁向辉 李学松 周利群 郭应禄.敲低卷曲螺旋结构域蛋白34表达对膀胱癌T24细胞增殖、迁移和侵袭能力的影响[J/CD].中华临床医师杂志:电子版,2017,11(3):452.

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