Capn4通过黏着斑激酶磷酸化激活核转录因子-κB信号调控人肾癌细胞增殖  

Calpain subunit - 1 induces human renal cancer cell proliferation by activating nuclear factor -κB signaling pathway through FAK phosphorylation

作　　者：庄乾锋[1] 沈杰[1] 王恺 钱曦 范敏[1] 王坤[1] 陆皓[1] 徐仁芳[1] 何小舟[1] Zhuang Qianfeng;Shen Jie;Wang Kai;Qian Xi;Fan Min;Wang Kun;Lu Hao;Xu Renfang;He Xiaozhou(Department of Urology,the Third Affiliated Hospital of Soochow University,Changzhou 213003,China（Zhuang QF,Shen J,Wang K,Fan M,Wang K,Lu H,Xu RF,He XZ;Department of Molecular Biology and Microbiology,Tufts University,Boston 02155,USA（Qian X)

机构地区：[1]苏州大学附属第三医院泌尿外科,常州213003 [2]美国塔夫茨大学医学院分子生物学与微生物学系,波士顿02155

出　　处：《中华实验外科杂志》2018年第10期1905-1907,共3页Chinese Journal of Experimental Surgery

基　　金：江苏省自然科学基金青年项目（BK20150251）;江苏省青年医学人才项目（QNRC2016292）;中国博士后科学基金项目（2018M632371）

摘　　要：目的探讨钙蛋白酶小亚基1（Capn4）的表达对肾透明细胞癌（ccRCC）增殖、迁移、侵袭的影响及其分子作用机制。方法选取Capn4高表达ccRCC细胞株786-0和低表达的Caki-1细胞株，采用慢病毒转染技术构建Capn4稳定低表达的786-0细胞株（786-0-shCapn4）和稳定过表达Capn4的Caki-1细胞株（Caki-1-Capn4），通过荧光定量聚合酶链反应（FQ-PCR）验证Capn4在转染肾细胞癌（RCC）细胞株中的表达。通过细胞增殖实验、划痕实验及Transwell实验观察Capn4对肾癌细胞增殖、侵袭迁移能力的影响。通过Westernblot法验证黏着斑激酶（FAK）、磷酸化FAK、核转录因子-κB（NF-κB）和磷酸化NF-κB的表达。结果细胞计数试剂盒（CCK-8）结果显示，Capn4过表达后细胞增殖能力过表达组高于对照组（P=0．010），而转染Capn4短发卡RNA（shRNA）后细胞增殖能力下调组低于对照组（P=0．010）；划痕实验结果表明，24hCakil-Capn4细胞的迁移距离过表达组高于对照组（P=0．001），786-O-shCapn4细胞的迁移距离下调组低于对照组（P=0．001）；Transwell结果表明，24hCakil-Capn4细胞的侵袭细胞数量过表达组高于对照组（P=0．001），786-0-shCapn4细胞的迁移距离下调组低于对照组（P=0．001）；Westernblot结果显示：Cakil-Capn4组FAK及NF-κB磷酸化水平较各自对照组上调（P=0．010），而786-0-shCapn4组FAK及NF-κB磷酸化水平较各自对照组下调（P=0．010）；FAK特异性抑制剂（PF573228）或者NF-κB抑制剂QNZ均可抑制由Capn4过表达引起的癌细胞生长速度的增加。结论Capn4通过激活FAK和下游信号通路，从而激活NF-κB，进而促进RCC细胞生长。Objective To study the effect of calpain subunit- 1 （Capn4） expression on the proliferation, migration and invasion of renal clear cell carcinoma （ccRCC） and to explore its molecular mechanism. Methods Upregulation of Capn4 was performed by using RNA interference （RNAi） method in the ccRCC cell line Caki - 1, Down - regulation of Capn4 was performed by using RNAi method in the ccRCC cell line 786 -0.The expression level of Capn4 was confirmed by using real - time fluorescent quantitative polymerase chain reaction （FQ- PCR） methods. The cell counting kit- 8 （CCK- 8） assay, scrape assay and Transwe11 assay were used to investigate the contribution of Capn4 on biological behavior of ccRCC cells. The expression of Focal Adhesion Kinase （FAK）, phosphorylated FAK, nuclear factor -κB （ NF -κB） and phosphorylated NF -κB was confirmed by Western blotting. We tested FAK and NF -κB phosphorylation levels after using FAK inhibitors and tested FAK and NF -κB phosphoryla- tion levels after using NF -κB inhibitors. Results The results of CCK - 8 showed that the cell prolifera- tion ability of Capn4 overexpression in overexpression group was increased more than control group （ P =0. 010）, and the cell proliferation ability of Capn4 short hairpin RNA （shRNA） in down group was de- creased more than control group （P = 0. 010）. The scrape assay results showed that the migration distance of Cakil -Capn4 ceils in overexpression group was significantly higher than control group （P = 0. 001 ） , the migration distance of 786 - 0 - shCapn4 cells in down group was significantly lower than control group （P = 0. 001 ）. Transwell invasion assay showed that 24 hours of Cakil - Capn4 cell invasion cell number in ovcrexpression group was higher than control group （ P = 0. 001 ） , the migration distance of 786 - 0 - shCapn4 cells in down group was significantly lower than control group （P = 0. 001 ）. Western blotting re- sults showed that： Cakil - Capn4 group＇ s FAK and NF -κB pho

关 键 词：肾细胞癌 钙蛋白酶小亚基1 黏着斑激酶 核因子活化B细胞K轻链增强子 

分 类 号：R737.25[医药卫生—肿瘤]