活化激酶C受体1对胆管癌细胞迁移和侵袭能力的影响

Effect of receptor for activated C kinase 1 on migration and invasion of cholangiocarcinoma cells

目的:检测活化激酶C受体1(RACK1)在胆管癌细胞中的表达,探讨其对胆管癌细胞生物学表型的影响。方法:实时荧光定量聚合酶链反应(qRT-PCR)和蛋白免疫印迹法(Western blot)检测RACK1在人正常胆管上皮细胞HIBEC和2株胆管癌细胞RBE、HUH28中的表达水平。采用t检验分析RACK1细胞水平表达差异。慢病毒构建RACK1低表达稳定株,分为sh-RACK1-1、sh-RACK1-2组和阴性对照组(NC-RACK1组),qPCR检测其转染效率;Transwell实验检测胆管癌细胞的迁移和侵袭能力。采用t检验分析敲低RACK1后胆管癌细胞的迁移和侵袭能力变化。结果:qPCR结果显示RACK1在RBE、HUH28胆管癌细胞株中的表达量(3.45±0.92、3.21±0.33)明显高于人正常胆管上皮细胞HIBEC(1.43±0.58,t=21.965、24.153,P<0.01),差异均有统计学意义;Western blot结果表明RACK1在RBE和HUH28胆管癌细胞株中的表达水平明显高于人正常胆管上皮细胞HIBEC;Transwell迁移实验结果提示RBE中sh-RACK1-1、sh-RACK1-2组细胞穿膜数量为[(261.89±8.65)、(231.07±9.73)个/视野],明显低于NC-RACK1组[(430.19±10.15)个/视野,t=30.684、24.380,P<0.01],差异均有统计学意义;HUH28中sh-RACK1-1、sh-RACK1-2组细胞穿膜数量[(253.57±7.28)、(210.11±6.09)个/视野]低于NC-RACK1组[(424.78±8.03)个/视野,t=35.442、36.781,P<0.01],差异均有统计学意义;侵袭实验结果表明RBE中sh-RACK1-1、sh-RACK1-2组细胞穿膜数量为[(52.15±1.43)、(60.58±2.01)个/视野]明显低于NC-RACK1组[(125.39±4.03)个/视野,t=9.935、12.566,P<0.05],差异均有统计学意义;HUH28中sh-RACK1-1、sh-RACK1-2组细胞穿膜数量[(60.91±1.30)、(51.19±0.96)个/视野]低于NC-RACK1组[(150.88±7.28)个/视野,t=13.554、15.328,P<0.01],差异均有统计学意义。结论:RACK1在胆管癌细胞中表达上调,降低RACK1表达后可抑制胆管细胞迁移及侵袭能力,RACK1可促进胆管癌发生发展。

Objective To detect the expression of receptor for activated C kinase 1(RACK1)in cholangiocarcinoma cells,and to explore its effect on the biological phenotype of cholangiocarcinoma cells.Methods Real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)and Western blotting were used to detect the expression level of RACK1 in human normal bile duct epithelial cells HIBEC,2 cholangiocarcinoma cell lines RBE and HUH28.The t test was used to analyze the difference in the RACK1 expression.Lentivirus was used to construct a stable low-expression strain of RACK1,which was divided into sh-RACK1-1,sh-RACK1-2 and negative control(NC-RACK1)group.The transfection efficiency was tested by qPCR.The invasion and migration abilities of cholangiocarcinoma cells were tested by Transwell experiment.T test was used to analyze the migration and invasion ability of cholangiocarcinoma cells after knocking down RACK1.Results The qPCR results showed that the expression level of RACK1 in RBE and HUH28 cholangiocarcinoma cell lines(3.45±0.92,2.96±0.33)was significantly higher than that in HIBEC cells(1.73±0.58,t=21.965,24.153,P<0.01).Western blotting showed that the expression level of RACK1 in RBE and HUH28 cells was significantly higher than that in HIBEC cells.The results of Transwell migration experiment suggested that the number of cells penetrating the membrane in the sh-RACK1-1 and RBE RACK1-2 groups in RBE cells was[(261.89±8.65),(231.07±9.73)/field of view],significantly less than that in the NC-RACK1 group[(430.19±10.15)/field of view,t=30.684,24.380,P<0.01].The number of cells penetrating the membrane in the sh-RACK1-1 and sh-RACK1-2 groups in HUH28 cells[(253.57±7.28)/field of view,(210.11±6.09)/field of view]was significantly less than in the NC-RACK1 group[(424.78±8.03)/field of view](t=35.442,36.781,P<0.01).The results of the invasion experiment showed that the number of cells in the sh-RACK1-1 and sh-RACK1-2 groups in RBE cells was[(52.15±1.43)and(60.58±2.01)/field of view]was significantly lower than that in NC-RACK1 group[(125.39±4.03)/field of view,t=9.935,12.566,P<0.05].The number of cells passing through the membrane in the sh-RACK1-1 and sh-RACK1-2 groups[(60.91±1.30)and(51.19±0.96)/field of view]in HUH28 cells was less than in the NC-RACK1 group[(150.88±7.28)/field of view](t=13.554,15.328,P<0.01).Conclusion RACK1 is up-regulated in cholangiocarcinoma cells.The down-regulation of the RACK1 expression can inhibit the migration and invasion of cholangiocarcinoma cells.RACK1 can promote the occurrence and development of cholangiocarcinoma.