特异性下调LKB1的表达对肝癌细胞侵袭迁移能力的影响

摘要

目的 研究特异性下调LKB1的表达对肝癌细胞系SK-hep-1迁移侵袭能力的影响.方法 Real-time PCR和Western B1ot检测人肝癌细胞株和人正常肝细胞HL7702中LKB1表达情况.设计并合成LKB1特异性双链小干扰RNA(LKB1-siRNA),转染到高表达LKB1的人肝癌细胞株SK-hep-1中靶向沉默LKB1基因表达,并设置阴性对照组(NC组).Real-time PCR和WesternBlot检测LKB1基因的mRNA水平与蛋白表达水平的变化.利用CCK-8法检测肝癌细胞增殖能力.通过体外Transwell小室基质侵袭和迁移实验,观察LKB1表达沉默后对人肝癌细胞株SK-hep-1侵袭迁移能力的影响.计量资料两组间比较采用t检验,多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验.结果 与正常人肝细胞HL7702相比,LKB1基因和蛋白在人肝癌细胞SK-hep-1中高表达(P值均＜0.05).与NC组相比,siLKB1-1处理组SK-hep-1细胞中的LKB1基因和蛋白表达均明显降低(P值均＜0.05).与NC组相比,siLKB1-1转染的SK-hep-1细胞增殖速度明显加快、侵袭和迁移能力明显增强(1.393±0.022 vs 1.128±0.032、147±19 vs 83±21、113±13 vs 75 ±17;t值分别为15.313、14.879、8.791;P值均＜0.001).结论 LKB1有可能参与抑制肝癌增殖、侵袭迁移的过程并影响肝癌的预后.%Objective To investigate the influence of specific down-regulation of LKB1 expression on invasion and migration of the hepatoma cell line SK-hep-1.Methods Quantitative real-time PCR (RT-PCR) and Western blot were used to measure the expression of LKB1 in the human hepatoma cell line SK-hep-1 and normal hepatocytes (HL7702).LKB1 specific double-strand small interfering RNA was designed,synthesized,and then transfected into the human hepatoma cell line SK-hep-1 with high expression of LKB1 to silence the expression of LKB1 gene.A negative control (NC) group was established.RT-PCR and Western blot were used to measure the changes in the mRNA and protein expression of LKB1.Cell Counting Kit-8 was used to measure the proliferative capacity of hepatoma cells.In vitro Transwell chamber invasion and migration assays were used to observe the influence of LKB1 gene silencing on invasion and migration of the human hepatoma cell line SK-hep-1.The t-test was used for comparison of continuous data between two groups;a one -way analysis of variance was used for comparison between multiple groups,and the least significant difference t-test was used for further comparison between any two groups.Results Compared with normal human hepatocytes (HL7702),SK-hep-1 cells had significantly higher mRNA and protein expression of LKB1 (both P ＜ 0.05).Compared with the NC group,the siLKB1-1 treatment group had significant reductions in the mRNA and protein expression of LKB1 in SK-hep-1 cells (both P ＜ 0.05).Compared with the NC group,the siLKB1-1 treatment group had a significantly higher cell proliferation rate and significant increases in invasion and migration (1.393 ± 0.022 vs 1.1284 ±0.032,t =15.313,P ＜0.001;147 ± 19 vs 83 ±21,t =14.879,P ＜0.001;113 ± 13 vs 75 ± 17,t =8.791,P ＜0.001).Conclusion LKB1 may be involved in inhibiting the proliferation,invasion,and migration of hepatoma cells and can affect the prognosis of patients with liver cancer.