BAG-1基因的小干扰RNA转染对肝癌细胞生物学特性的影响

Effects of small interfering RNA transfection of B cell lymphoma/leukemia - 2 - associated athano- gene - 1 gene on biological characteristics of hepatocellular carcinoma cells

目的观察RNA干扰BAG-1基因表达对肝癌细胞增殖凋亡及Wnt/β-连环蛋白（β-catenin）信号通路的影响。 方法以人正常肝癌细胞L02作为对照细胞，反转录-聚合酶链反应（RT-PCR）及Western blot分别检测BAG-1在无转移能力的HepG2、Huh7人肝癌细胞株及转染潜能依次增高的MHCC97L、MHCC97H、HCCLM3人肝癌细胞株中的表达；将阴性小干扰RNA（siRNA）和BAG-1-siRNA转染HCCLM3细胞，不转染的细胞为空白对照组，转染48 h后检测转染效果；分别于转染后的24、48、72 h通过细胞计数试剂盒（CCK-8）实验检测细胞的增殖；流式细胞仪检测转染48 h细胞的凋亡；Western blot检测Wnt/β-catenin信号通路β-catenin、生存素（Survivin）、c-Myc的蛋白表达。 结果BAG-1在肝癌细胞中的表达均显著高于对照细胞L02（mRNA表达：PHepG2＝0.000；PHuh7＝0.000；PMHCC97L＝0.000；PMHCC97H＝0.000；PHCCLM3＝0.000；蛋白表达：PHepG2＝0.030；PHuh7＝0.001；PMHCC97L＝0.000；PMHCC97H＝0.000；PHCCLM3＝0.000）；BAG-1-siRNA转染HCCLM3细胞后BAG-1的蛋白表达（0.097±0.010）显著低于空白对照组（0.489±0.039）（P＝0.000）；BAG-1-siRNA转染HCCLM3细胞48 h（0.312±0.024）和72 h（0.398±0.036）后细胞的增殖显著低于空白对照组（0.448±0.032）、（0.673±0.051）（P48 h＝0.004；P72 h＝0.002）；与空白对照组（2.13±0.11）%、（0.512±0.048）、（0.349±0.032）、（0.166±0.015）比较，BAG-1-siRNA组细胞凋亡率[（13.47±1.05）%]显著升高，β-catenin（0.247±0.028）、Survivin（0.153±0.017）、c-Myc（0.089±0.009）蛋白显著下调表达（Pβ-catenin＝0.001；PSurvivin＝0.001；Pc-Myc＝0.002）。 结论抑制BAG-1表达可通过下调Wnt/β-catenin信号通路降低肝癌细胞增殖，促进细胞凋亡。

Objective To investigate the effect of B cell lymphoma/leukemia- 2 -associated ath- anogene - 1 gene （ BAG - 1 ） gene expression by RNA interference on the proliferation, apoptosis and Wnt/β - catenin signaling pathway in hepatocellular carcinoma cells. Methods Human normal liver cells L02 as control cells, reverse transcriptase - polymerase chain reaction （ RT - PCR） and Western blotting were used to detect the expression of BAG - 1 in human hepatocellular carcinoma cell lines HepG2, Huh7, MHCC97L, MHCC97H and HCCLM3 ; the negative small interfering RNA （siRNA） and BAG - 1 - siRNA transfected HCCLM3 cells, and not transfected cells for the blank control group, transfection effect was de- tected after cells transfected for 48 h; the proliferation of the cells was detected by cell counting kit - 8 （ CCK - 8） after ceils were transfected at 24, 48 and 72 h ; the cells apoptosis after cells were transfected for 48 h was detected by flow cytometry; β- catenin, Snrvivin, c - Myc protein expression were detected by Western blotting. Results The expression of BAG - 1 in hepatocellular carcinoma cells were signifi- cantly higher than those of control cells L02 （ （ mRNA ： PHepG2 = 0. 000, Pnuh7 = 0. 000, PMHCC97L = 0. 000, PMHCC97H=0. 000 , PHCCLM3 = 0.000; protein： PHepG2 = 0.030, PHuh7=0.001, PMHCC97L= 0.000, PMHCC97H = 0. 000, PSCCLM3 = 0. 000） ; the expression of BAG - 1 protein （0. 097 ± 0. 010） after BAG - 1 - siRNA were transfected into HCCLM3 cells was significantly lower than the control Group （0. 489± 0. 039 ）（ P = 0. 000） ; the cells proliferation after BAG - 1 - siRNA transfected HCCLM3 cells for 48 h （0. 312 ± 0. 024） and 72 h （ 0. 673 ± 0. 051 ） were significantly lower than the control group （ 0. 448± 0. 032 ）, （0.673 ±0.051） （Pash =0.004, P72h =0.002） ; compared with the control group （2. 13±0. 11）%, （0.512±0.048）, （0.349 ±0.032）, （0.166 ±0.015）, the apoptosis rate （13.47 ±1.05）% in BAG - 1 - siRNA group increased significantly, β- catenin （ 0. 247 ± 0. 028 ）, Survivin （ 0. 153 ± 0. 017）, c - Myc （0. 089 ±0. 009）, protein expression was down regulated （Pβ-catenin =0. 001, Psurvivin=0. 001, Pc-Myc =0. 002）. Conclusion Inhibiting of BAG - 1 expression can reduce the proliferation of hepatoma cells and promote apoptosis by inhibiting the Wnt/β - catenin signaling pathway.