联合干扰转化生长因子-βRⅡ和卷曲蛋白-7基因对肝癌细胞增殖和转移的影响

Short hairpin RNA silencing of transforming growth factor - βRⅡ and frizzled - 7 synergisticly suppresses the proliferation and metastasis of hepatocellular carcinoma

目的探讨联合抑制转化生长因子-βRⅡ受体（TGF-βRⅡ）和卷曲蛋白-7（FZD-7）对肝癌细胞增殖和转移的影响及机制。方法实验分5组：空白组（Blank）、阴性对照组（shNC）、TGF-βRⅡ干扰组（shTGF-βRⅡ）、FZD-7干扰组（shFZD-7）及TGF-βRⅡ和FZD-7共同干扰组（shTGF-βRⅡ＋shFZD-7）。用shTGF-βRⅡ和（或）shFZD-7转染HepG2和Huh-7细胞，应用细胞计数试剂盒（CCK-8）检测细胞增殖能力，Transwell实验检测细胞迁移、侵袭能力，流式细胞仪检测细胞周期的变化，Westernblot检测β-连环蛋白（β-catenin）、C-myc、细胞周期蛋白D1（CyelinD1）的表达。结果转染后96h，共同干扰组HepG2、Huh-7细胞吸光度（A）值（0．87±0．09、0．63±0．03）较TGF-βRⅡ（1．12±0．02、0．82±0．08）或FZD-7（1．06±0．04、0．77±0．04）单基因干扰组明显下降（P〈0．05）；Transwell实验表明，与TGF-βRⅡ或FZD-7单基因干扰组比较，共同干扰组HepG2、Huh-7细胞迁移、侵袭的细胞数明显降低（P〈0．01）；Huh-7细胞共同干扰组G1期细胞百分比为（85．74±0．91）％，较TGF-βRⅡ的（69．70±2．45）％或FZD-7的（74．86±1．02）％单基因干扰组明显增加（P〈0．01）；灰度分析显示HepG2、Huh-7细胞共同干扰组较TGF-βRⅡ或FZD-7单基因干扰组，β-catenin、C-mye、CyclinD1蛋白表达量明显降低，差异均有统计学意义（P〈0．05）。结论联合干扰TGF-βRⅡ和FZD-7基因可以通过下调β-catenin、C-myc、CyelinD1的表达对肝癌细胞的增殖和转移产生协同抑制作用。

Objective To study whether simultaneously blocking of transforming growth factor （TGF） -13R ]] and frizzled-7 （FZD -7） genes could exert synergistic inhibition effects on the prolifera- tion and metastasis as well as their potential molecular mechanism in hepatocellular carcinomas. Methods The experiment was divided into five groups： blank group, shNC group, shTGF - βRⅡ group, shFZD -7 group and shTGF - βRⅡ ＋ shFZD -7 group. The short hairpin RNA （shRNA） eukaryotic expression vectors specific to TGF - βRⅡ and FZD - 7 were transfected into HepG2 and Huh - 7 cells. The proliferation abilities were measured by cell counting kit - 8 （ CCK - 8 ） assay. The migration and invasion abilities of the trans- letted cells were examined by cell migration and invasion assay. The cell cycle of hepatoma cells was detec- ted by flow cytometry. Western blotting was used to detect the expression of β - catenin, C - mye and Cyclin D1 in transfected cells to illustrate the probable mechanisms. Results After transfection by 96 h, the A val- ues of co - transfection groups in HepG2 and Huh -7 （0. 87 ±0. 09, and 0. 63 ±0. 03） were significantly re- duced as compared with the shTGF - βRⅡ （ 1.12 ± 0. 02, and 0. 82 ± 0. 08 ） or shFZD - 7 （ 1.06 ± 0. 04, and 0. 77 ± 0. 04） groups （P 〈 0. 05 ） ; the amount of metastatic ceils in the co -transfection groups was sig- nificantly reduced as compared with the single - transfected groups （ P 〈 0. 01 ） ; the percent of ceils in Gl phase in co - transfection Huh - 7 group [ （ 85.74 ± 0. 91 ） % ] was significantly increased as compared with the shTGF - βRⅡ （69. 70 ±2. 45 ） % ] or shFZD - 7 [ （74. 86 ± 1.02） % ] groups, and the protein levels of -catenin, C -myc and Cyclin D1 were significantly suppressed in the co -transfection groups （P 〈 0. 05）. Conclusion Our results demonstrate that simultaneously targeting of TGF - βRⅡ and FZD - 7 can inhibit the proliferation and metastasis in hepatoma cells more effectively than blocking either pathway alone by downregulating the expression of β -catenin, C -myc and Cyclin D1.