GPR49基因对肝癌细胞增殖侵袭能力的影响

Effect of GPR49 gene on proliferation and invasive ability of hepatoma cells

目的探讨肝癌细胞系Huh7细胞中G蛋白偶联受体49(GPR49)基因对肝癌细胞侵袭能力的影响及其分子生物学机制。方法根据转染的小干扰RNA(si-RNA)不同,将Huh7细胞分为GPR49-siRNA(si-GPR49)组和阴性对照NC-siRNA(si-NC)组,另设未转染Huh7细胞为对照组(control组)。采用逆转录聚合酶链反应(RT-PCR)法和Western blot法分别检测3组细胞GPR49、细胞周期素D1(cyclin D1)、基质金属蛋白酶9(MMP9)的信使核糖核酸(mRNA)和蛋白的表达。采用MTT法和Transwell法分别检测各组细胞增殖能力和侵袭能力。结果 si-GPR49组GPR49 mRNA的相对表达量为control组的(23.8±3.1)%(P<0.05)。与control组相比,si-GPR49组GPR49、cyclin D1、MMP9蛋白的表达量均明显降低(均为P<0.05)。MTT检测细胞增殖能力实验结果显示,si-GPR49组细胞在72 h的吸光度(OD)值(0.53±0.12)明显低于control组(1.35±0.28),差异有统计学意义(P<0.05)。si-GPR49组平均穿膜细胞数为(13.6±2.5)个,明显低于control组的(65.3±6.1)个,差异有统计学意义(P<0.05)。结论 GPR49-siRNA可抑制Huh7细胞GPR49基因表达。其机制可能是通过降低cyclin D1水平抑制Huh7细胞的增殖能力,通过影响MMP9蛋白表达水平抑制Huh7细胞的迁移和侵袭能力。

Objective To discuss the effect of G-protein-coupled receptor 49 （GPR49）gene on proliferation and invasive ability of hepatoma cell line Huh7 and its molecular biological mechanism.Methods According to the different transfected small interfering RNA（si-RNA）,Huh7 cells were divided into the GPR49-siRNA（si-GPR49）group and the NC-siRNA （si-NC）group.Untransfected Huh7 cells were set as the control group. Messenger RNA （mRNA ）and protein expression of GPR49, cyclin D1 and matrix metalloproteinase 9 （MMP9）in the cells of the three groups were respectively detected by reverse transcription-polymerase chain reaction （RT-PCR）and Western blot method.The proliferation and invasive ability of the cells of each group were respectively detected by MTT method and Transwell method.Results The relative expression of GPR49 mRNA of the si-GPR49 group was （23.8 ±3.1）% of the control group （P 〈0.05）. Compared with the control group,the protein expression of GPR49,cyclin D1 and MMP9 of the si-GPR49 group decreased significantly （all in P 〈0.05）.The proliferation experiment results by MTT indicated that the optical density（OD）of the cells of the si-GPR49 group at 72 h was （0.53 ±0.12）,which was significantly lower than that of the control group （1.35 ±0.28）.The difference had statistical significance （P 〈0.05）. The average invaded cell counts of the si-GPR49 group were （13.6 ±2.5）,which was significantly lower than （65.3 ±6.1 ）of the control group.The difference had statistical significance （P 〈0.05 ）.Conclusions GPR49-siRNA may inhibit the gene expression of GPR49 in Huh7 cells.Its mechanism may be that the proliferation of Huh7 cells is inhibited by reducing the level of cyclin D1;the migration and invasive ability of Huh7 cells is inhibited by affecting the expression level of MMP9.