tRNA-ValAAC-5促进肝癌细胞增殖和迁移作用机制研究

Study on the mechanism of tRNA-ValAAC-5 in promoting the proliferation and migration of hepatocellular carcinoma cells

目的:本研究论证肝细胞癌(hepatocellular carcinoma,HCC)细胞中tRNA-ValAAC-5表达发挥作用及机制。方法:使用Real-time PCR法检测tRNA-ValAAC-5在人正常细胞肝癌细胞(THLE2,THLE3)和HCC细胞株(Hep3B,HuH7,SNU398,Hep3G2)和中的表达。Hep3B和Hep3G2细胞分别转染tRNA-ValAAC-5-inhibitor及对照tRNA-ValAAC-5-NC即为inhibitor组和NC组。两组细胞分别通过CCK-8法检测细胞增殖,Transwell小室实验检测细胞侵袭转移能力,Western blot法检测p21、基质金属蛋白酶2(MMP2)和基质金属蛋白酶9(MMP9)的表达。结果:相较于THLE2、THLE3细胞,tRNA-ValAAC-5在Hep3B、HuH7、SNU398、Hep3G2细胞中相对表达量均有升高,差异有统计学意义(P<0.05)。与tRNAValAAC-NC相比,转染tRNA-ValAAC-5-inhibitor的Hep3B、Hep3G2细胞tRNA-ValAAC-5表达均明显降低,两组差异有统计学意义(t=36.52、27.45,P<0.001),表示转染成功。与tRNA-ValAAC-5-NC相比,Hep3B、Hep3G2细胞转染tRNA-ValAAC-5-inhibitor后24、48、72、96 h增殖能力受到抑制,差异均有统计学意义(t=5.25、8.23、7.33、14.16,P<0.001)、(t=4.25、5.11、9.39、7.59,P<0.001)。转染tRNA-ValAAC-5-inhibitor组Hep3B、Hep3G2细胞侵袭转移减少,两组差异有统计学意义(t=14.01、21.85,P<0.001)。转染tRNA-ValAAC-5-inhibitor组Hep3B和Hep3G2细胞p21表达升高、MMP2和MMP9的表达量降低,差异均有统计学意义(t=8.96、12.80、4.652,P<0.01)、(t=15.17、22.36、12.61,P<0.01)。结论:tRNA-ValAAC-5能够有效促进肝癌增殖,侵袭和转移,其可能的作用机制与调控p21、MMP2和MMP9的表达有关。

Objective:To demonstrate the role and mechanism of tRNA-ValAAC-5 expression in hepatocellular carcinoma(HCC)cells.Methods:The expression levels of tRNA-ValAAC-5 in HCC(Hep3B,HuH7,SNU398,Hep3G2)and human hepatocellular carcinoma(THLE2,THLE3)were detected by real-time PCR.HEP3B and Hep3G2 cells were respectively transfected with tRNA-ValAAC-5-inhibitor and tRNA-ValAAC-5-NC as the inhibitor group and the NC group.Then the ability of cell proliferation was detected by CCK-8 assay and the ability of invasion and metastasis was detected by Transwell assay.The protein expression levels of p21,Matrix metalloproteinase 2(MMP2)and Matrix metalloproteinase 9(MMP9)were determined by Western blot.Results:The relative expression of tRNA-ValAAC-5 in Hep3B,HuH7,SNU398 and Hep3G2 cells were significantly higher than THLE2 and THLE3 cells,the differences were statistically significant(P<0.05).After tRNA-ValAAC-5-inhibitor transfection,the expression of tRNA-ValAAC-5 in Hep3B and Hep3G2 cells were reduced than tRNA-ValAAC-NC group.Both of the differences were statistically significant(t=36.52,27.45,P<0.001),which indicated the transfection was successful.The proliferative ability of Hep3B and Hep3G2 cells transfected with tRNA-ValAAC-5-inhibitor after 24、48、72、96 h were inhibited effectively compared with tRNA-ValAAC-5-NC group.All of the differences were statistically significant in Hep3B(t=5.25,8.23,7.33,14.16,P<0.001)and Hep3G2(t=4.25,5.11,9.39,7.59,P<0.001)cells.The number of invasion and metastasis of Hep3B and Hep3G2 cells were reduced in tRNA-ValAAC-5-inhibitor group compared with tRNA-ValAAC-5-NC group,there was significant difference(t=14.01,21.85,P<0.001).The protein expression levels of P21 were lower,MMP2and MMP9 were higher in tRNA-ValAAC-5-inhibitor group compared with tRNA-ValAAC-5-NC group,the differences were statistically significant in Hep3B(t=8.96,12.80,4.652,P<0.001)cells and Hep3G2(t=15.17,22.36,12.61,P<0.001)cells.Conclusion:tRNA-ValAAC-5 can effectively promote the proliferation,invasion and metastasis of HCC,and its possible mechanism is related to regulating the expression of p21,MMP2 and MMP9.