RhoA短发夹状双链RNA对肝癌细胞HepG2生物学行为的调控研究

The regulation role of short double-stranded hairpin RNA of RhoA inbiologic behavior of hepatoma cell

[目的]探讨肝癌细胞HepG2中RhoA的表达水平,以及载体表达的短发夹状双链RNA(shRNA)特异性抑制RhoA在HepG2中的表达后,对肝癌细胞侵袭迁移和增殖能力的影响。[方法]RT-PCR和Western-blot方法检测RhoA在HepG2及L02中的表达;构建RhoA shRNA真核表达载体;脂质体法转染至高表达RhoA的HepG2细胞中;筛选得到稳定低表达RhoA的HepG2细胞株,用侵袭小室法(transwell)检测该细胞株侵袭和迁移能力的改变;并用染料羧基荧光素乙酰乙酸琥珀酰亚胺酯(CFSE)标记该细胞株,流式细胞仪检测细胞增殖能力的变化。[结果]HepG2中RhoA mRNA和蛋白的表达水平均显著高于正常人肝胚胎细胞系L02(两者均P<0.05)。转染pshRNARhoA真核载体特异性抑制RhoA的表达后,HepG2细胞分别在侵袭实验和迁移实验中穿过微孔膜的细胞个数均远少于对照组细胞(两者均P<0.05),增殖能力也明显减弱(P<0.05)。[结论]抑制肝癌细胞HepG2中RhoA基因的表达能降低细胞的侵袭迁移和增殖能力,部分逆转肝癌细胞的恶性表型,为进一步研究肝癌侵袭转移和增殖机制提供了实验基础。

[Objective]To study the expression of RhoA in human hepatoma cell line HepG2 and the reg- ulation role of RhoA shRNA in cell invasion, migration and proliferation. [Methods]The expression of RhoA in human liver cell line L02 and hepatoma cell line HepG2 was detected by RT-PCR and Western- blot. ShRNA plasmid of RhoA was constructed by Pgenesil-2 and stably transferred into HepG2 by METAFECTENETM. Cell invasion and migration was detected by Transwell in vitro, and cell proliferation was examined by flow cytometry before which cells were stained by CFSE. [Results] The expression of RhoA gene in HepG2 was much higher than that in L02（P〈0.05）. After being transfected with pshR- NARhoA,the invasion and migration of cells was decreased obviously（P〈0.05）,and the growth rate of HepG2 was also inhibited with the decrease in RhoA gene expression（P〈0.05）. [Conclusion]Some of the malignant characteristics of hepatoma cell line can be partially reversed by inhibiting RhoA expres- sion. These provide potent theoretical ground for studying the mechanism not only of cell invasion and mo- tility but also proliferation in hepatoma.