干扰乳酸脱氢酶A表达对肝癌HepG2细胞增殖与迁移的影响

Effects of LDHA knockdown on proliferation and migration of HepG2 cells by RNA interference

目的:观察靶向乳酸脱氢酶A(lactate dehydrogenase-A,LDHA)的短发夹RNA(short hairpin RNA,shRNA)对肝癌Hep G2细胞系的干扰效率及其对细胞增殖与迁移的影响。方法:设计合成LDHA及对照e GFP shRNA序列,克隆至真核表达质粒p LKO.1-TRC,质粒抽提纯化后测序鉴定。重组质粒用脂质体转染肝癌细胞系,采用反转录(RT)-PCR和免疫印迹检测LDHA的表达,验证shRNA的干扰效率。采用CCK-8和克隆形成实验检测干扰LDHA后细胞增殖的变化,划痕实验检测干扰LDHA对细胞迁移的影响。结果:成功构建针对LDHA和对照e GFP的shRNA表达质粒。与转染sh-e GFP组相比,转染sh-LDHA组Hep G2细胞中LDHA mRNA和蛋白表达水平明显降低(P均<0.05)。与sh-e GFP组相比,sh-LDHA组72,96 h细胞增殖明显降低,克隆形成数减少,细胞迁移距离短(P均<0.05)。结论:LDHA的干扰质粒能有效干扰LDHA在肝癌Hep G2细胞中的表达,明显抑制Hep G2细胞的增殖活力和迁移能力。

Objective： To explore the proliferation and migration effects of lactate dehydrogenase-A （LDHA） short hairpin RNA （shRNA） on hepatocellular carcinoma HepG2 cells. Methods： LDHA and eGFP shRNA sequences were designed and inserted into pLKO. 1-TRC plasmid. HepG2 cells were transfected with the recombinant plasmids that were identified by sequencing. Reverse transcription （RT）-PCR and Western blotting were used to evaluate the silencing effect, CCK-8 experiment and clone formation assay for proliferation, wound healing assay for migration. Results： In HepG2 cells, LDHA shRNA significantly reduced the expression of LDHA, the efficiency of silencing were 76.6% and 82.6% on the transcription of LDHA gene and the expression of LDHA protein respectively. Compared with the sh-eGFP group, sh-LDHA group showed decresed celluar vitablitly, lower levels of the formed clones, shorter migration distance （ both P 〈0.05）. Conclusion： LDHA shRNA was successfully constructed and could effectively knockdown target gene in HepG2 cells. Inhibition of LDHA expression evidently resulted in inhibition of HepG2 proliferation and migration.