短发夹RNA抑制肝癌细胞HepG2 COX-2基因表达的探讨

目的：研究shRNA对肝癌细胞株HepG2中COX-2基因表达的抑制作用,同时检测对细胞周期和细胞生长的影响。方法：设计、合成一对环氧化酶-2（COX-2）基因编码的反向重复序列,中间间隔9个核苷酸序列,定向克隆至质粒载体pGenesil-1,构建抑制COX-2基因的短发夹RNA（short hairpin RNA shRNA）的重组表达质粒pshRNA-COX-2,用脂质体Lipofectamine^TM2000转染肝癌细胞HepG2,经RT-PCR和Westernblot分别检测pshRNA-COX-2重组表达质粒对COX-2mRNA和蛋白抑制效应,流式细胞仪检测细胞周期、细胞计数检测细胞生长变化。结果：pshRNA-COX-2重组表达质粒能够抑制COX-2基因表达。与未转染肝癌细胞HepG2相比,pshRNA-COX-2重组表达质粒转染肝癌细胞后,COX-2 mRNA和蛋白表达明显降低,抑制率分别为69.9%和50.3%;G0-G1期细胞由55.4%上升为77.9%,S期细胞由31.1%下降为16.2%;细胞生长明显减慢。结论：构建的pshRNA-COX-2重组表达载体能够显著抑制COX-2基因表达;引起G0-G1期细胞增多,S期细胞减少,从而阻止肝癌细胞生长。

The Studies on Depressant Effect of Short-Hairpin RNA of COX-2 Gene Expression in HepG2 Cells

Objective:To investigate the inhibitory action of short hairpin RNA (ShRNA) targeting at the COX-2 gene expression in HepG2 cells and to determine the effect of ShRNA on the change of cell cycle and cell growth. Methods:A pair of reverse repeated sequence targeting COX-2 mRNA were contrived and synthesized, with 9 nucleotide sequences between them, and were directionally cloned to the pGenesil-1, a plasmid vector to construct a recombinant expression plasmid pshRNA-COX-2, and LipofectamineTM2000, a liposome was used to transfect the hepatocelluar carcinoma cell HepG2. Suppressing effects of the pshRNA-COX-2 on the COX-2 mRNA and proteins were detected by the RT-PCR and Western blot methods, respectively. Flow cytometry was conducted to assay the cell cycle and change of the cell growth. Results:Recombinant expression plasmid pshRNA-COX-2 could inhibit the expression of COX-2 gene. Compared to the group without transfecting the hepatoma carcinoma cell (HCC) HepG2, the expressions of COX-2 mRNA and proteins were apparently lower in the group with recombinant expression plasmid pshRNA-COX-2's transfecting the HCC. The inhibition rates were 69.9% and 50.3% respectively. The cell counts of the G0 and G1 phase were increased from 5.4% to 77.9%, and that of stage S was decreased from 31.1%to16.2 %. The cell growth became significantly slow. Conclusion:Construction of the recombinant expression vector pshRNA-COX-2 could significantly inhibit the expression of COX-2 gene, which result in an increase of the cells in the G0 and G1 phase and a decrease of the cells in the S phase, thus suppressing the proliferation of hepatocelluar cells.