携带人mda-7/IL-24基因溶瘤腺病毒SG600-IL24构建及其选择性杀伤肝癌细胞的机制研究

Construction of oncolyic adenovirus SG600-IL24 carrying human mda-7/IL-24 gene and mechanism for its selective killing hepatocullular carcinoma cells

目的:探讨携带人mda-7/IL-24基因的溶瘤腺病毒SG600-IL24对肝癌细胞选择性杀伤效应的机制。方法:构建携带人mda-7/IL-24基因的溶瘤腺病毒SG600-IL24后,用其分别感染肝癌细胞株HepG2、HCCLM3和正常肝细胞株L02。用RT-PCR和Westernblot分别检测感染后,各细胞株STAT3以及其信号通路下游相关信号分子基因与蛋白的表达变化,以及磷酸化STAT3(p-STAT3)蛋白的表达变化。结果:成功构建携带人mda-7/IL-24基因的溶瘤腺病毒SG600-IL24载体。SG600-IL24感染后,3种细胞的mda-7/IL-24基因及蛋白表达均明显升高(均P<0.05)。两种肝癌细胞感染后,STAT3的表达水平明显下调,其下游信号分子c-myc、Bcl-xl、Bcl-2、cyclin D2、survivin、MMP-2、MMP-9、XIAP、OPN、VEGF的表达水平明显下调,Bax表达明显上调,均呈一定的时间依赖趋势(均P<0.05);p-STAT3蛋白在感染后上升,并在感染2 h达到峰值,随后下降。感染后,L02细胞中未见STAT3及其下游信号分子表达的变化(均P>0.05)。结论:携带人mda-7/IL-24基因溶瘤腺病毒SG600-IL24选择性杀伤肝癌细胞的机制可能与其选择性抑制肝癌细胞STAT3信号通路,而对正常肝细胞不产生明显影响。

Objective: To investigate the mechanism for the selective killing effect of oncolyic adenovirus SG600-IL24 bearing human mda-7/IL-24 on l hepatocullular carcinoma(HCC) cells. Methods: The oncolytic adenovirus SG600-IL24 carrying human MDA-7/IL-24(SG600-IL24) was constructed, and then was infected into the HCC cell lines HepG2 and HCCLM3 and normal liver cell line L02, respectively. In each cell line after infection, the changes in gene and protein expressions of STAT3 and its downstream signaling molecules as well as the protein expression of phosphorylated STAT3 were determined by RT-PCR and Western blot, respectively. Results: The oncolytic adenovirus SG600-IL24 carrying human mda-7/IL-24 was constructed successfully. After SG600-IL24 infection, the expressions of human mda-7/IL-24 gene and protein were significantly increased in all the cell lines(all P<0.05). In the two types of HCC cells, the STAT3 expression level was significantly downregulated, and its downstream signaling molecule c-myc, Bcl-xl, Bcl-2, cyclin D2, survivin, MMP-2, MMP-9, XIAP, OPN, and VEGF were downregulated while Bax were upregulated significantly, and all presented with a time-dependent trend(all P<0.05);the protein expression of p-STAT3 was increased after infection and reached to a peak in 2 hours, and then decreased. No significant changes were noted in expressions of STAT3 and its downstream molecules in L02 cells after infection(all P>0.05).Conclusion: The mechanism for the selective killing effect of oncolyic adenovirus SG600-IL24 bearing human mda-7/IL-24 on HCC cells may probably be associated with its selectively inhibiting STAT3 signaling pathway in HCC cells, with no influence on normal liver cells.