黑色素瘤分化相关基因-7/白细胞介素-24促进阿霉素杀伤肝癌细胞MHCC-97L机制的研究

The MDA-7/IL-24 promote ADM to arrest the growth of hepatoma carcinoma cell lines MHCC-97L

目的 探讨黑色紊瘤分化相关基因-7/白细胞介素-24（MDA-7/IL-24）基因促进阿霉素（ADM）杀伤肝癌细胞,逆转肝癌细胞多药耐药（MDR）的机制.方法 以人肝癌细胞株MHCC-97L为实验对象,使用噻唑蓝（MTT）比色法和流式细胞仪比较Ad. MDA-7联合ADM处理组与ADM组、Ad. MDA-7组对肝癌细胞MHCC-97L和正常肝细胞L02的作用差异.观察MDA-7/IL-24对多药耐药的逆转作用.荧光定量聚合酶链反应（PCR）检测MDR-1、STAT-3、bcl-2、bax mRNA的变化.Western blot检测gp-170、STAT-3、bcl-2、bax蛋白的表达的变化.结果 MTT表明Ad. MDA-7对正常肝细胞LO2无生长抑制作用（P〉0.05）.LO2细胞Ad. MDA-7联合ADM组与ADM组细胞生存率差异无统计学意义（P〉0.05）.低浓度（100 VP/cell）的Ad. MDA-7联合正常肝细胞的IC50浓度的ADM（1.5 mg/L）使得细胞抑制率从ADM组的17.46%上升到79.50%,生长抑制逆转4.55倍（P〈0.05）.MDR-1mRNA相对表达量从（16.49±0.11）下降至（5.48±0.05）.STAT-3 mRNA相对表达量从（13.17±0.08）上升至（21.57±0.11）.bcl-2及BAX表达与其他实验组比较差异均有统计学意义（P〈0.05）.联合实验组P-170蛋白的表达量较其他组明显降低,而磷酸化STAT-3蛋白的表达量亦增加.结论 Ad. MDA-7具有逆转肝癌细胞MHCC-97L多药耐药的作用,其下调MDR-1 mRNA的表达的同时,并通过活化STAT-3信号通路的表达促进肝癌细胞凋亡.

Objective To explore the mechanism of the melanoma differentiation associated gene-7 （MDA-7/IL-24） promoting adriamycin （ADM） to kill the hepatoma carcinoma cell and reversing multidrug resistance （MDR）. Methods By using the human hepatoma carcinoma cell line MHCC-97L and normal hepatic cell line LO2, MTT assay and flow cytometry （FCM） were applied to compare the cell growth among the combined （ Ad. mda-7 ＋ ADM ） group, ADM group and Ad. mda-7 group. The expression levels of MDR-1, STAT-3, bcl-2, and bax mRNA were examined by using real-time polymerase chain reaction （PCR）. Western blotting was performed to observe the change in the expression of gp-170, STAT-3, bcl-2, and bax among those groups. Results MTT showed that Ad. mda-7 had no toxic effect on LO2 cells, and there was no significant difference in growth rate between adriamycin group and combined group （P〉0.05）. The ratio of growth suppression in MHCC-97L cells only treated with ADM （1.5 mg/L） was 17.46%, but in combined group subject to treatment of 100 VP/cell Ad. mda-7 and ADM （1.5 mg/L） it was increased to 79.50% （P〈0.05 ）. Real-time PCR revealed the expression of MDR-1 mRNA was decreased from （16.49±0.11） in ADM group to （5.48±0.05） in combined group, and that of STAT-3 mRNA increased from （13.17±0.08） to （21.57±0.11） correspondingly （P〈0.05）. Western blotting also demonstrated that the expression of P-170 in combined group was decreased and that of phosphorylated STAT-3 was increased as compared with other two groups. Conclusion Ad. mda-7 can reverse the MDR of ADM to MHCC-97L cells, inhibit the expression of MDR-1 mRNA and activate the signaling of STAT-3 to induce apoptosis of hepatoma carcinoma cell MHCC-97L.