腺病毒介导的黑色素瘤分化相关基因-7抑制内质网应激蛋白表达阻滞MHCC-97H血管形成及转移机制的研究  

Melanoma differentiation associated gene-7/interleukin-24 suppress the angiogenesis of hepatoma carcinoma cell line MHCC-97H by inhibiting endoplasmic reticulum stress proteins

作　　者：郑建伟[1] 张晓梅 李雁[3] 秦仁义[1] 申铭[4] 吴在德[1] 薛新波[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院普外科,武汉430030 [2]武汉市第五医院消化内科 [3]武汉大学附属中南医院肿瘤科 [4]华中科技大学同济医学院附属同济医院胆胰外科,武汉430030

出　　处：《中华实验外科杂志》2013年第12期2566-2569,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30872510）;湖北省自然科学基金资助项目（2008CDB127）

摘　　要：目的 探讨黑色素瘤分化相关基因（MDA）-7抑制内质网应激蛋白表达,以及阻滞MHCC-97H血管形成的机制.方法 以人肝癌细胞株MHCC-97H为实验对象,使用噻唑蓝（MTT）法和流式细胞仪比较Ad.MDA-7及对照组、缺氧组及正氧组的细胞生存率,比较缺氧环境中,MHCC-97H凋亡率的变化.荧光定量聚合酶链反应（FQ-PCR）检测MDA-7、细胞外信号调节激酶（ERK）、E-钙黏蛋白（E-cad）、血管内皮生长因子（VEGF）、基质金属蛋白酶（MMP）-9 mRNA的变化.Western blot检测MDA-7、蛋白激酶样内质网激酶（PERK）、E-cad、VEGF、MMP-9蛋白表达的变化.结果 MTT表明缺氧条件下,正氧组正常肝细胞LO2生长抑制率分别为（12.60±4.85）％,（7.80±0.61）％,Ad.MDA-7对正常肝细胞LO2无生长抑制作用.肝癌细胞株MHCC-97H生长抑制率缺氧组,正氧组分别为（5.10±0.09）％、（4.80±0.05）％.缺氧环境中,ERK、VEGF和MMP-9空白组及空白对照组mRNA表达量为[（18.042 ±5.915）/（17.512 ±6.354）、（22.654±5.976）/（22.587 ±7.741）、（22.654±2.789）/（22.587±8.551）]高于正氧组[（10.987±2.514）/（12.347±4.385）、（15.321 ±4.262）/（15.634 ±8.521）、（15.321 ±6.384）/（15.634±4.423）].缺氧环境中,E-cad mRNA空白组及空白对照组[（6.427 ±0.512）/（6.042±1.972）]表达低于正氧组[（10.987±2.514）/（12.347 ±4.385）],而Ad.MDA-7干预后,ERK、VEGF和MMP-9 mRNA缺氧组及正氧组表达量降低[（4.117±1.843）/（5.683±0.794）、（5.371 ±0.541）/（4.209 ±0.348）、（5.371 ±0.941）/（4.209±0.265）],E-cad表达量明显增加[（19.573 ±5.971）/（21.309±8.544）].PERK、VEGF和MMP-9蛋白表达与mRNA一致.结论 缺氧环境中,肝癌细胞株MHCC-97H生长抑制率与正氧环境中差异无统计学意义,MDA-7基因明显抑制PERK表达,抑制肝癌细胞血管生成作用.同时,MDA-7基因增强E-cad表达,协同其作用,抑制肿瘤细胞转移.Objective To explore the mechanism of the melanoma differentiation associated gene-7/ interleukin-24 （MDA-7/IL-24） suppressing the angiogenesis of hepatoma carcinoma cell line MHCC-97H by inhibiting endoplasmic reticulum stress proteins.Methods By using the human hepatoma carcinoma cell line MHCC-97L as the experimental subject,MTT assay and flow cytometry were applied to examine the difference of tumor cell survival rate among different groups.The apoptosis of MHCC-97H cells was compared in the anoxic environments.The expression levels of MDA-7,extracellular signal-regulated kinase （ERK）,E-cadherin （E-cad）,vascular endothelial growth factor （VEGF）,and matrix metalloproteinase （MMP）-9 mRNA were detected by using real-time fluorescent quantitative polymerase chain reaction （FQ-PCR）.Western blotting was perform to observe the changes of MDA-7,protein kinase like endoplasmic reticulum kinase （PERK）,E-cad,VEGF,and MMP-9 proteins among those groups.Results MTT showed that the growth inhibition rate of liver cells LO2 in hypoxia group and normoxia group was （12.60 ± 4.85）％ and （7.80 ± 0.61）％ respectively.Ad.MDA-7 had no the significant inhibitory effect on the growth of LO2 cells.Growth inhibition rate of MHCC-97H cells in hypoxia group and normoxia group was （5.10 ±0.09）％ and （4.80 ±0.05）％ respectively.Real-time PCR showed the expression of ERK,VEGF and MMP-9 mRNA in blank group and control blank group under hypoxia [（18.042 ± 5.915）/（17.512 ± 6.354）,（22.654 ±5.976）/（22.587 ± 7.741）,and （22.654 ± 2.789）/（22.587-± 8.551）] was higher than in normoxia group [（10.987 ±2.514）/（12.347 ±4.385）,（15.321 ±4.262）/（15.634 ±8.521）,and （15.321 ±6.384）/（15.634 ±4.423）].In hypoxic environments,E-cad mRNA expression in blank group and control blank group [（6.427 ± 0.512）/（6.042 ± 1.972）] was lower than in normoxia group [（10.987 ±2.514）/（12.347 ±4.385）].After Ad.MDA-7 interference,the expression of ER

关 键 词：癌 肝细胞 黑色素瘤分化相关基因-7 白细胞介素-24 内质网应激蛋白 血管形成 转移 

分 类 号：R735.7[医药卫生—肿瘤]