钙激活中性蛋白酶-2对整合素β4水解的影响  

Research on the Effect of Calpain-2 on the Proteolysis of Integrin β4 in MCF7 Cell

作　　者：胡晓霞[1] 霍建云 张金娟[2] 潘娅[1] 王晋星一[1] 陈妮[1] 张祥令[3] 陈腾祥[1] 

机构地区：[1]贵阳医学院生理学教研室,贵州贵阳550004 [2]贵阳医学院机能学实验室,贵州贵阳550004 [3]贵阳医学院组织胚胎学教研室,贵州贵阳550004

出　　处：《贵阳医学院学报》2015年第1期1-5,共5页Journal of Guiyang Medical College

基　　金：国家自然科学基金资助项目(NO.81060176);贵州省科技厅自然基金项目[黔科合J字(2008)2280];贵阳市科技局社会发展公关计划项目[筑科农字(2010)1号]

摘　　要：目的:观察乳腺癌细胞系MCF7细胞中,钙激活中性蛋白酶2(calpain-2)对整合素(integrin)β4水解的影响。方法:利用"短发夹"RNA(shRNA)和微小RNA(mirRNA)技术结合的calpain-2基因沉默(gene silencing)慢病毒质粒(GIPZ lentiviral shRNAmir)转染乳腺癌细胞株MCF7,嘌呤霉素筛选稳定沉默calpain-2基因的细胞株,细胞株传4代,用荧光显微镜观察转染效率,用蛋白质印迹(Western blot)实验检测calpain-2基因沉默效率及integrinβ4水解的情况。结果:嘌呤霉素筛选、细胞传4代,荧光显微镜下观察显示转染和筛选后,EGFP表达阳性的MCF7细胞的比例分别达到(95.6±2.6)%(空shRNAmir载体)、(97.1±1.7)%(Calpain-2 shRNAmir1)和(97.7±0.2)%(Calpain-2 shRNAmir 2),差异无统计学意义(P>0.05);Western blot结果显示,基因沉默的MCF7细胞中calpain-2的表达被明显抑制,相对于空shRNAmir载体转染的MCF7细胞,差异有统计学意义(P<0.01),calpain-2的表达下调到21.5%(Calpain-2 shRNAmir 1)和18.8%(Calpain-2 shRNAmir 2),空shRNAmir载体转染的MCF7细胞中calpain-2的表达与未转染的MCF7细胞比较,差异无统计学意义(P>0.05);比较calpain-2基因沉默和空shRNAmir载体转染的MCF7细胞,发现integrinβ4均被水解,水解片段的分子量主要有200 k D、130和95 k D;calpain-2基因沉默后,calpain-2基因沉默MCF7细胞中200 k D的水解片段比空shRNAmir载体转染的MCF7细胞减少(P<0.01)。结论:在乳腺癌细胞MCF7中,calpain-2可能参与integrinβ4的200 k D片段的形成,从而参与调整integrinβ4的构象变化。Objective： To observe the effect of calcium-activated neutral proteases 2 （calpain-2） on proteolysis of integrin β4 in breast cancer cell line, MCF7. Methods： Plasmids of GIPZ lentiviral shRNAmir combined with shRNA and microRNA （miRNA） technology were transfected into MCF7 cells to silence the expression of calpain-2. The puromycin was used to screen MCF7 with 4 passages to acquire the stable calpain-2-silenced cell lines. The transfected rates were evaluated with immunofluo- recence method under microscope, and efficiency of calpain-2 silencing and integrin β4 proteolysis condition were detected with Western blot. Results： As immunofluorescence microscope result show- ing, the ratios of the cells expressing green fluorescence protein （GFP） were （95.6 ± 2.6） % （ empty shRNAmir）, （97.1 ± 1.7 ） % （ calpain-2 shRNAmir 1 ） and （97.7 ± 0.2） % （ calpain-2 shRNAmir 2 ）, without statistic diversity （P 〉 0.05 ） . The expression of calpain-2 had no distinctive diversity （P 〉 0. 05 ） in empty shRNAmir transfected MCF7 cells and in control, while was decreased to 21.5%, 18.8% in calpain-2 shRNAmir 1 and 2 transfected ceils respectively V.S. control （ P 〈 0.01 ）. In MCF7 cell, integrin β4 were cut into low molecular weight （LMW） fragment as 200 kD, 130 kD and 95 kD. However, in calpain-2 silenced MCF7 cell, 200 kD fragments were decreased. Conclusion： Formation of 200 kD fragments of integrin β4 is associated with calpain-2.

关 键 词：乳腺肿瘤 钙激活中性蛋白酶2 整合素β4 蛋白水解 基因沉默 

分 类 号：R737.9[医药卫生—肿瘤]