胡晓霞,霍建云,张金娟,潘娅,王晋星一,陈妮,张祥令,陈腾祥
HU Xiaoxia,HE Jianyun,ZHANG Jinjuan,PAN Ya,WANG Jingxingyi,CHEN Ni,ZHANG Xiangling,CHEN Tengxiang

• 1:贵阳医学院生理学教研室
• 2:贵阳医学院机能学实验室
• 3:贵阳医学院组织胚胎学教研室

摘要(Abstract)：

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 immunofluorecence method under microscope,and efficiency of calpain-2 silencing and integrin β4 proteolysis condition were detected with Western blot. Results: As immunofluorescence microscope result showing,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 cells respectively V. S. control( P < 0. 01). In MCF7 cell,integrin β4 were cut into low molecular weight( LMW) fragment as 200 k D,130 k D and95 k D. However,in calpain-2 silenced MCF7 cell,200 k D fragments were decreased. Conclusion:Formation of 200 k D fragments of integrin β4 is associated with calpain-2.

关键词(KeyWords)： 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
breast neoplasms;calcium-activated neutral proteases 2;integrein β4;proteolysis;gene silencing

Abstract:

Keywords:

基金项目(Foundation): 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]

作者(Author): 胡晓霞,霍建云,张金娟,潘娅,王晋星一,陈妮,张祥令,陈腾祥
HU Xiaoxia,HE Jianyun,ZHANG Jinjuan,PAN Ya,WANG Jingxingyi,CHEN Ni,ZHANG Xiangling,CHEN Tengxiang

DOI: 10.19367/j.cnki.1000-2707.2015.01.001

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