摘要
目的:研究构建靶向ING1基因的miR-622真核表达载体并验证其转染人胃癌细胞株MKN-45细胞后对ING1基因的干扰效果及其功能.方法:将外源性重组真核表达载体pSuper/miR-622转染到人胃癌细胞株MKN-45内,经G418筛选并建立高表达miR-622的稳定转染胃癌细胞株.稳定表达该miR-622的胃癌细胞为:MKN-45-pSuper/miR-622组,转染空质粒细胞及未处理细胞为对照组(MKN-45-pSuper组和MKN-45组),采用实时荧光定量PCR验证miR-622在稳定转染细胞的表达,蛋白印迹检测其对ING1基因表达的干扰效果,通过细胞增殖和周期实验验证miR-622在胃癌细胞MKN-45中的功能.结果:与pSuper空载体组相比,转染了pSuper/miR-622高表达质粒的MKN-45细胞中ING1蛋白表达明显减少,降低了4.63倍(1.83±0.86vs8.47±1.43,P<0.05);与转染pSuper空载体的MKN-45细胞对照组相比,转染了pSuper/miR-622高表达质粒的MKN-45细胞地促进了胃癌细胞增殖(P<0.05),而转染了pSuper空载体的MKN-45细胞组与正常组组间无统计学意义(P>0.05).pSuper/miR-622组在胃癌细胞G0/G1期为21.45±0.16而pSuper空载体组48.21±0.34;pSuper/miR-622组在胃癌细胞G2/M期为53.67±0.41而pSuper空载体组20.27±0.18,与pSuper空载体组细胞相比较,miR-622的高表达促进了胃癌细胞周期的演化.结论:miR-622真核表达载体构建和稳定表达胃癌细胞筛选成功,为继续深入的研究miR-622在胃癌中的功能奠定了基础.
AIM： To investigate the function of miR-622 in human gastric cancer cell line MKN-45 by constructing a miR-622 eukaryotic expression vector targeting the ING1 gene and to explore the po- tential role of miR-622 in gastric carcinogenesis.METHODS： A recombinant plasmid carrying miR-622 （pSuper/miR-622） was transfected into MKN-45 cells using lipofectin-mediated method. Cells stably expressing miR-622 were selectedusing G418. MKN-45 cells untransfected and those transfected with empty pSuper plasmid were used as controls. The expression levels of miR-622 were detected by TaqMan real-time PCR in stably transfected MKN-45 cells, and Western blot was used to detect the expression of ING1 protein. RESULTS： Compared to untransfected MKN-45 cells, the expression of ING1 protein showed an average 4.63-fold decrease （1.83 ± 0.86 vs 8.47 ± 1.43, P 〈 0.05）. MKN-45 cells tranfected with pSuper/miR-622 showed higher cell growth activity than control cells （P 〈 0.05）. Over-expression of miR-622 in MKN-45 cells promoted cell cycle progression （G0/G1 phase： 21.45 ± 0.16 vs 48.21 ±0.34; G2 / M phase： 53.67± 0.41 vs 20.27± 0.18） compared to cells transfected with pSuper empty vector. CONCLUSION： A MiR-622 eukaryotic expression vector that can stably express miR-622 in MKN-45 cells has been successfully constructed and can be used to study the functions of miR-622 in human gastric cancer.
出处
《世界华人消化杂志》
CAS
北大核心
2011年第27期2810-2815,共6页
World Chinese Journal of Digestology
基金
山东省自然科学基金资助项目
No.Y2007C127~~
