微小RNA-10b对人胰腺癌细胞RhoC表达的影响

Effect of transfection of microRNA-10b and antisense microRNA-10b on the expression of RhoC in human pancreatic cancer cell line AsPC-1

目的 观察微小RNA（miR）-10b对人胰腺癌细胞AsPC-1细胞（以下简称AsPC-1）表达RhoC蛋白的影响.方法 以脂质体转染法将重组正、反义真核表达载体pPG-CMV-增强绿色荧光蛋白（EGFP）-miR-10b转染AsPC-1细胞株（分为正、反义组）,实时荧光定量聚合酶链反应（FQ-PCR）和荧光显微镜检测载体瞬时表达的效率,Matrigel实验评价不同组间细胞迁移侵袭能力的差异；Western blot检测细胞RhoC蛋白的表达.结果 转染正义组miR-10b表达量增加10倍,反义组减少至对照组的14％.差异均有统计学意义（P＜0.05）.细胞RhoC蛋白的表达水平：正义组为（95.7±8.5）像素单位；反义组为（28.5±5.7）像素单位,差异均有统计学意义（P＜0.05）；细胞移行实验：正义组移行细胞数为（85±7）个,反义组为（32±5）个,差异有统计学意义（P＜0.05）；细胞侵袭实验：正义组细胞穿膜细胞数为（35±9）个,反义组为（8±3）个,差异有统计学意义（P＜0.05）.结论 miR-10b可促进胰腺癌细胞RhoC蛋白的表达,并影响胰腺癌细胞侵袭迁移的能力.

Objective To observe the effect of microRNA （miR）-10b on the expression of RhoC in human pancreatic cancer cell line AsPC-1.Methods The recombinant eukaryotic expression plasmid vector with miR-10b sequence [pPG-CMV-enhanced green fluorescent protein （EGFP）-miR-10b] or the antisense sequence was transfected into human pancreatic cancer cell line （AsPC-1） with lipofectamine,and the efficiency of the instant expression was confirmed by real-time fluorescent quantitative polymerase chain reaction （FQ-PCR） and fluorescence microscope.The difference of the migration and invasion of the cells was evaluated by matrigel assay,and the expression of RhoC was evaluated by Western blotting.Results The expression of miR-10b in the sense group was increased by 10 folds,and in antisense group,the expression was decreased to 14% compare to the control （P 〈 0.05）.The expression of RhoC protein in the sense group was （95.7 ± 8.5） unit,and that in the antisense group （28.5 ± 5.7） unit （P 〈0.05）.As compared to the antisense group,the number of migrating and penerating cells was decreased from （85 ±7） and （35 ±9） to （32±5） and （8 ±3） in the sense group （P〈0.05） Conclusion miR10b can up-regulate the expression of RhoC,and promote the migration and invasion of the AsPC-1 cells.