下调人乳腺癌MCF-7细胞系miRNA-221和miRNA-222表达对抑制肿瘤细胞增殖及迁移能力的探讨

摘要

目的：通过敲低微小RNA（microRNA，miRNA）-221和miRNA-222的表达上调组织金属蛋白酶抑制剂3（TIMP3）的方法来研究人乳腺癌 MCF-7细胞系的增殖和迁移能力。方法根据 miRBase 数据库中 Homo sapiens（hsa）-miRNA-221和hsa-miRNA-222的寡核苷酸序列和一段无义序列分别设计并重组成质粒：反义抑制miRNA-221（antisense-miRNA-221，AS-miRNA-221）、反义抑制miRNA-222（antisense-miRNA-222，AS-miRNA-222）、共同反义抑制miRNA-221/222（antisense-miRNA-221/222，AS-miRNA-221/222）和无义抑制（Scramble），并将其分别利用脂质体LipofectamineTM2000转染进人乳腺癌MCF-7细胞中，经G418筛选后建立稳定表达的对照细胞株[未转染正常细胞（Control组）和无义抑制细胞（Scramble组）]和低表达miRNA-221、miRNA-222细胞株（即AS-miRNA-221组、AS-miRNA-222组和AS-miRNA-221/222组）。转染24 h后于荧光显微镜下观察转染效率；采用实时荧光定量聚合酶链反应（PCR）检测各组细胞中miRNA-221和miRNA-222的表达量，采用逆转录PCR检测各组细胞中质粒载体上携带的抗性neo 基因的表达情况，以此来验证转染是否成功；分别采用实时荧光定量PCR、免疫印迹法检测TIMP3和金属蛋白酶17（ADAM17）的表达差异；采用细胞计数试剂盒（CCK-8）检测并绘制生长曲线，观察低表达miRNA-221、miRNA-222各组细胞的生长能力；采用划痕修复实验观察转染后细胞的迁移能力。结果转染24 h之后，在荧光显微镜下观察到各转染组细胞的绿色荧光蛋白表达较多，即转染效率较高。检测转染后各组细胞中 neo 基因、miRNA-221和 miRNA-222的表达。与 Control 组比较，Scramble 组、AS-miRNA-221组、AS-miRNA-222组、AS-miRNA-221/222组neo基因表达明显升高；与Scramble组比较，AS-miRNA-221组、AS-miRNA-222组、AS-miRNA-221/222组miRNA-221和miRNA-222的表达量明显降低，即证实转染了反义抑制miRNA-221/222的低表达稳定细胞株构建成功。与Scramble组比较，AS-miRNA-221组、AS-miRNA-222组、AS-miRNA-221/222组细胞TIMP3 mRNA表达升高，而其调控的ADAM17水平降低。生长曲线显示低表达miRNA-221、miRNA-222各组细胞的生长能力明显受到抑制。划痕修复实验结果显示低表达 miRNA-221、miRNA-222各组细胞的迁移能力降低。结论通过敲低miRNA-221、miRNA-222、上调TIMP3表达可以抑制人乳腺癌细胞系MCF-7的增殖和迁移能力。%Objective To study the proliferation and migration abilities of human breast cancer MCF-7 cell line through knocking down microRNA (miRNA )-221 and miRNA-222 expressions and up-regulating tissue metalloproteinase inhibitor 3 (TIMP3 ).Methods Antisense-miRNA-221 (AS-miRNA-221 ),antisense-miRNA-222 (AS-miRNA-222),antisense-miRNA-221/222 (AS-miRNA-221/222)and scramble were designed based on Homo sapiens (hsa )-miRNA-221 and hsa-miRNA-222 oligonucleotide sequence and nonsense sequence in miRBase. LipofectamineTM2000 was used to transfect AS-miRNA-221,AS-miRNA-222,AS-miRNA-221/222 and scramble into MCF-7 cell line .They were classified into control groups with stable expression [untransfected normal cell group (Control group)and scramble group(Scramble group)]and down-regulated miRNA-221 and miRNA-222 groups(AS-miRNA-221 group,AS-miRNA-222 group and AS-miRNA-221/222 group),respectively,according to G418 screening. After transfecting for 24 h,transfection efficiency was determined by fluorescence microscopy.Real-time fluorescence quantitation polymerase chain reaction (PCR)was used to determine the expressions of miRNA-221 and miRNA-222. The neo gene expression on the plasmid vector was detected in each cell line by reverse transcription PCR.The TIMP3 mRNA level was assessed by real-time fluorescence quantitation PCR,and the expression difference of metalloproteinase 17(ADAM17)protein was determined by Western blotting.The growth curves to describe the low-expressed miRNA-221 and miRNA-222 cells′growth were evaluated by CCK-8.The wound healing model was used to represent the migration ability.Results After transfecting for 24 h,there was an increasing expression of green fluorescence,and the transfection efficiency was high.The expressions of neo gene in Control group were lower than those in Scramble,AS-miRNA-221,AS-miRNA-222 and AS-miRNA-221/222 groups.Compared to Scramble group,the expressions of miRNA-221 and miRNA-222 decreased in AS-miRNA-221,AS-miRNA-222 and AS-miRNA-221/222 groups,and the establishment of AS-miRNA-221/222 down-regulated expression-stable cell line had succeeded. Compared with Scramble group,the expressions of TIMP3 mRNA of AS-miRNA-221 group,AS-miRNA-222 and AS-miRNA-221/222 groups increased,and the level of ADAM17 decreased.The growth curves showed that the proliferation of down-regulated miRNA-221 and miRNA-222 groups had been inhibited.The wound healing model showed that the migration of down-regulated miRNA-221 and miRNA-222 groups had been decreased.Conclusions Through up-regulatingp TIMP3,knocking down miRNA-221 and miRNA-222 can inhibit the proliferation and migration of human breast cancer MCF-7 cell line.