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
하조인유선암MCF-7세포계miRNA-221화miRNA-222표체대억제종류세포증식급천이능력적탐토
Inhibition of down-regulated miRNA-221 and miRNA-222 in human breast cancer MCF-7 cell line to tumor cell proliferation and migration
  • 万方数据
    检验医学 검험의학
    LABORATORY MEDICINE
    2014年 5期 452-459 ,共8页

    甘蓉%杨晓%杨英梅%赵苓旭%孟庆贺

    감용%양효%양영매%조령욱%맹경하

    微小RNA-221%微小RNA-222%转染%组织金属蛋白酶抑制剂3%金属蛋白酶17%人乳腺癌MCF-7细胞 微小RNA-221%微小RNA-222%轉染%組織金屬蛋白酶抑製劑3%金屬蛋白酶17%人乳腺癌MCF-7細胞
    미소RNA-221%미소RNA-222%전염%조직금속단백매억제제3%금속단백매17%인유선암MCF-7세포
    MicroRNA-221%MicroRNA-222%Transfection%Tissue metalloproteinase inhibitor 3%Metalloproteinase 17%Human breast cancer MCF-7 cell
    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。
    목적：통과고저미소RNA（microRNA，miRNA）-221화miRNA-222적표체상조조직금속단백매억제제3（TIMP3）적방법래연구인유선암 MCF-7세포계적증식화천이능력。방법근거 miRBase 수거고중 Homo sapiens（hsa）-miRNA-221화hsa-miRNA-222적과핵감산서렬화일단무의서렬분별설계병중조성질립：반의억제miRNA-221（antisense-miRNA-221，AS-miRNA-221）、반의억제miRNA-222（antisense-miRNA-222，AS-miRNA-222）、공동반의억제miRNA-221/222（antisense-miRNA-221/222，AS-miRNA-221/222）화무의억제（Scramble），병장기분별이용지질체LipofectamineTM2000전염진인유선암MCF-7세포중，경G418사선후건립은정표체적대조세포주[미전염정상세포（Control조）화무의억제세포（Scramble조）]화저표체miRNA-221、miRNA-222세포주（즉AS-miRNA-221조、AS-miRNA-222조화AS-miRNA-221/222조）。전염24 h후우형광현미경하관찰전염효솔；채용실시형광정량취합매련반응（PCR）검측각조세포중miRNA-221화miRNA-222적표체량，채용역전록PCR검측각조세포중질립재체상휴대적항성neo 기인적표체정황，이차래험증전염시부성공；분별채용실시형광정량PCR、면역인적법검측TIMP3화금속단백매17（ADAM17）적표체차이；채용세포계수시제합（CCK-8）검측병회제생장곡선，관찰저표체miRNA-221、miRNA-222각조세포적생장능력；채용화흔수복실험관찰전염후세포적천이능력。결과전염24 h지후，재형광현미경하관찰도각전염조세포적록색형광단백표체교다，즉전염효솔교고。검측전염후각조세포중 neo 기인、miRNA-221화 miRNA-222적표체。여 Control 조비교，Scramble 조、AS-miRNA-221조、AS-miRNA-222조、AS-miRNA-221/222조neo기인표체명현승고；여Scramble조비교，AS-miRNA-221조、AS-miRNA-222조、AS-miRNA-221/222조miRNA-221화miRNA-222적표체량명현강저，즉증실전염료반의억제miRNA-221/222적저표체은정세포주구건성공。여Scramble조비교，AS-miRNA-221조、AS-miRNA-222조、AS-miRNA-221/222조세포TIMP3 mRNA표체승고，이기조공적ADAM17수평강저。생장곡선현시저표체miRNA-221、miRNA-222각조세포적생장능력명현수도억제。화흔수복실험결과현시저표체 miRNA-221、miRNA-222각조세포적천이능력강저。결론통과고저miRNA-221、miRNA-222、상조TIMP3표체가이억제인유선암세포계MCF-7적증식화천이능력。
    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.
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