微小RNA-99a靶向作用哺乳动物雷帕霉素靶蛋白对乳腺癌细胞生长和迁移的影响

Effect on the growth and migration of breast cancer cell targets of microRNA -99a on mammalian target of rapamycin

目的探讨微小RNA（miRNA，miR）-99a作用于靶蛋白哺乳动物雷帕霉素靶蛋白（mTOR）对乳腺癌细胞生长和迁移的影响。方法构建miR-99a转染乳腺癌细胞、miR-99a和mTORcDNA共同转染乳腺癌细胞模型，采用实时定量聚合酶链反应（Real—timePCR）检测乳腺癌组织及癌旁组织中miR-99a和mTORmRNA的表达，Westernblot检测乳腺癌组织中及细胞中mTOR蛋白水平，分析乳腺癌组织中miR-99a与mTOR表达的相关性。流式细胞术检测细胞周期。膜联蛋白V-异硫氰酸荧光素（AnnexinV—FITC）／碘化丙锭（PI）测定细胞凋亡水平。Transwell实验检测细胞迁移水平。结果乳腺癌组织中miR-99a的相对表达量为0．31±0．08显著低于癌旁正常组织（P〈0．05）。mTOR蛋白在正常组织相对表达量为0．51±0．07，癌组织中其相对表达量为1．03±0．11，mTOR蛋白在乳腺癌组织中表达量显著升高（P〈0．05）。乳腺癌组织中miR-99a的表达量与roTOR蛋白表达呈负相关。与正常对照组比较，miR-99a模拟物转染组乳腺癌细胞中mTOR蛋白表达水平显著下降，mRNA相对表达量为0．42±0．06显著低于正常对照组（P〈0．01）。miR．99a模拟物转染组乳腺癌细胞处于G0／G1比率为（20．05±1．23）％显著高于正常对照组的（2．854-0．76）％（P〈0．01）。miR-99a模拟物转染组乳腺癌细胞凋亡率为（24．66±2．14）％显著高于正常对照组[（4．81±3．37）％，P〈0．01h且细胞迁移数为（27±4）个显著低于正常对照组细胞的（124±18）个（P〈0．01）。结论miR-99a可通过靶向作用于mTOR蛋白以抑制乳腺癌细胞的生长和迁移，其机制与调节细胞周期、促进细胞凋亡、抑制细胞迁移有关。

Objective Study the targets of microRNA （miRNA, miR） - 99a on mammalian target of rapamycin （roTOR） to explore its effect on the growth and migration of breast cancer cell. Methods Build the model of miR -99a transfection breast cancer cells, miR -99a and mTOR cDNA transfection breast cancer cells model, real - time quantitative polymerase chain reaction （ Real - time PCR） to detect the mTOR mRNA expression, and Western blotting to detect the difference of protein expression level, flow cytometry to detect the cell cycle, Annexin V - fluoresceine isothiocyanate （FITC）/propidium iodide （PI） to detect cell apoptosis, Transwell experiments to detect cell migration. Results In breast cancer ceils, relative expression level of miR -99a is 0. 31 ±0. 08, which was significantly decreased than miR -99a in normal cells. After miR - 99a mimic transfection, relative expression of mTOR mRNA level 0. 42 ± 0.06 which was significantly decreased than that in normal breast cells, and the protein level of roTOR was sig- nificantly contrast to that in adjacent normal cells （P 〈 0. 01 ）. The G0/G1 ratio was （20. 05 ± 1.23） % in miR -99a mimic treated breast cancer cells and apoptosis rate was （24. 66 ± 2. 14）% significantly higher than that of （2. 85 ± 0. 76） % and （4. 81 ± 3.37 ） % in normal cells, respectively （ P 〈 0. 01 ）, whereas the migration number of miR -99 - treated breast cells was 27 ± 4 significantly lesser than that of 124 ± 18 in normal cells （P 〈 0. 01 ）. Conclusion miR -99 a can be targeted on the mTOR protein to inhibit the growth of breast cancer ceils and migration and its mechanism were associated with regulating the cell cycle, promoting apoptosis, inhibition of cell migration.