CAJ | 학술논문

目的：探讨抑制miR?23a对EGFR T790M突变非小细胞肺癌H1975细胞吉非替尼耐药性的影响及其可能机制。方法选取吉非替尼耐药的对数生长期H1975细胞为研究样本，合成miR?23a抑制物（ miR?23a inhibitor），应用脂质体转染H1975细胞以抑制其miR?23a表达（抑制组），同时设未经转染处理的非转染组和转染无关序列的阴性对照组，实时荧光定量PCR（ qRT?PCR）验证转染效果；采用CCK?8法检测转染24、48、72及96 h后各组的增殖情况，同时于转染48 h后用吉非替尼（0?03～300μmol／L）处理各组细胞以评价对吉非替尼的药物敏感性变化情况；分别用流式细胞术检测各组的凋亡率（ FITC?Annexin V／PI双染）和细胞周期（PI单染）情况，免疫印迹法检测各组多药耐药相关蛋白1（MRP1）、肺耐药相关蛋白（LRP）及谷胱甘肽转移酶π（ GST?π）的表达变化。结果抑制组在转染24～96 h出现miR?23a水平的持续下降，其miR?23a水平均低于其余两组，转染96 h后的miR?23a水平分别为非转染组的（32?06±4?68）％和阴性对照组的（31?77±3?18）％，且吉非替尼对抑制组的半数抑制浓度（IC50）为（2?82±0?46）μmol／L，均低于其余两组，以上差异均有统计学意义（P＜0?05）；与其余两组相比，抑制组转染后增殖抑制率、凋亡率及G0／G1期比例均升高，S期、G2／M期比例均下降且耐药蛋白MRP1、LRP及GST?π蛋白水平亦下调，以上差异均有统计学意义（ P＜0?05）。结论下调miR?23a可逆转H1975细胞的吉非替尼耐药性，同时可抑制细胞增殖、诱导凋亡及细胞周期阻滞，可能与降低耐药蛋白表达有关。
목적：탐토억제miR?23a대EGFR T790M돌변비소세포폐암H1975세포길비체니내약성적영향급기가능궤제。방법선취길비체니내약적대수생장기H1975세포위연구양본，합성miR?23a억제물（ miR?23a inhibitor），응용지질체전염H1975세포이억제기miR?23a표체（억제조），동시설미경전염처리적비전염조화전염무관서렬적음성대조조，실시형광정량PCR（ qRT?PCR）험증전염효과；채용CCK?8법검측전염24、48、72급96 h후각조적증식정황，동시우전염48 h후용길비체니（0?03～300μmol／L）처리각조세포이평개대길비체니적약물민감성변화정황；분별용류식세포술검측각조적조망솔（ FITC?Annexin V／PI쌍염）화세포주기（PI단염）정황，면역인적법검측각조다약내약상관단백1（MRP1）、폐내약상관단백（LRP）급곡광감태전이매π（ GST?π）적표체변화。결과억제조재전염24～96 h출현miR?23a수평적지속하강，기miR?23a수평균저우기여량조，전염96 h후적miR?23a수평분별위비전염조적（32?06±4?68）％화음성대조조적（31?77±3?18）％，차길비체니대억제조적반수억제농도（IC50）위（2?82±0?46）μmol／L，균저우기여량조，이상차이균유통계학의의（P＜0?05）；여기여량조상비，억제조전염후증식억제솔、조망솔급G0／G1기비례균승고，S기、G2／M기비례균하강차내약단백MRP1、LRP급GST?π단백수평역하조，이상차이균유통계학의의（ P＜0?05）。결론하조miR?23a가역전H1975세포적길비체니내약성，동시가억제세포증식、유도조망급세포주기조체，가능여강저내약단백표체유관。
Objective To explore the effect of miR?23a downregulation on the gefitinib resistance in non?small cell lung cancer cell with the epidermal growth factor receptor ( EGFR) T790M mutation. Methods The gefitinib resistance cell H1975 at logarithmic growth phase was chosen and the miR?23a inhibitor was synthesized. H1975 cells were transfected with liposome to inhibit its miR?23a ex?pression ( inhibition group) , and the non?transfection group without transfection and the negative control ( NC) group treated with the un?related sequence were also prepared. The transfection effect was verified by real time fluorescence quantitative PCR (qRT?PCR). CCK?8 method was used to detect the proliferation at 24, 48, 72 and 96 h, and the drug sensitivity for gefitinib was evaluated after exposure to different doses of gefitinib (0?03~300μmol/L). Flow cytometry was used to detect the apoptosis rate (FITC?Annexin V/PI staining) and cell cycle (PI staining) of each group. Western blotting was used to detect the level of multidrug resistance associated protein 1 (MRP1), lung resistance related protein (LRP) and glutathione S?transferase?π(GST?π). Results The level of miR?23a continued to decline in inhibition group for 24?96 h, all lower than those of other two groups with significant difference ( P<0?05) . The level of miR?23a was (32?06±4?68)% of non?transfection group and (31?77±3?18)% of NC group at 96 h, respectively. The half maximal inhibitor concentration (IC50) value of gefitinib for the inhibition group was (2?82±0?46) μmol/L, all lower than those of other two groups with significant difference ( P<0?05) . Compared with the other two groups, there were increased proliferation inhibition rate, apoptosis rate and the percentage of G0/G1 phase but decreased S?G2/M phase and level of resistance proteins in inhibition group ( P<0?05) . Conclu?sion Downregulation of miR?23a overcomes gefitinib resistance in H1975 cell, and inhibits cell proliferation, induces apoptosis and cell cycle arrest, with the possible mechanism of influencing the expressions of drug resistance proteins.