CAJ | 학술논문

目的：探讨miR?490?3p在阿霉素（ ADM）耐药乳腺癌细胞系MCF?7／ADM中的表达情况及其对MCF?7／ADM细胞增殖、凋亡和ADM耐药的影响。方法以野生型人乳腺癌细胞系MCF?7及其耐药型细胞系MCF?7／ADM为研究对象，用实时荧光定量PCR（qPCR）比较两种细胞中miR?490?3p的表达水平，将miR?490?3p的过表达载体pcDNA3?1（＋）?miR?490?3p瞬时转染至MCF?7／ADM细胞（过表达组）后采用qPCR检测转染效果，同时设pcDNA3?1（－）空载体对照组和空白对照组；采用MTT法测定转染pcDNA3?1（＋）?miR?490?3p对各组MCF?7／ADM细胞增殖能力的影响，测定ADM对MCF?7／ADM细胞的增殖抑制率并计算半数抑制浓度（ IC50）及逆转倍数以评价对ADM敏感性的变化；分别于瞬时转染48 h后用Annexin V／FITC流式细胞术检测各组 MCF?7／ADM 细胞的凋亡率， Western blotting 检测耐药蛋白 P?糖蛋白（ P?gp ），乳腺癌耐药蛋白（BCRP）和多药耐药相关蛋白1（MRP1）的表达，荧光分光光度计检测胞内ADM药物浓度，流式细胞仪检测P?gp活性。结果MCF?7／ADM细胞中miR?490?3p的表达水平为0?24±0?07，显著低于野生型MCF?7细胞的1?02±0?03（P＜0?05）；过表达组瞬时转染24～96 h的miR?490?3p水平持续升高，均高于空载体对照组和空白对照组（ P＜0?05）；过表达组的增殖抑制率随转染时间的延长而增加，转染24、48、72和96 h的增殖抑制率依次为（17?52±1?87）％、（31?67±2?79）％、（45?09±1?88）％和（61?82±2?52）％，高于空载体对照组（P＜0?05）；ADM对过表达组的IC50值为（11?27±2?34）μg／ml，低于空白对照组的和空载体对照组（ P＜0?05），且过表达组相对于空白对照组和空载体对照组的逆转倍数分别为3?35倍和3?39倍；与其余两组相比，过表达组的MCF?7／ADM细胞早、晚期凋亡率和细胞内药物浓度均升高，P?gp、BCRP和MRP1表达及P?gp活性均降低，差异均有统计学意义（ P＜0?05）。结论上调miR?490?3p可逆转MCF?7／ADM细胞对ADM的耐药性，可能通过降低耐药蛋白表达及抑制P?gp活性有关，且升高其水平可抑制细胞增殖并诱导凋亡。目的：探討miR?490?3p在阿黴素（ ADM）耐藥乳腺癌細胞繫MCF?7／ADM中的錶達情況及其對MCF?7／ADM細胞增殖、凋亡和ADM耐藥的影響。方法以野生型人乳腺癌細胞繫MCF?7及其耐藥型細胞繫MCF?7／ADM為研究對象，用實時熒光定量PCR（qPCR）比較兩種細胞中miR?490?3p的錶達水平，將miR?490?3p的過錶達載體pcDNA3?1（＋）?miR?490?3p瞬時轉染至MCF?7／ADM細胞（過錶達組）後採用qPCR檢測轉染效果，同時設pcDNA3?1（－）空載體對照組和空白對照組；採用MTT法測定轉染pcDNA3?1（＋）?miR?490?3p對各組MCF?7／ADM細胞增殖能力的影響，測定ADM對MCF?7／ADM細胞的增殖抑製率併計算半數抑製濃度（ IC50）及逆轉倍數以評價對ADM敏感性的變化；分彆于瞬時轉染48 h後用Annexin V／FITC流式細胞術檢測各組 MCF?7／ADM 細胞的凋亡率， Western blotting 檢測耐藥蛋白 P?糖蛋白（ P?gp ），乳腺癌耐藥蛋白（BCRP）和多藥耐藥相關蛋白1（MRP1）的錶達，熒光分光光度計檢測胞內ADM藥物濃度，流式細胞儀檢測P?gp活性。結果MCF?7／ADM細胞中miR?490?3p的錶達水平為0?24±0?07，顯著低于野生型MCF?7細胞的1?02±0?03（P＜0?05）；過錶達組瞬時轉染24～96 h的miR?490?3p水平持續升高，均高于空載體對照組和空白對照組（ P＜0?05）；過錶達組的增殖抑製率隨轉染時間的延長而增加，轉染24、48、72和96 h的增殖抑製率依次為（17?52±1?87）％、（31?67±2?79）％、（45?09±1?88）％和（61?82±2?52）％，高于空載體對照組（P＜0?05）；ADM對過錶達組的IC50值為（11?27±2?34）μg／ml，低于空白對照組的和空載體對照組（ P＜0?05），且過錶達組相對于空白對照組和空載體對照組的逆轉倍數分彆為3?35倍和3?39倍；與其餘兩組相比，過錶達組的MCF?7／ADM細胞早、晚期凋亡率和細胞內藥物濃度均升高，P?gp、BCRP和MRP1錶達及P?gp活性均降低，差異均有統計學意義（ P＜0?05）。結論上調miR?490?3p可逆轉MCF?7／ADM細胞對ADM的耐藥性，可能通過降低耐藥蛋白錶達及抑製P?gp活性有關，且升高其水平可抑製細胞增殖併誘導凋亡。
목적：탐토miR?490?3p재아매소（ ADM）내약유선암세포계MCF?7／ADM중적표체정황급기대MCF?7／ADM세포증식、조망화ADM내약적영향。방법이야생형인유선암세포계MCF?7급기내약형세포계MCF?7／ADM위연구대상，용실시형광정량PCR（qPCR）비교량충세포중miR?490?3p적표체수평，장miR?490?3p적과표체재체pcDNA3?1（＋）?miR?490?3p순시전염지MCF?7／ADM세포（과표체조）후채용qPCR검측전염효과，동시설pcDNA3?1（－）공재체대조조화공백대조조；채용MTT법측정전염pcDNA3?1（＋）?miR?490?3p대각조MCF?7／ADM세포증식능력적영향，측정ADM대MCF?7／ADM세포적증식억제솔병계산반수억제농도（ IC50）급역전배수이평개대ADM민감성적변화；분별우순시전염48 h후용Annexin V／FITC류식세포술검측각조 MCF?7／ADM 세포적조망솔， Western blotting 검측내약단백 P?당단백（ P?gp ），유선암내약단백（BCRP）화다약내약상관단백1（MRP1）적표체，형광분광광도계검측포내ADM약물농도，류식세포의검측P?gp활성。결과MCF?7／ADM세포중miR?490?3p적표체수평위0?24±0?07，현저저우야생형MCF?7세포적1?02±0?03（P＜0?05）；과표체조순시전염24～96 h적miR?490?3p수평지속승고，균고우공재체대조조화공백대조조（ P＜0?05）；과표체조적증식억제솔수전염시간적연장이증가，전염24、48、72화96 h적증식억제솔의차위（17?52±1?87）％、（31?67±2?79）％、（45?09±1?88）％화（61?82±2?52）％，고우공재체대조조（P＜0?05）；ADM대과표체조적IC50치위（11?27±2?34）μg／ml，저우공백대조조적화공재체대조조（ P＜0?05），차과표체조상대우공백대조조화공재체대조조적역전배수분별위3?35배화3?39배；여기여량조상비，과표체조적MCF?7／ADM세포조、만기조망솔화세포내약물농도균승고，P?gp、BCRP화MRP1표체급P?gp활성균강저，차이균유통계학의의（ P＜0?05）。결론상조miR?490?3p가역전MCF?7／ADM세포대ADM적내약성，가능통과강저내약단백표체급억제P?gp활성유관，차승고기수평가억제세포증식병유도조망。
Objective To investigate the expression of miR?490?3p in adriamycin ( ADM) resistant human breast cancer cell line MCF?7/ADM and its effect on the cell proliferation, apoptosis and resistance to ADM of MCF?7/ADM cell line. Methods Cul?tured wild?type MCF?7 human breast cancer cells and its resistant cells MCF?7/ADM were selected as research objectives. The real?time fluorescent quantitative PCR ( qPCR) was used to compare the level of miR?490?3p in both cell lines. The recombinant plasmid pcDNA3?1(+)?miR?490?3p to over?express miR?490?3p was transiently transfected into MCF?7/ADM cells ( over?expression group) and then the transfection effect was verified by qPCR. Meanwhile, the pcDNA3?1 (-) empty control group and blank control group were set up. MTT method was used to determine the effect of pcDNA3?1 (+)?miR?490?3p on the proliferation of each group. The inhib?itory rate of ADM on the proliferation of MCF?7/ADM cells was determined, and the inhibition concentration ( IC50 ) and the reversal factor were calculated to evaluate the sensitivity to ADM. The cells in each group were collected at 48 h post?transfection for the detec?tion of apoptotic rate by flow cytometry with Annexin FITC/PI double staining. Meanwhile, the expression of resistance proteins inclu?ding P?glycoprotein (P?gp), breast cancer resistance protein (BCRP) and multidrug resistance associated protein 1 (MRP1) were tested by Western blotting, intracellular ADM concentration was detected by fluorescence photometer and P?gp activity by flow cytome?try. Results The level of miR?490?3p in MCF?7/ADM cells was 0?24±0?07, significantly lower than 1?02±0?03 of wild type MCF?7 cells ( P<0?05) . The levels of miR?490?3p in the over?expression group were higher than those in the other two groups during 24?96 h post?transfection (P<0?05). The proliferation inhibition rates were (17?52 ±1?87)%, (31?67±2?79)%, (45?09±1?88)% and (61?82±2?52)% at 48, 24, 72 and 96 h post?transfection, all higher than those of empty control group (P<0?05). IC50 value of ADM for MCF?7/ADM cells was (11?27±2?34)μg/ml, lower than that of blank control group and empty control group (P<0?05). Moreover, reversal index was 3?35?and 3?39?fold of blank control group and empty control group for the over?expression group. Com?pared with the other two groups, there were increased apoptotic rates and intracellular drug concentration but decreased expression of P?gp, BCRP and MRP1 as well as P?gp activity in over?expression group ( P<0?05) . Conclusion Upregulation of miR?490?3p exhibits the reversal effect of resistance to ADM in MCF?7/ADM cell, possibly by reducing the resistance gene protein expression and inhibition of P?gp activity. Elevated level of miR?490?3p can inhibit proliferation and induce apoptosis.