中国癌症杂志
中國癌癥雜誌
중국암증잡지
CHINA ONCOLOGY
2015年
3期
190-198
,共9页
李智敏%罗喜平%曾俐琴%彭秀红%王意%王泽华
李智敏%囉喜平%曾俐琴%彭秀紅%王意%王澤華
리지민%라희평%증리금%팽수홍%왕의%왕택화
卵巢癌%乳腺癌%紫杉醇%阿霉素%微小RNA%耐药
卵巢癌%乳腺癌%紫杉醇%阿黴素%微小RNA%耐藥
란소암%유선암%자삼순%아매소%미소RNA%내약
Ovarian cancer%Breast cancer%Paclitaxel%Doxorubicin%MicroRNA%Drug resistance
背景与目的：肿瘤细胞耐药是临床化疗失败的主要原因，微小RNA(microRNA，miRNA)在肿瘤细胞中的异常表达与耐药关系密切。本研究旨在探讨卵巢癌及乳腺癌细胞中hsa-miRNA27a和hsa-miRNA451的表达差异及其与耐药的关系。方法：用浓度递增法建立卵巢癌耐紫杉醇细胞系A2780/Taxol；颈环状引物实时定量聚合酶链反应(stem-loop quantitative real-time PCR，stem-loop RT-PCR)检测卵巢癌耐紫杉醇细胞A2780/Taxol和亲本细胞A2780以及乳腺癌耐阿霉素细胞MCF-7/ADM和亲本细胞MCF-7中hsa-miRNA27a和hsa-miRNA451的表达；利用LipofectamineTM 2000分别将成熟miRNA27a的模拟物、阻遏物及阴性对照(negative control，NC)RNA转染A2780和A2780/Taxol细胞，将成熟miRNA451的模拟物及NC转染MCF-7/ADM细胞；RT-PCR技术检测细胞MDR1 mRNA表达；蛋白[质]印迹法(Western blot)检测细胞中P-糖蛋白(P-glycoprotein，P-gp)的表达；采用四甲基偶氮唑蓝(MTT)法检测细胞增殖情况。结果：miRNA27a在A2780/Taxol细胞中高表达，与A2780细胞相比，表达增高2.2±0.30倍，差异有统计学意义(P<0.05)；miRNA451在MCF-7/ADM细胞中低表达，与MCF-7细胞相比，表达降低84%，差异有统计学意义(P<0.05)。A2780/Taxol细胞转染miRNA27a阻遏物后，MDR1 mRNA表达明显下降，与转染NC组相比，表达下降(39±0.14)%，差异有统计学意义(P<0.05)。P-gp相对表达量[(26±5.3)%)]与转染NC组的P-gp相对表达量[(43±6.7)%]比较，下降39%，差异有统计学意义(P<0.05)。对紫杉醇的敏感性增加，半数抑制浓度(IC50)为0.53μmol/L，与转染NC组IC50(6.8μmol/L)相比，差异有统计学意义(P<0.05)。A2780细胞转染miRNA27a模拟物后，细胞的MDR1 mRNA表达升高，与转染NC组相比，升高(121±0.11)%，差异有统计学意义(P<0.05)；细胞对紫杉醇的敏感性下降，IC50为0.2μmol/L，与转染NC组IC50(0.06μmol/L)相比，差异有统计学意义(P<0.05)。MCF-7/ADM细胞转染miRNA451模拟物后，MDR1 mRNA表达明显下降，与转染NC组细胞相比，表达下降(65±12)%，差异有统计学意义(P<0.05)；P-gp相对表达量[(31±19)%)]与转染NC组细胞P-gp相对表达量[(83±12)%]相比，下降62%，差异有统计学意义(P<0.05)；对阿霉素的敏感性增加，IC50为4.61μmol/L，与转染NC组细胞IC50(26μmol/L)相比，差异有统计学意义(P<0.05)。结论：在卵巢癌耐紫杉醇细胞A2780/Taxol和乳腺癌耐阿霉素细胞MCF-7/ADM中，miRNA27a和miRNA451分别异常表达，它们可能分别通过间接或直接作用于MDR1/P-gp，参与肿瘤细胞耐药的发生、发展。
揹景與目的：腫瘤細胞耐藥是臨床化療失敗的主要原因，微小RNA(microRNA，miRNA)在腫瘤細胞中的異常錶達與耐藥關繫密切。本研究旨在探討卵巢癌及乳腺癌細胞中hsa-miRNA27a和hsa-miRNA451的錶達差異及其與耐藥的關繫。方法：用濃度遞增法建立卵巢癌耐紫杉醇細胞繫A2780/Taxol；頸環狀引物實時定量聚閤酶鏈反應(stem-loop quantitative real-time PCR，stem-loop RT-PCR)檢測卵巢癌耐紫杉醇細胞A2780/Taxol和親本細胞A2780以及乳腺癌耐阿黴素細胞MCF-7/ADM和親本細胞MCF-7中hsa-miRNA27a和hsa-miRNA451的錶達；利用LipofectamineTM 2000分彆將成熟miRNA27a的模擬物、阻遏物及陰性對照(negative control，NC)RNA轉染A2780和A2780/Taxol細胞，將成熟miRNA451的模擬物及NC轉染MCF-7/ADM細胞；RT-PCR技術檢測細胞MDR1 mRNA錶達；蛋白[質]印跡法(Western blot)檢測細胞中P-糖蛋白(P-glycoprotein，P-gp)的錶達；採用四甲基偶氮唑藍(MTT)法檢測細胞增殖情況。結果：miRNA27a在A2780/Taxol細胞中高錶達，與A2780細胞相比，錶達增高2.2±0.30倍，差異有統計學意義(P<0.05)；miRNA451在MCF-7/ADM細胞中低錶達，與MCF-7細胞相比，錶達降低84%，差異有統計學意義(P<0.05)。A2780/Taxol細胞轉染miRNA27a阻遏物後，MDR1 mRNA錶達明顯下降，與轉染NC組相比，錶達下降(39±0.14)%，差異有統計學意義(P<0.05)。P-gp相對錶達量[(26±5.3)%)]與轉染NC組的P-gp相對錶達量[(43±6.7)%]比較，下降39%，差異有統計學意義(P<0.05)。對紫杉醇的敏感性增加，半數抑製濃度(IC50)為0.53μmol/L，與轉染NC組IC50(6.8μmol/L)相比，差異有統計學意義(P<0.05)。A2780細胞轉染miRNA27a模擬物後，細胞的MDR1 mRNA錶達升高，與轉染NC組相比，升高(121±0.11)%，差異有統計學意義(P<0.05)；細胞對紫杉醇的敏感性下降，IC50為0.2μmol/L，與轉染NC組IC50(0.06μmol/L)相比，差異有統計學意義(P<0.05)。MCF-7/ADM細胞轉染miRNA451模擬物後，MDR1 mRNA錶達明顯下降，與轉染NC組細胞相比，錶達下降(65±12)%，差異有統計學意義(P<0.05)；P-gp相對錶達量[(31±19)%)]與轉染NC組細胞P-gp相對錶達量[(83±12)%]相比，下降62%，差異有統計學意義(P<0.05)；對阿黴素的敏感性增加，IC50為4.61μmol/L，與轉染NC組細胞IC50(26μmol/L)相比，差異有統計學意義(P<0.05)。結論：在卵巢癌耐紫杉醇細胞A2780/Taxol和乳腺癌耐阿黴素細胞MCF-7/ADM中，miRNA27a和miRNA451分彆異常錶達，它們可能分彆通過間接或直接作用于MDR1/P-gp，參與腫瘤細胞耐藥的髮生、髮展。
배경여목적：종류세포내약시림상화료실패적주요원인，미소RNA(microRNA，miRNA)재종류세포중적이상표체여내약관계밀절。본연구지재탐토란소암급유선암세포중hsa-miRNA27a화hsa-miRNA451적표체차이급기여내약적관계。방법：용농도체증법건립란소암내자삼순세포계A2780/Taxol；경배상인물실시정량취합매련반응(stem-loop quantitative real-time PCR，stem-loop RT-PCR)검측란소암내자삼순세포A2780/Taxol화친본세포A2780이급유선암내아매소세포MCF-7/ADM화친본세포MCF-7중hsa-miRNA27a화hsa-miRNA451적표체；이용LipofectamineTM 2000분별장성숙miRNA27a적모의물、조알물급음성대조(negative control，NC)RNA전염A2780화A2780/Taxol세포，장성숙miRNA451적모의물급NC전염MCF-7/ADM세포；RT-PCR기술검측세포MDR1 mRNA표체；단백[질]인적법(Western blot)검측세포중P-당단백(P-glycoprotein，P-gp)적표체；채용사갑기우담서람(MTT)법검측세포증식정황。결과：miRNA27a재A2780/Taxol세포중고표체，여A2780세포상비，표체증고2.2±0.30배，차이유통계학의의(P<0.05)；miRNA451재MCF-7/ADM세포중저표체，여MCF-7세포상비，표체강저84%，차이유통계학의의(P<0.05)。A2780/Taxol세포전염miRNA27a조알물후，MDR1 mRNA표체명현하강，여전염NC조상비，표체하강(39±0.14)%，차이유통계학의의(P<0.05)。P-gp상대표체량[(26±5.3)%)]여전염NC조적P-gp상대표체량[(43±6.7)%]비교，하강39%，차이유통계학의의(P<0.05)。대자삼순적민감성증가，반수억제농도(IC50)위0.53μmol/L，여전염NC조IC50(6.8μmol/L)상비，차이유통계학의의(P<0.05)。A2780세포전염miRNA27a모의물후，세포적MDR1 mRNA표체승고，여전염NC조상비，승고(121±0.11)%，차이유통계학의의(P<0.05)；세포대자삼순적민감성하강，IC50위0.2μmol/L，여전염NC조IC50(0.06μmol/L)상비，차이유통계학의의(P<0.05)。MCF-7/ADM세포전염miRNA451모의물후，MDR1 mRNA표체명현하강，여전염NC조세포상비，표체하강(65±12)%，차이유통계학의의(P<0.05)；P-gp상대표체량[(31±19)%)]여전염NC조세포P-gp상대표체량[(83±12)%]상비，하강62%，차이유통계학의의(P<0.05)；대아매소적민감성증가，IC50위4.61μmol/L，여전염NC조세포IC50(26μmol/L)상비，차이유통계학의의(P<0.05)。결론：재란소암내자삼순세포A2780/Taxol화유선암내아매소세포MCF-7/ADM중，miRNA27a화miRNA451분별이상표체，타문가능분별통과간접혹직접작용우MDR1/P-gp，삼여종류세포내약적발생、발전。
Background and purpose: Resistance of cancer cells to chemotherapy is a major clinical obstacle to successful treatment and leads to poor prognosis for the patients. MicroRNA (miRNA) plays a vital role in tumor cells response to chemotherapeutic agents. This study plans to investigate the expressions of miRNA27a and miRNA451 in ovarian cancer and breast cancer cells and its correlation with drug resistance. Methods:A2780/Taxol cells were established using stepwise selection;Stem-loop quantitative real-time PCR (stem-loop RT-PCR) was used to detect expression of miRNA27a and miRNA451 in ovarian cancer and breast cancer cells. The A2780 and A2780/Taxol cells were transfected with the mimics or inhibitors of miRNA27a or negative control (NC) RNA and the mimics of miRNA451 or NC were transfected into MCF-7/ADM cells by LipofectamineTM 2000. The expression levels of MDR1 mRNA and P-glycoprotein (P-gp) were examined using RT-PCR and Western blot respectively. MTT method was used to analyze drug sensitivity. Results:The expression of miRNA27a was an average of (2.2±0.30) times higher in A2780/Taxol cells than in A2780 cells, with a significant difference between the two groups (P<0.05). The expression of miRNA451 was lower by 84%in MCF-7/ADM cells than in MCF-7 cells, with a signiifcant difference between the two groups (P<0.05). A2780/Taxol cells transfection with inhibitors of miRNA27a showed that the levels of MDR1 mRNA was decreased by (39±0.14)%, P-gp level [(26±5.3)%] decreased compared with the NC group [(43±6.7)%], the IC50 (0.53μmol/L) was less than the NC group (6.8μmol/L), and there was a signiifcant difference between two groups (P<0.05). Transfection of A2780 cells with mimics of miRNA27a led to increase MDR1 mRNA expression by (121±0.11)%and decrease the sensitivity to paclitaxel (IC50:0.2μmol/L vs 0.06μmol/L). There was a signiifcant difference between two groups (P<0.05). Transfection of MCF-7/ADM cells with mimics of miRNA451 showed that expression of MDR1 mRNA was decreased by (65±12)%, P-gp [(31±19)%] was less than the NC group [(83±12)%], the sensitivity of cells to adriamycin enhanced and the IC50 of adriamycin (4.61μmol/L) was less than the NC group (26μmol/L), and there was a signiifcant difference between two groups (P<0.05). Conclusion:The expressions of miRNA27a and miRNA451 are deregulated in A2780/Taxol and MCF-7/ADM cells respectively, which may play vital roles in drug resistance by regulating MDR1/P-gp expression directly or indirectly.
