miR-214-3p对人卵巢癌细胞顺铂耐药及EGR1表达的影响

Effect of miR-214-3p on cisplatin resistant of human ovarian cancer cells and EGR1 expression

目的探讨微小RNA-214-3p(miR-214-3p)与人卵巢癌细胞顺铂耐药的关系,并分析miR-214-3p对早期生长反应因子1(EGR1)表达影响。方法脂质体方法行瞬时转染,CCK-8法检测细胞增殖能力和顺铂半数抑制浓度(IC50);流式细胞术检测细胞凋亡率;实时定量PCR(qRT-PCR)分析miR-214-3p及EGR1表达;蛋白质印迹法检测细胞EGR1及着色性干皮病基因(XPD)蛋白表达。结果人卵巢癌顺铂敏感细胞OV2008中miR-214-3p表达高于人卵巢癌顺铂耐药细胞C13K(P<0.001)。转染48 h,miR-214-3p在OV2008 inhibitor组细胞(0.11±0.09)、C13K mimics组细胞(22.42±4.27),与OV2008空白对照组细胞(7.92±2.22)及C13K空白对照组细胞(0.33±0.16)比较,差异有统计学意义(P<0.001);C13K mimics组细胞增殖减慢,细胞凋亡上升,细胞对顺铂敏感性增加,OV2008 inhibitor组细胞增殖加快,细胞凋亡下降,对顺铂敏感性降低(P<0.01);miR-214-3p上调后EGR1在mRNA及蛋白水平表达升高,XPD蛋白降低;miR-214-3p下调使EGR1在mRNA及蛋白水平表达降低,XPD蛋白升高。结论miR-214-3p与卵巢癌细胞顺铂耐药有关,EGR1蛋白可能是miR-214-3p的下游靶点。

Objective To investigate the influence on cisplatin resistant of ovarian cancer regulating the expression of miR-214-3 p and analyze the effects of miR-214-3 p on early growth response protein 1(EGR1). Methods Lipofection transfection technology was used for transient inhibition or increasing of miR-214-3 p expression. Cell viability and the half concentration(IC50) of DDP were determined by CCK-8 method. Cell apoptosis was detected by flow cytometry. The mRNA expressions of miR-214-3 p and EGR1, the protein levels of EGR1 and xeroderma pigmentosum(XPD) were measured by using Real-time quantitantive PCR(qRT-PCR) and Western blot, respectively. Results miR-214-3 p in C13 K cells was down-regulated than those in OV2008 cells(P<0.001). After 48 h of transfection, qRT-PCR results revealed that the expression of miR-214-3 p was higher in mimics groups C13 K cells(22.42±4.27) compared to the blank control C13 K cells(0.33±0.16)(P<0.001). The expression of miR-214-3 p in inhibitor groups OV2008 cells(0.11±0.09) was lower than that of blank control OV2008 cells(7.92±2.22)(P<0.001). The decreasing cell proliferation and promoted apoptosis in C13 K cells after transfected with miR-214-3 p mimics was detected(P<0.01), while the increasing of cell proliferation and decreasing of apoptosis in OV2008 cells after transfected with miR-214-3 p inhibitor was detected in the same time(P<0.01). The IC50 of DDP in mimics group C13 K cells was decreased, while was promoted in inhibitor group OV2008 cells after transfected(P<0.01). With increased miR-214-3 p expression by mimics transfected, the mRNA and protein expressions of EGR1 greatly increased, and the protein levels of XPD decreased in C13 K cells. By down-regulation of miR-214-3 p, the mRNA and protein expressions of EGR1 greatly decreased, and the protein levels of XPD increased in OV2008 cells. Conclusion miR-214-3 p plays the role of ovarian cancer cisplatin resistant. EGR1 protein is a possible downstream target of miR-214-3 p in ovarian cancer cells.