微小RNA-99a对食管癌细胞生物学行为和顺铂敏感性的影响

Effects of microRNA-99a on biological behavior and cisplatin sensitivity of esophageal cancer cells

目的探讨微小RNA-99a(miR-99a)对食管癌细胞生物学行为和顺铂敏感性的影响。方法实时定量PCR(qPCR)检测食管癌细胞EC109及其顺铂(CDDP)耐药细胞株EC109/CDDP的miR-99a水平。实验分为两部分:第一部分的EC109/CDDP细胞经脂质体转染miR-99a模拟物(mimics)或阴性对照(NC),经不同浓度CDDP(0~30 mg/L)处理后用CCK-8法测定细胞活力以计算半数抑制浓度(IC50)。第二部分EC109/CDDP细胞根据处理情况分为4组:空白对照组(仅脂质体处理)、CDDP组(脂质体联合CDDP处理)、NC组(脂质体转染NC联合CDDP处理)和过表达组(脂质体转染mimics联合CDDP处理),CCK-8法、流式细胞术、Transwell小室实验和划痕实验评估细胞增殖、凋亡、侵袭和迁移能力,qPCR与Western blotting检测Bcl-2、caspase-3和p-Akt的表达情况。结果EC109/CDDP细胞的miR-99a水平较EC109细胞下调(0.408±0.051 vs.1.023±0.074,P<0.01)。第一部分中转染mimics的EC109/CDDP细胞较转染NC的增殖抑制率升高(P<0.05),且CDDP的IC50由未转染细胞的(18.972±1.653)mg/L和转染NC细胞的(19.294±1.810)mg/L降至转染mimics细胞的(11.062±1.417)mg/L(P<0.05)。第二部分中,与其余3组相比,过表达组转染后的细胞活力降低,凋亡率升高且划痕愈合率和穿膜细胞数减少,差异有统计学意义(P<0.05)。过表达组的caspase-3水平升高,而Bcl-2和p-Akt水平降低,且与其余3组的差异均有统计学意义(P<0.05)。结论miR-99a可能通过调控Akt信号通路活性,抑制EC109/CDDP细胞的增殖、侵袭和迁移能力并诱导细胞凋亡,进而提高耐药细胞对CDDP的敏感性,该因子可能为食管癌的化疗耐药提供了一种新解释。

Objective To investigate the effects of microRNA-99a(miR-99a)on biological behavior and cisplatin sensitivity of esophageal cancer cells.Methods Real-time quantitative PCR(qPCR)was used to detect the miR-99a level in esophageal cancer cell EC109 and its cisplatin(CDDP)resistant cell line EC109/CDDP.The experiment was divided into two parts.In the first part,EC109/CDDP cells were transfected with miR-99a mimics or negative control(NC)mediated by liposome.After exposure to different concentrations of CDDP(0-30 mg/L),cell viability was determined by CCK-8 assay to calculate the half inhibitory concentration(IC50).In the second part,EC109/CDDP cells were divided into four groups:blank control group(liposome only),CDDP group(liposome plus CDDP treatment),NC group(NC plus CDDP treatment)and overexpression group(mimics plus CDDP treatment).Cell proliferation,apoptosis,invasion and migration were evaluated by CCK-8 assay,flow cytometry,Transwell chamber test and scratch test.The expressions of Bcl-2,caspase-3 and p-Akt were detected by qPCR and Western blotting.Results MiR-99a level was down regulated in EC109/CDDP cells as compared with EC109 cells(0.408±0.051 vs.1.023±0.074,P<0.01).The first part showed that the proliferation inhibition rate of EC109/CDDP cells transfected with mimics was higher than those transfected with NC(P<0.05),and the IC50 of CDDP decreased from(18.972±1.653)mg/L of untransfected cells and(19.294±1.810)mg/L of cells transfected with NC to(11.062±1.417)mg/L of cells transfected with mimics(P<0.05).In the second part,compared with other three groups,the cell viability was decreased in the overexpression group together with increased apoptotic rate and decreased wound healing rate and number of cells penetrating the membrane(P<0.05).The elevated level of caspase-3 and decreased levels of Bcl-2 and p-Akt in overexpression group were observed rather than those in other three groups(P<0.05).Conclusion MiR-99a may inhibit the proliferation,invasion and migration of EC109/CDDP cells and induce apoptosis by regulating the activity of Akt signaling pathway,thus increasing the sensitivity of EC109/CDDP cells to CDDP.MiR-99a may provide a new explanation for chemotherapy resistance of esophageal cancer.