microRNA-21调节宫颈鳞癌SiHa细胞中PDCD4的表达研究

Programmed cell death 4(PDCD4) is an important functional target of microRNA-21 in cervical cancer cell

目的探讨microRNA(miR)-21-反义寡核苷酸(ASODN)对宫颈鳞癌SiHa细胞中PDCD4基因表达、细胞增殖与凋亡的影响。方法宫颈鳞癌SiHa细胞分3组,采用LipofectamineTM2000转染试剂,miR-21调控组转染miR-21抑制剂的ASODN,miR-21抑制剂阴性对照的ASODN,空白对照组不转染。Cell Titer和荧光TUNEL分别检测转染前后SiHa细胞增殖和凋亡的变化,流式细胞仪分析细胞周期,实时RT-PCR检测miR-21和PDCD4mRNA水平表达,Western blot检测靶蛋白PDCD4表达。结果 AS-miR-21组与阴性、空白对照组相比,SiHa细胞生长明显抑制,而阴性和空白对照组比较无明显差异;细胞凋亡率分别为(9.7±0.52)%、(3.6±0.17)%和(3.4±0.36)%;细胞周期结果显示AS-miR-21组G0/G1期细胞明显增多,S期细胞减少,与对照组间差异显著(P<0.05);实时RT-PCR结果显示,miR-21表达下降而PDCD4表达升高。Western blot检测结果显示,PDCD4表达AS-miR-21组明显高于空白对照组和阴性对照组(P<0.05)。结论 AS-miR-21可有效抑制miR-21在宫颈鳞癌SiHa细胞中的表达并上调PDCD4基因表达,发挥抑制细胞增殖和诱导细胞凋亡的作用。

Objective To study the effect of microRNA-21-ASODN on PDCD4 expression in vitro in cultured SiHa cells.Methods The cellular growth activity was assayed by CellTiter,and the apoptosis was tested by fluorometric TUNEL.The miR-21 and PDCD4 expression was detected by real-time PCR.Fluorescence activated cell sorting（FACS）was used to analyze the cell cycle.The protein expression of PDCD4 was measured by Western blot analysis.Results The miR-21 expression in miR-21 modulated cells discreased.The cellular growth ratio,apoptosis ratio had significant difference between miR-21 modulated group and two control groups（P0.05）.FCM analysis showed that cells at G0 /G1 phase in the AS-miR-21 group were significantly increased,while cells at S phase were decreased（P 0.05）;the PDCD4 protein expression of miR-21 modulated group was 1.31±0.12,blank control group was 0.68±0.04 and negative control group was 0.64±0.04.Conclusions The miR-21-ASODN up-regulates the expression of PDCD4 and could suppress the cellular proliferation and induce apoptosis in SiHa cervical squamous cell cancer cells.