反义miR-21抑制宫颈鳞状细胞癌CaSki细胞系生长体外研究

目的探讨敲低miR-21表达对宫颈鳞状细胞癌细胞系CaSki增殖活性与凋亡的影响。方法实时荧光定量RT-PCR法检测正常宫颈组织、CaSki细胞转染前后miR-21的表达水平,采用MTT法、流式细胞术等技术检测转染后CaSki细胞增殖活性与凋亡的变化。结果 miR-21在CaSki细胞中表达水平为正常宫颈组织的3.25倍。CaSki细胞中转染反义miR-21(AS-miR-21)可显著抑制其表达。转染后24h、48h、72h、96h,MTT检测提示10nmol与20nmolAS-miR-21转染组中细胞增殖率较对照组明显降低(P<0.05),自48h抑制作用显现,且随着转染浓度增加,抑制作用增强,呈现剂量依赖性。细胞周期检测结果显示AS-miR-21转染组的G0/G1期细胞明显增多,S期细胞减少(P<0.05)。AnnexinⅤ联合PI技术检测细胞凋亡,结果显示AS-miR-21转染组早期凋亡细胞率(23.40±2.16)%]明显高于空白对照组(5.1±2.16)%]、阴性对照组(6.87±0.55)%](P=0.000)。结论 AS-miR-21转染可敲低CaSki细胞中miR-21表达,并有效抑制细胞增殖活性,促进细胞凋亡。

AS-miR-21 inhibit the growth of cervical squamous carcinoma cell line CaSki in vitro

Objective To investigate the effects of down-regulated expression of microRNA-21（miR-21）on cell proliferation and apoptosis of cervical carcinoma cell lines CasKi. Methods Synthetic miR-21 antisense oligonucleotides （AS-miR-21）was transfected into CasKi cell line by lipofectamine. The expression change of miR-21 in the CasKi cell line was measured by real time RT-PCR. Cellular growth and proliferation capacity were assayed by the methyl thiazolyl tetrazolium（MTT） assay and flow cytometry（FCM）. The apoptosis rate were tested by Annexin-V and PI assay. Results The expression of miR-21 in CasKi cell was 3.25 times up-regulation compared to the normal cervical tissue. Compared to that of blank control group, the expression of miR-21 in AS-miR-21 group decreased significantly. The cell growth inhibition of AS-miR-21 was demonstrated for 48 hours after cells treated with transfection by MTY results. FCM analysis showed that cells at GO/G1 phase in the AS-miR-21 group were significantly increased, while cells at S phase were decreased （P 〈 0. 05 ） ;the apoptosis rate in the AS-miR-21 group （9.45 ±2. 31 ）% ] was higher than the negative control group （4.03 ± 1.35 ）% ] and the blank control group （3.85 ±1.61）%] （P〈 0.05）. Conclusions Up-regulated expression of miR-21 can not only inhibit the proliferation but also induce the apoptosis capacity of CasKi cell.