岳晓雪 苗劲蔚 路攀

[摘要] 目的 探討miR21影响宫颈癌细胞株Hela及顺铂耐药细胞株Hela/DDP顺铂敏感性的分子机制。 方法 利用riboFECTTM CP转染试剂分别将成熟miR21 mimic及其阴性对照试剂NC转染至Hela细胞，miR21 inhibitor及其阴性对照试剂NC转染至Hela/DDP细胞，并将细胞分为mimic组、inhibitor组、相应NC组及Blank组。Real-time PCR检测PTEN mRNA的表达水平；流式细胞仪检测细胞周期（PI法）及经顺铂处理后的凋亡率（AnnexinⅤ/PI法）。 结果 Real-time PCR法检测PTEN mRNA在Hela/DDP中低表达，为Hela的（0.410±0.046）倍（P < 0.01）；转染mimic后，Hela中PTEN mRNA表达明显低于NC组及Blank组（P < 0.01），NC组与Blank组比较差异无统计学意义（P > 0.05）；转染inhibitor后，Hela/DDP中miR21表达明显高于NC组及Blank组（P < 0.01），NC组与Blank组比较差异无统计学意义（P > 0.05）。AnnexinⅤ/PI检测结果显示，mimic组凋亡率与NC组、Blank组比较明显减少（P < 0.05），inhibitor组凋亡率与NC组、Blank组比较显著提高（P < 0.05）。PI法检测结果显示，mimic组S期所占比例与NC组、Blank组比较明显增加（P < 0.05），NC组与Blank组比较差异无统计学意义（P > 0.05）；inhibitor组S期所占比例与NC组、Blank组比较明显减少（P < 0.05），NC组与Blank组比较差异无统计学意义（P > 0.05）。 结论 PTEN mRNA在Hela/DDP中低表达，在Hela中高表达。上调miR21在Hela中的表达能明显降低PTEN mRNA的表达，减少凋亡，增加细胞周期中S期所占比例，导致细胞耐药；下调miR21在Hela/DDP中的表达能明显增加PTEN mRNA的表达，增加凋亡率，降低细胞周期中S期所占比例，从而达到增加化疗敏感性的效果。

[关键词] 宫颈癌细胞；宫颈癌顺铂耐药细胞；miR21；PTEN；顺铂；化疗敏感性

[中图分类号] R737.33 [文献标识码] A [文章编号] 1673-7210（2018）02（c）-0013-06

Study on the mechanism of Cisplatin chemosensitivity in cervical cancer cells based on miR21

YUE Xiaoxue MIAO Jinwei LU Pan

Department of Gynecological Oncology， Beijing Obstetrics and Gynecology Hospital， Capital Medical University， Beijing 100006， China

[Abstract] Objective To detect the mechanism of miR21 gene on the sensitivity of Hela and Cisplatin-resistant Hela/DDP cells to Cisplatin. Methods Mature miR21 mimic and negative control （NC） miRNA were transfected into Hela cells， while miR21 inhibitor and negative control （NC） miRNA were transfected into Hela/DDP cells by riboFECTTM CP. Therefore， Hela cells were divided into mimic group， inhibitor group， NC group and blank group. Real-time PCR was used to measure the expression of PTEN mRNA in each group. The cell cycle was measured through PI method and apoptosis rate of cells after Cisplatin treatment was detected through AnnexinⅤ/PI by fluorescene activated cell sorter. Results Real-time PCR results showed that the expression of PTEN mRNA was an average of （0.410±0.046） fold higher in Hela/DDP than in Hela （P < 0.01）. The expression of PTEN mRNA in mimic group was obviously lower than those in NC group and blank group （P < 0.01）. The expression of PTEN mRNA in inhibitor group was significantly higher than those in NC group and blank group （P < 0.01）. There was no statistical difference between NC group and blank group （P > 0.05） in Hela and Hela/DDP cells. The results of Annexin Ⅴ/PI showed that the apoptosis rate of mimic group was lower than NC group and blank group （P < 0.05）， while the inhibitor group showed that the apoptosis rate was more than NC group and blank group （P < 0.05）. The of cell proportion of S period in mimic group was higher than those of NC group and blank group （P < 0.05）， and there was no significant difference between NC group and blank group （P > 0.05）. In the meantime， the cell proportion of S period in inhibitor group was less than those of NC group and blank group （P < 0.05）， and there was no significant difference between NC group and blank group （P > 0.05）. Conclusion PTEN mRNA is low expressed in Hela/DDP cells and highly expressed in Hela cells. The up-regulated expression of miR21 in Hela can significantly reduce the expression of PTEN mRNA， reduce apoptosis and increase the proportion of S phase in the cell cycle， thereby resulting in Cisplatin resistance. The down-regulated expression of miR21 in Hela/DDP can increase the expression of PTEN mRNA， increase the apoptosis rate and reduce the proportion of S phase in the cell cycle， so as to increase the chemosensitivity of Cisplatin.

[Key words] Hela； Hela/DDP； miR21； PTEN； Cisplatin； Cheosensitivity

宫颈癌在我国女性恶性肿瘤中发病率仅次于乳腺癌，严重威胁妇女健康[1]。化疗是许多恶性肿瘤的主要治疗手段，但在宫颈癌中目前仅作为术前新辅助治疗或晚期复发的姑息性治疗。化疗在宫颈癌应用中受到限制的主要原因是化疗耐药[2]。因此阐明宫颈癌化疗耐药的分子机制，逆转化疗耐药对中晚期宫颈癌患者预后意义重大。微小RNA（microRNAs）的发现为攻克肿瘤化疗难题开启了一条新的途径。microRNAs是一类单链非编码的微小RNA，长度为21～23个核苷酸，通过调控不同的靶点在肿瘤细胞对化疗药物敏感性方面发挥重要作用[3-4]。……