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shRNA介导的Rac1基因下调对人宫颈癌细胞生物学行为的影响

Effect of Rac1 downregulation mediated by shRNA on the biological behaviour of human cervical cancer cells.

作者信息

Xu Ai Li, Yu Guo Qiang, Kong Xian Chao, Qiu Xiao Hong, Li Pei Ling

机构信息

Department of Obstetrics and Gynaecology, Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China.

出版信息

J Int Med Res. 2013 Aug;41(4):1037-48. doi: 10.1177/0300060513479875. Epub 2013 Jun 12.

Abstract

OBJECTIVE

The function of Ras-related C3 botulinum toxin substrate1 (Rac1) in the progression of cervical cancer is unclear. This study used RNA interference technology to explore the involvement of Rac1 in the regulation of cervical cancer cells.

METHODS

A short hairpin (sh) RNA plasmid targeting Rac1 was constructed and transfected into HeLa cells. Rac1 mRNA and protein levels were investigated by reverse transcription-polymerase chain reaction and Western blot, respectively. Cell proliferation and cisplatin chemosensitivity were determined using the methyl thiazolyl tetrazolium assay. The Matrigel™ assay and flow cytometry were used to assess cell invasion and apoptosis, respectively. The concentration of matrix metalloproteinase (MMP)-2 in cell supernatants was detected by enzyme-linked immunosorbent assay.

RESULTS

Rac1 expression was significantly downregulated at the mRNA and protein levels in HeLa cells transfected with Rac1 shRNA, and the cell proliferation and invasion capability of cells was decreased. Rac1 downregulation was associated with a decrease in MMP-2 secretion, and increased cell chemosensitivity to cisplatin and cisplatin-induced apoptosis.

CONCLUSIONS

Rac1 may play an important role in cervical cancer progression and could be a potential target for anticancer therapy.

摘要

目的

Ras相关的C3肉毒杆菌毒素底物1(Rac1)在宫颈癌进展中的作用尚不清楚。本研究采用RNA干扰技术探讨Rac1在宫颈癌细胞调控中的作用。

方法

构建靶向Rac1的短发夹(sh)RNA质粒并转染至HeLa细胞。分别通过逆转录-聚合酶链反应和蛋白质免疫印迹法检测Rac1 mRNA和蛋白水平。采用甲基噻唑基四氮唑法测定细胞增殖和顺铂化疗敏感性。分别用基质胶实验和流式细胞术评估细胞侵袭和凋亡。采用酶联免疫吸附测定法检测细胞上清液中基质金属蛋白酶(MMP)-2的浓度。

结果

用Rac1 shRNA转染的HeLa细胞中,Rac1表达在mRNA和蛋白水平均显著下调,细胞增殖和侵袭能力降低。Rac1下调与MMP-2分泌减少有关,并增加了细胞对顺铂的化疗敏感性和顺铂诱导的凋亡。

结论

Rac1可能在宫颈癌进展中起重要作用,可能成为抗癌治疗的潜在靶点。

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