靶向Skp2的短发夹RNA质粒构建及其对Tca8113细胞抑制作用的观察

[Construction of targeting-Skp2 shRNA plasmids and observation of their inhibitory effect on Tca8113 cells].

作者信息

Fang Liang, Hu Qin-gang, Hua Zi-chun, Li Shu-feng

机构信息

Department of Oral and Maxillofacial Surgery, Nanjing Stomatological Hospital, The Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China.

出版信息

Zhonghua Kou Qiang Yi Xue Za Zhi. 2007 Oct;42(10):624-8.

PMID:18215375

Abstract

OBJECTIVE

To construct the recombinant plasmids expressing Skp2 short hairpin RNA (shRNA) by pRNAT-U6.1/Neo plasmid vector and observe the effects of RNAi-mediated Skp2 gene silencing on Tca8113 cells.

METHODS

Five recombinant eukaryotic expression vectors were successfully constructed using pRNAT-U6.1/Neo plasmid vector separately. After they were transfected into Tca8113 cells with PEI, the interference effects no Skp2 and p27 were detected by RT-PCR and Western blot. The cell cycle of Tca8113 cells were tested by flow cytometry. The proliferation of Tca8113 cells were examined by MTT.

RESULTS

In Skp2shRNA-2 and Skp2shRNA-3 vectors, the expression of Skp2 protein of Tca8113 cells was down-regulated and p27 protein up-regulated (P < 0.01). The cell number during G1/G 0 phases increased 22% (P < 0.01) and during G(2)/M and S phases the number decreased 10% and 12% (P < 0.01). The proliferation of Tca8113 cells slowed down and the cells number decreased (P < 0.01).

CONCLUSIONS

Skp2shRNA-2 and Skp2shRNA-3 vectors of shRNA for Skp2 were successfully constructed. They could influence expression of Skp2 and p27 gene. Skp2 may be a promising target of gene therapy on human tongue squamous cell carcinoma.

摘要

目的

利用pRNAT-U6.1/Neo质粒载体构建表达Skp2短发夹RNA（shRNA）的重组质粒，并观察RNA干扰介导的Skp2基因沉默对Tca8113细胞的影响。

方法

分别利用pRNAT-U6.1/Neo质粒载体成功构建5种重组真核表达载体。用聚乙烯亚胺将其转染至Tca8113细胞后，采用RT-PCR和蛋白质免疫印迹法检测Skp2和p27的干扰效果。采用流式细胞术检测Tca8113细胞的细胞周期。采用MTT法检测Tca8113细胞的增殖情况。

结果

在Skp2shRNA-2和Skp2shRNA-3载体中，Tca8113细胞Skp2蛋白表达下调，p27蛋白表达上调（P<0.01）。G1/G0期细胞数量增加22%（P<0.01），G2/M期和S期细胞数量分别减少10%和12%（P<0.01）。Tca8113细胞增殖减缓，细胞数量减少（P<0.01）。