Naa10基因RNA干扰慢病毒载体的构建及其对口腔鳞癌细胞生长的影响

doi:10.13701/j.cnki.kqyxyj.2017.07.005

口腔医学研究 ›› 2017, Vol. 33 ›› Issue (7): 707-711.DOI: 10.13701/j.cnki.kqyxyj.2017.07.005

Naa10基因RNA干扰慢病毒载体的构建及其对口腔鳞癌细胞生长的影响

杨洋¹, 郑军², 徐江², 顾永清³, 黄瑾¹, 王丹妮¹, 朱茂祥³, 曾妍^1*

1. 新疆石河子大学新疆地方与民族高发病教育部重点实验室/医学院生化教研室新疆石河子 832000;
2. 新疆石河子大学医学院第一附属医院口腔科新疆石河子 832000;
3. 军事医学科学院放射与辐射医学研究所北京 100850

收稿日期:2017-03-22 出版日期:2017-07-20 发布日期:2017-07-27
通讯作者: 曾妍,E-mail:yzeng910@163.com
作者简介:杨洋,女,河南人,硕士在读,研究方向为口腔肿瘤发生机制及临床应用研究。
基金资助:
国家自然科学基金(编号:81560473,81560442,U1603117,31470827)兵团基金资助项目(编号:2014BB021,2015AD003

Construction of Lentiviral Vector of Naa10 Small-interfering RNA and Its Effect on Growth of Oral Squamous Cell Carcinoma.

YANG Yang¹, ZHENG Jun², XU Jiang², GU Yong-qing³, HUANG Jin¹, WANG Dan-ni¹, ZHU Mao-xiang³, ZENG Yan^1*.

1. Key Laboratory of Xinjiang Endemic and Ethnic Disease, School of Medicine, Shihezi University, Shihezi 832000, China;
2. Department of Stomatology, The First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi 832000,China;
3. Department of Radiation Toxicology and Oncology, Beijing Institute of Radiation Medicine, Beijing 100850, China.

Received:2017-03-22 Online:2017-07-20 Published:2017-07-27

摘要/Abstract

摘要： 目的:构建人N-α-乙酰基转移酶10(N-α-acetyltransferase 10, Naa10)基因有效的慢病毒RNAi载体并转染人口腔鳞癌细胞SCC-15,研究其对口腔鳞癌细胞生长的影响。方法:根据人Naa10基因mRNA序列设计合成3个siRNA片段,转染人口腔鳞癌SCC-15细胞株并进行验证,将最有效的干扰序列克隆至pLV-shRNA载体上,测序正确后进行包装,测定病毒颗粒滴度后,将慢病毒感染SCC-15细胞以测定Naa10的表达情况,并通过生长曲线研究干扰Naa10对SCC-15细胞生长的影响。结果:3个靶点的siNaa10 均能有效抑制Naa10基因的表达,其中2号靶点最为有效(P<0.005);重组RNAi质粒pLV-shNaa10经测序证实构建成功, pLV-shNaa10可在293T细胞中成功包装;测定病毒颗粒LV-shNaa10、LV-NC滴度分别为1.2×10⁹ TU/mL和1.0×10⁹ TU/mL;SCC-15细胞感染LV-shNaa10后,Naa10蛋白表达明显降低(P<0.005);干扰Naa10可促进人口腔鳞癌细胞SCC-15的生长。结论:成功构建了Naa10 shRNA慢病毒表达载体,感染人口腔鳞癌细胞SCC-15后,有效抑制了内源性Naa10基因的表达,干扰Naa10可促进SCC-15细胞的生长。

关键词: N-α-乙酰基转移酶10, 慢病毒, RNA干扰, 口腔鳞癌

Abstract: Objective: To construct the effective lentiviral vector of Naa10 small-interfering RNA, and transfect it into oral squamous cell carcinoma SCC-15 cells so as to investigate its effect on the growth of SCC-15 cells. Methods: Three pairs of siRNA sequences against different parts of Naa10 mRNA were separately transfected into SCC-15 cells. The transfection efficiency was verified, and then the most effective shNaa10 sequence was cloned into pLV-shRNA. pLV-shNaa10 was verified by sequencing and co-transfected into 293T cells to obtain packaged lentivirus particles LV-shNaa10. After viral titer determination, SCC-15 cells were infected with LV-shNaa10 and the expression of Naa10p was detected by Western Blot. Growth curves were used to study the effect of Naa10 knockdown on the growth of SCC-15 cells. Results: Naa10 siRNAs could suppress Naa10 expression and the second siRNA was the most effective (P<0.005). LV-shNaa10 and LV-NC harvested from 293T cells had a titer of 1.2×10⁹ TU/mL and 1.0×10⁹ TU/mL, respectively. After infection with LV-shNaa10, the expression of Naa10 protein in SCC-15 cells was down-regulated (P<0.005), which could in turn markedly facilitate the growth of SCC-15 cells. Conclusion: The lentivirus-mediated Naa10 shRNA was successfully constructed, and knocking down of Naa10 could promote the growth of SCC-15 cells.

Key words: Naa10 , Lentivirus, RNA interference, Oral squamous cell carcinoma

中图分类号:

R739.8

杨洋, 郑军, 徐江, 顾永清, 黄瑾, 王丹妮, 朱茂祥, 曾妍. Naa10基因RNA干扰慢病毒载体的构建及其对口腔鳞癌细胞生长的影响[J]. 口腔医学研究, 2017, 33(7): 707-711.

YANG Yang, ZHENG Jun, XU Jiang, GU Yong-qing, HUANG Jin, WANG Dan-ni, ZHU Mao-xiang, ZENG Yan.. Construction of Lentiviral Vector of Naa10 Small-interfering RNA and Its Effect on Growth of Oral Squamous Cell Carcinoma.[J]. Journal of Oral Science Research, 2017, 33(7): 707-711.

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Naa10基因RNA干扰慢病毒载体的构建及其对口腔鳞癌细胞生长的影响

Construction of Lentiviral Vector of Naa10 Small-interfering RNA and Its Effect on Growth of Oral Squamous Cell Carcinoma.

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