microRNA-330-3p调控口腔鳞癌细胞增殖和凋亡的实验研究

doi:10.3877/cma.j.issn.1674-1366.2015.01.003

中华口腔医学研究杂志(电子版) ›› 2015, Vol. 09 ›› Issue (01) : 14 -20. doi: 10.3877/cma.j.issn.1674-1366.2015.01.003

所属专题：文献；

基础研究

microRNA-330-3p调控口腔鳞癌细胞增殖和凋亡的实验研究

赵珍¹, 陈万涛², 张建军², 秦星², 孙强¹, 李新明¹^,^†(

)

^1. 450052　郑州大学第一附属医院口腔颌面外科
^2. 200011　上海交通大学医学院附属第九人民医院口腔颌面-头颈肿瘤科，上海市口腔医学研究所/上海市口腔医学重点实验室

收稿日期:2014-12-29 出版日期:2015-02-01
通信作者: 李新明
基金资助:
国家自然科学基金重大研究计划（培育）项目(91229103); 教育部高等学校博士学科点专项科研基金(20110073110078)

MicroRNA-330-3p regulates the cell proliferation and apoptosis of oral squamous cell carcinoma

Zhen Zhao¹, Wantao Chen², Jianjun Zhang², Xing Qin², Qiang Sun¹, Xinming Li¹^,^†()

^1. Department of Oral & Maxillofacial Surgery, Zhengzhou University, Zhengzhou 450052, China
^2. Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Research Institute of Stomatology and Shanghai Key Laboratory of Stomatology, Shanghai 200011, China

Received:2014-12-29 Published:2015-02-01
Corresponding author: Xinming Li
About author:
Corresponding author: Li Xinming, Email: lxming@zzu.edu.cn, Tel: 0371-66913177

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引用本文：

赵珍, 陈万涛, 张建军, 秦星, 孙强, 李新明. microRNA-330-3p调控口腔鳞癌细胞增殖和凋亡的实验研究[J/OL]. 中华口腔医学研究杂志(电子版), 2015, 09(01): 14-20.

Zhen Zhao, Wantao Chen, Jianjun Zhang, Xing Qin, Qiang Sun, Xinming Li. MicroRNA-330-3p regulates the cell proliferation and apoptosis of oral squamous cell carcinoma[J/OL]. Chinese Journal of Stomatological Research(Electronic Edition), 2015, 09(01): 14-20.

目的

探讨微小RNA-330-3p（miR-330-3p）对口腔鳞癌细胞增殖和凋亡的调控作用。筛选并验证相关靶基因。

方法

实时荧光定量聚合酶链反应（PCR）检测口腔鳞癌细胞系、口腔鳞癌组织与癌旁正常上皮组织中miR-330-3p的表达情况。应用miR-330-3p抑制物（miR-330-3p inhibitor）和miR-330-3p模拟物（miR-330-3p mimics），分别转染口腔鳞癌细胞系（HN13）细胞，实时荧光定量PCR检测miR-330-3p的转染效率；然后应用细胞增殖、细胞克隆形成以及流式细胞术等实验技术，分别检测miR-330-3p上调和下调对口腔鳞癌细胞增殖及凋亡的影响。通过生物信息网站miRanda和TargetScan与本课题组前期基因芯片数据比对预测了9个可能的靶基因，并进一步用相关实验验证miR-330-3p对靶基因的负向调控作用。

结果

与癌旁正常上皮组织相比，口腔鳞癌组织中miR-330-3p的表达显著上调，平均表达水平为癌旁正常组织的1.75倍（n=29，t=5.282，P=0.0045）；miR-330-3p抑制物和miR-330-3p模拟物，能分别显著降低或升高HN13细胞中miR-330-3p的表达量；转染miR-330-3p抑制物组HN13细胞增殖能力明显降低、凋亡率升高；而miR-330-3p模拟物组细胞的增殖能力明显升高、凋亡率降低。miR-330-3p能够结合PDCD4的3’UTR，负向调控PDCD4的表达。

结论

miR-330-3p能够与PDCD4的3’UTR结合抑制PDCD4的转录后翻译作用，从而促进了口腔鳞癌细胞的生长；其抑制物能发挥有效的抗癌作用，研究结果为口腔鳞癌的靶向基因治疗提供候选分子。

关键词: 微小RNA-330-3p, 口腔鳞癌, 增殖, 凋亡

Objective

To investigate the regulatory function of microRNA-330-3p (miR-330-3p) on the cell growth and apoptosis in oral squamous cell carcinoma (OSCC) , and to screen and verify the revelant target genes.

Methods

The expressions of miR-330-3p were measured in OSCC tissues, adjacent normal tissues and OSCC cell lines by quantitative real-time PCR. Then miR-330-3p inhibitor and mimics were transfected into OSCC cell line HN13 cells, and their transfection efficiency was detected by quantitative real-time PCR. And then, cell proliferation assay, cell cloning experiments and flow cytometry were conducted to determine the effects of miR-330-3p on the proliferation and apoptosis of OSCC cells. We found 9 putative targets through miRanda and TargetScan that matching the data of related gene chip. Then quantitative real-time PCR, Western blot test and Dual-Luciferase Reporter assay were used to verify the putative targets of miR-330-3p.

Results

compared with adjacent normal tissues, the expression of miR-330-3p was upregulated in OSCC tissues and cell lines. The inhibitor and mimics of miR-330-3p could significantly downregulate or upregulate the expression of miR-330-3p in transfected HN13 cells. miR-330-3p-inhibitor-transfected HN13 cells had lower proliferation rate and higher apoptosis rate. And HN13 cells showed an opposite change of proliferation rate and apoptosis when transfected with miR-330-3p mimics. MiR-330-3p can negatively regulate the translation of PDCD4.

Conclusions

miR-330-3p is a tumor promoter in OSCC by repressing the post-transcriptional translation of PDCD4 through binding with 3’UTR, and its inhibitor has the anticancer potential. So our study provides a new candidate molecule to gene-targeting treatment in OSCCs.

Key words: MicroRNA-330-3p, Oral squamous cell carcinoma, Proliferation, Apoptosis

图1 miR-330-3p在口腔鳞癌组织及癌旁正常组织中的表达差异

图2 口腔鳞癌细胞系细胞中miR-330-3p的表达情况

图3 实时荧光定量PCR法检测miR-330-3p干扰及过表达效率

图4 不同转染组对HN13细胞增殖的影响

图5 细胞克隆形成实验检测转染不同序列后HN13细胞的克隆形成能力比较

图6 流式细胞术检测转染不同序列后HN13细胞的凋亡情况

图7 转染miR-330-3p抑制物及模拟物后PDCD4 mRNA的表达量

图8 Western blot实验及PDCD4蛋白相对灰度值

图9 荧光素报告载体体外转染实验结果

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