微小RNA-181a抑制唾液腺腺样囊性癌细胞株增殖能力的研究

doi:10.3877/cma.j.issn.1674-1366.2015.06.004

中华口腔医学研究杂志(电子版) ›› 2015, Vol. 09 ›› Issue (06) : 455 -460. doi: 10.3877/cma.j.issn.1674-1366.2015.06.004

所属专题：文献；

基础研究

微小RNA-181a抑制唾液腺腺样囊性癌细胞株增殖能力的研究

何倩婷¹, 陈丹¹, 赵婷婷¹, 刘中华¹, 王安训¹^,^†(

)

^1. 510080　广州，中山大学附属第一医院口腔科

收稿日期:2015-08-28 出版日期:2015-12-01
通信作者: 王安训
基金资助:
高校基本科研业务费中山大学青年教师重点培育计划(11ykz09); 广东省科技计划国际合作项目(2010B050700015)

The inhibition of miR-181a on proliferation of salivary adenoid cystic carcinoma

Qianting He¹, Dan Chen¹, Tingting Zhao¹, Zhonghua Liu¹, Anxun Wang¹^,^†()

^1. Department of Stomatology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China

Received:2015-08-28 Published:2015-12-01
Corresponding author: Anxun Wang
About author:
Corresponding author: Wang Anxun, Email: wang_anxun@aliyun.com

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目的

探讨微小RNA（miR）-181a对唾液腺腺样囊性癌（SACC）细胞增殖能力的体内外作用影响。

方法

通过过表达或沉默miR-181a在SACC-LM和SACC-83细胞中的表达。实时荧光定量PCR检测转染后SACC细胞株中miR-181a的表达水平。MTT法检测miR-181a对SACC细胞体外增殖能力的影响。Western blot检测转染后生长因子因子表达的改变。裸鼠皮下移植瘤模型检测miR-181a对SACC细胞体内增殖能力的影响。采用SPSS 17.0软件对数据进行统计学处理，以P<0.05为差异具有统计学意义。

结果

实时荧光定量PCR结果示，转染后SACC-LM细胞中miR-181a表达升高（t = -9.198，P = 0.012），而SACC-83细胞中miR-181a表达降低（t = -7.241，P = 0.019）；SACC-LM细胞增殖能力降低（t = -4.58，P = 0.045），而SACC-83细胞增殖能力提高（t = 3.016，P = 0.03）；SACC-LM细胞中转化生长因子β2（TGF-β2）、神经生长因子（NGF）和血管内皮生长因子（VEGF）的表达水平均下调，而SACC-83细胞中TGF-β2、NGF和VEGF的表达水平均升高。裸鼠皮下移植瘤模型结果显示，miR-181a mimics组移植瘤瘤体明显小于mimics NC组（t = -4.692，P = 0.043）。

结论

miR-181a能抑制SACC细胞体内体外的增殖能力。

关键词: 微小RNA-181a, 涎腺, 癌，腺样囊性, 增殖, 体外, 体内

Objective

To investigate the effect of microRNA (miR) -181a in proliferation of salivary adenoid cystic carcinoma (SACC) in vitro and in vivo.

Methods

The SACC cell lines SACC-LM and SACC-83 were transfected with miR-181a mimics and miR-181a LNA respectively. QRT-PCR analysis was used to detect the expression of miR-181a in the paired SACC cell lines after transfection. MTT assay was performed to analyze the effect of miR-181a on proliferation after transfection. Western blot analysis was used to demonstrate the proliferation related genes after transfection. Tumorigenesis model in nude mice was performed to analyze the effect of miR-181a on proliferation of SACC cell lines after transfection in vivo. Data were analyzed using the Statistical Package for the Social Science (SPSS) , Version 17.0. For all statistical analyses, P<0.05 was considered statistically significant.

Results

Ectopic transfection of the miR-181a mimics to the SACC-LM cells led to increased miR-181a expression (t = -9.198, P = 0.012) while the miR-181a expression was decreased in SACC-83 cells treated with miR-181a LNA (t = -7.241, P = 0.019) . After transfection, the proliferation rate of SACC-LM cells was reduced (t = -4.58, P = 0.045) , while it was increased in SACC-83 cells (t = 3.016, P = 0.03) . Gene expression of TGF-β2, NGF and VEGF was decreased in SACC-LM cells after transfection, while increased in transfected SACC-83 cells. Ectopic transfection of the miR-181a mimics to the SACC-LM cells led to decrease in tumorigenesis ability (t = -4.692, P = 0.043) .

Conclusion

MiR-181a can suppress the proliferation of SACC in vitro and in vivo.

Key words: MicroRNA-181a, Salivary glands, Carcinoma, adenoid cystic, Proliferation, In vitro, In vivo

表1 miR-181a的引物序列

mircroRNA	引物	引物序列
Hsa-miR-181a	HmiR-181a-RT	GTCGGGTCCAGAGCAGGGTCCGAGGTA
?	?	CACGTTCGCTCTGGACCCGACACTCAC
?	HmiR-181a-FO-1	TGCCGAACATTCAACGCT
?	HmiR-RE-4	AGAGCAGGGTCCGAGGTA
U6snRNA	U6snRNA FO	ATTGGAACGATACAGAGAAGATT
?	U6snRNA RE	GGAACGCTTCACGAATTTG

表1 miR-181a的引物序列

mircroRNA	引物	引物序列
Hsa-miR-181a	HmiR-181a-RT	GTCGGGTCCAGAGCAGGGTCCGAGGTA
?	?	CACGTTCGCTCTGGACCCGACACTCAC
?	HmiR-181a-FO-1	TGCCGAACATTCAACGCT
?	HmiR-RE-4	AGAGCAGGGTCCGAGGTA
U6snRNA	U6snRNA FO	ATTGGAACGATACAGAGAAGATT
?	U6snRNA RE	GGAACGCTTCACGAATTTG

图1 miR-181a mimics/LNA转染后SACC细胞中miR-181a的表达

图2 miR-181a mimics/LNA转染后对SACC细胞增殖能力的影响

图3 miR-181a mimics/LNA转染后对SACC细胞中生长因子表达的影响

图4 过表达miR-181a对SACC-LM细胞体内成瘤能力的影响

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The inhibition of miR-181a on proliferation of salivary adenoid cystic carcinoma

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