ssc-miR-148a启动子克隆及其特征分析

doi:10.11843/j.issn.0366-6964.2016.10.004

Abstract

Abstract:

In order to understand the transcriptional regulatory mechanism of ssc-miR-148a，we have cloned and analyzed its promoter.Firstly，we designed specific PCR primers to amplify 3 upstream fragments of ssc-miR-148a precursors，then inserted them into pGL3-Basic expression vector.Based on the amplified fragments，ssc-miR-148a promoter region，methylation sites and transcription factor binding sites were predicted by bioinformatics.The plasmid was transfected into 293T cells to analyze the promoter activity.Expression of ssc-miR-148a and DNA methylation transferase 1 (DNMT1) in treated porcine fibroblasts by Basic Fibroblast Growth Factor（bFGF）with different concentrations was detected.The results showed that the cloned 2 043 bp sequence had promoter activity，which had 5 CpGs and transcriptional factor binding sites，such as Sp1，AP2.After treated porcine fibroblasts with 0，5 and 10 ng•mL^-1 bFGF，the expression of ssc-miR-148a was decreased significantly（P<0.05），DNMT1 mRNA level increased significantly (P<0.05).The promoter activity significantly reduced (P<0.05)，but no significant difference between 5 and 10 ng•mL^-1 concentrations (P>0.05).The results indicate that the promoter region of ssc-miR-148a locate at －2 043 bp，which has Sp1 transcription factor binding sites.The expression of ssc-miR-148a is regulated by the bFGF.

Key words: ssc-miR-148a, promoter, cloning, bFGF, DNMT1

CLC Number:

S828
S813.3

WANG Ping，HAO Wen-yan，CAO Li-hua，SHEN Kai-yuan，DAI Xiao-li，LI Hai-yan，LIANG Xian-wei，SHI De-shun，LI Xiang-ping. Cloning and Analysis of ssc-miR-148a Promoter[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(10): 1969-1976.

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Cloning and Analysis of ssc-miR-148a Promoter

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