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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:

26卷

期数:

2020年03期

页码:

204-208

栏目:

基础医学

出版日期:

2020-06-10

文章信息/Info

Title:

Screening of the target gene and verification of its targeting relationship with miR-20a-5p to regulate osteoblast differentiation

文章编号:

1006-8147(2020)03-0204-05

作者:

李亚冲¹; 刘颖¹; 朱恩东²

（1. 天津医科大学口腔医院牙体牙髓科，天津 300070；2. 天津医科大学朱宪彝纪念医院生化与分子生物学研究室，国家卫生健康委员会激素与发育重点实验室, 天津市代谢性疾病重点实验室，天津市内分泌研究所，天津 300134）

Author(s):

LI Ya-chong¹; LIU Ying¹; ZHU En-dong²

（1.Department of Endodontics, Stomatological Hospital, Tianjin Medical University, Tianjin 300070, China；2. Department of Biochemistry and Molecular Biology， Chu Hsien-I Memorial Hospital，Tianjin Medical University， NHC Key Laboratory of Hormones and Development，Tianjin Key Laboratory of Metabolic Diseases, Tianjin Institute of Endocrinology, Tianjin 300134，China)

关键词:

微小RNAs; miR-20a-5p; Smad7; 双荧光素酶活性检测; GFP抑制检测

Keywords:

microRNAs; miR-20a-5p;  Smad7; dual luciferase activity assay; GFP repression assay

分类号:

Q78

DOI:

-

文献标志码:

A

摘要:

目的：筛选miR-20a-5p调控成骨分化的靶基因，深入研究miR-20a-5p调控成骨分化的分子机制。方法：通过靶基因预测软件预测miR-20a-5p的靶基因，将靶基因的3′UTR及点突变序列插入荧光素酶或绿色荧光蛋白（GFP）报告载体中，并与miR-20a-5p的模拟物或阴性对照共转染HEK-293T细胞，通过双荧光素酶实验和GFP抑制实验检测荧光素酶活性及GFP阳性细胞比例，确定miR-20a-5p与靶基因的靶向关系。结果：通过生物信息学预测到Smad7是miR-20a-5p的靶基因，miR-20a-5p在不同物种的Smad7基因3′UTR区都有保守的结合种子序列；荧光素酶、GFP报告基因及点突变载体经PCR及测序鉴定均正确；双荧光素酶活性检测结果显示：miR-20a-5p显著降低Smad7 3′UTR报告基因载体的荧光素酶活性；GFP抑制实验及流式细胞仪检测结果显示：miR-20a-5p可以显著降低GFP阳性细胞的比例。结论：miR-20a-5p可以直接靶作用于Smad7 3′UTR的种子序列，提示其可能通过直接靶向Smad7基因来发挥促进成骨分化的调控作用。

Abstract:

Objective: To screen the target gene of miR-20a-5p to regulate osteogenic differentiation, and study the molecular mechanism of miR-20a-5p regulating osteoblast differentiation. Methods：The target gene of miR-20a-5p was predicted by online software. The 3′UTR sequence and its mutant sequence of the target gene was cloned into the pMIR-REPORT and pMIR-GFP-REPORT. Then, HEK-293T cells were transfected with luciferase(or GFP) reporter vector and either miR-20a-5p mimics or miR-NC. Finally, determine the targeting relationship between miR-20a-5p and the target genes by luciferase activity and the GFP expression through dual luciferase reporter system and GFP repression assay. Results：As predicted by bioinformatics, Smad7 was the target gene of miR-20a-5p, and it contains a conserved seed sequence in 3′UTR element complementary to miR-20a-5p in different species. Results from PCR and DNA sequencing showed that the sequence of inserted Smad7 3′UTR in luciferase reporter constructions, GFP reporter constructions and all mutant constructions were correct. Dual luciferase reporter system showed that miR-20a-5p significantly repressed the reporter activity of Smad7 3′UTR reporter vector. GFP repression assay and FACS indicated that miR-20a-5p significantly reduced the proportion of GFP positive cells. Conclusion：miR-20a-5p can directly target the seed sequence of Smad7 3′UTR, suggesting that it may play a role in promoting osteogenic differentiation by targeting the Smad7 gene directly.

参考文献/References:

[1] Wu R, Shen D, Sohun H, et al. miR186, a serum microRNA, induces endothelial cell apoptosis by targeting SMAD6 in kawasaki disease[J]. Int J Mol Med, 2018, 41(4):1899
[2] 王珊, 李晓霞, 周杰, 等. Nfic基因3′UTR双荧光素酶报告质粒的构建及其与miR-20a靶向关系的验证[J].天津医药，2016，44(9):1065
[3] Lin S C, Wang C C, Wu M H, et al. Hypoxia-induced microRNA-20a expression increases ERK phosphorylation and angiogenic gene expression in endometriotic stromal cells[J]. J Clin Endocrinol Metab, 2012, 97(8):E1515
[4] Zhou J, Guo F, Wang G, et al. miR-20a regulates adipocyte differentiation by targeting lysine-specific demethylase 6b and transforming growth factor-β signaling[J]. Int J Obes, 2015, 39(8):1282
[5] Zhu E D, Zhang J J, Zhou J, et al. miR-20a-5p promotes adipogenic differentiation of murine bone marrow stromal cells via targeting kruppel-like factor 3[J]. J Mol Endocrinol, 2018, 60(3):225
[6] Zhang J F, Fu W M, He M L, et al. MiRNA-20a promotes osteogenic differentiation of human mesenchymal stem cells by co-regulating BMP signaling[J]. RNA Biol, 2011, 8(5):829
[7] 张月红.骨质疏松发病机制研究进展[J].解放军预防医学杂志,2004, 22(2):151
[8] Yang D H, Yang M Y. The role of macrophage in the pathogenesis of osteoporosis[J]. Int J Mol Sci, 2019, 20(9):2093
[9] Khojasteh A, Behnia H, Naghdi N A, et al. Effects of different growth factors and carriers on bone regeneration: a systematic review[J]. Oral Surg Oral Med Oral Pathol Oral Radiol, 2013, 116(6):E405
[10] Yousefi A M, James P F, Akbarzadeh R, et al. Prospect of stem cells in bone tissue engineering: a review[J]. Stem Cells Int, 2016(2016): 6180487
[11] Valenti M T, Carbonare L D, Mottes M. Role of microRNAs in progenitor cell commitment and osteogenic differentiation in health and disease (Review)[J].Int J Mol Med, 2018,41(5):2441
[12] Tu M L, Tang J J, He H B, et al. MiR-142-5p promotes bone repair by maintaining osteoblast activity[J]. J Bone Miner Metab, 2017, 35(3):255
[13] Lin Z Y, He H B, Wang M, et al. MicroRNA-130a controls bone marrow mesenchymal stem cell differentiation towards the osteoblastic and adipogenic fate[J]. Cell Prolif, 2019, 52(6):e12688
[14] Macias M J, Martin-Malpartida P, Massagué J. Structural determinants of Smad function in TGF-β signaling[J]. Trends Biochem Sci, 2015, 40(6):296

相似文献/References:

[1]张自立,石文霞,李 霖,等.肝癌血清中miRNA-183的表达及临床意义[J].天津医科大学学报,2017,23(06):519.
　ZHANG Zi-li,?SHI Wen-xia,LI Lin,et al.Expression and significance of serum microRNA-183 in hepatocellular carcinoma[J].Journal of Tianjin Medical University,2017,23(03):519.

备注/Memo

备注/Memo:

基金项目 国家自然科学基金面上项目基金资助（81871741）；天津市自然科学基金资助项目（18JCYBJC94300）
作者简介 李亚冲（1993-），女，硕士在读，研究方向：口腔临床医学；通信作者：朱恩东，E-mail：ezhu@tmu.edu.cn。

常用功能

更新日期/Last Update: 2020-06-13