miR-378促进脂质生成相关靶基因鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2020-0848

摘要/Abstract

摘要：

旨为验证miR-378在脂肪细胞中的功能,及其脂质相关靶基因的筛选和鉴定。利用miR-378类似物转染3T3-L1细胞,验证miR-378在脂肪细胞中的功能;根据靶标位点的保守性以及促脂功能确定miR-378潜在靶基因;采用microRNA pulldown技术验证miR-378与靶基因的靶标关系;运用双荧光素报告基因实验确定miR-378与靶基因的结合位点。结果发现,miR-378可以通过增加脂质合成和减少脂质分解两条途经来促进脂肪细胞中脂质生成,确定了miR-378与Runx1t1、Galnt3、和RAB10的靶标关系以及结合的靶位点。

关键词: miR-378, 脂质生成, microRNA pulldown, 靶基因, 靶位点

Abstract:

The purpose of this study was to investigate the function of miR-378 in adipocytes and to filter and verify its target genes. 3T3-L1 cells were transfected with miR-378 mimic to validate the function of mir-378 in adipocytes. The potential target genes of miR-378 were identified according to the conservation of target sites and lipid promoting function. The relationship between miR-378 and its targets was verified by microRNA pulldown technology,and then the binding sites of miR-378 and its targets were determined by Dual-Luciferase Reporter Assay System. Some important results were found that miR-378 promoted lipogenesis in adipocytes by increasing lipid synthesis and decreasing lipid decomposition,and the relationship between miR-378 and Runx1t1,Galnt3,RAB10 and the binding sites were determined.

Key words: miR-378, lipogenesis, microRNA pulldown, target genes, target sites

张廷焕, 龙熙, 郭宗义, 柴捷. miR-378促进脂质生成相关靶基因鉴定[J]. 生物技术通报, 2021, 37(2): 80-87.

ZHANG Ting-huan, LONG Xi, GUO Zong-yi, CHAI Jie. miR-378 Promoting Lipogenesis and Identification of Target Genes[J]. Biotechnology Bulletin, 2021, 37(2): 80-87.

图/表 9

表1 RT-PCR扩增引物

Primer name	Primer sequence	Length/bp
GDF6	F：CTCGAGCTACTAAATGACAG	20
GDF6	R：TCTCCTTCCTCACTGCCTGT	20
Runx1t1	F：ATCGGGAATTCCTTCACAGGC	21
Runx1t1	R：GCTTTTTGCAGCTCCGTCAT	20
Galnt3	F：ACGCAGGTGATTGCTCGTAA	20
Galnt3	R：AGGTCTGGCACATACGCTTC	20
RAB10	F：ATGTACTTGCTCAGCTCAACT	21
RAB10	R：AGGGACTCAAGCACATTATCCA	22

表2 PCR扩增引物

primer name	DNA primer sequence	Length/bp
GDF6	F：AGGGGACACAAGTCCCTGTC	20
GDF6	R：AGAACTGCCCGCCACCAACG	20
Runx1t1	F：GCCAAAGACTGATCTGAGGGA	21
Runx1t1	R：GGAGAAGTGTCTTCCCAGCC	20
Galnt3	F：TAGAACCGCTGCAGAAACCC	20
Galnt3	R：TGAGTGTGTGGATGCAGGTG	20
RAB10	F：CTCGAGTCCCACAGACAATTC	20
RAB10	R：CAAAGCCCTAATAGTAAGCAG	20

图1 miR-378增加3T3-L1脂肪细胞脂质生成 A：油红O染色脂肪细胞,标尺：50 μm;B：定量分析脂质含量。**代表差异显著（P<0.01）,下同

图2 miR-378调控脂质相关基因的表达 *代表差异显著（P<0.05）,下同

图3 miR-378与候选靶基因结合位点在不同物种间的序列对比红色区域表示结合位点

图4 Biotin-miRNA捕获靶基因的富集分析 NS代表差异不显著,下同

图5 候选基因PCR目的片段的电泳图

图6 四个报告基因插入片段的测序峰图

图7 报告基因载体双荧光素酶活性检测

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