胰高血糖素样肽-1激动剂Exendin-4 刺激小鼠胚胎成骨细胞前体细胞MC3T3-E1的转录组学体外研究

doi:10.16150/j.1671-2870.2022.03.013

摘要/Abstract

摘要：

目的：观察胰高血糖素样肽-1（glucagon-like peptide-1,GLP-1）受体激动剂Exendin-4对体外培养小鼠胚胎成骨细胞前体细胞增殖和向成骨分化的影响。方法：根据前期预实验选用浓度为30 nmol/L的 Exendin-4 处理小鼠胚胎成骨细胞前体细胞MC3T3-E1细胞72 h 后,收集细胞进行测序,获得的 RNA-seq 数据,筛选差异表达变化在2倍以上的基因（差异表达基因）,用KEGG PATHWAY富集分析和 GO 富集分析其基因功能,确定相关生物通路。结果：用Exendin-4 处理 MC3T3-E1细胞后,细胞出现增殖,共筛选得到418个差异表达基因,其中表达上调的基因有236个,表达下调的基因有182个。经GO富集分析显示,上调的差异表达基因主要富集在免疫系统、细胞黏着和蛋白质水解等通路中。KEGG PATHWAY富集分析显示,下调的差异表达基因主要富集在PI3K-Akt 信号通路、癌症中异常转录、哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin,mTOR）受体信号通路、缺氧诱导因子-1（hypoxia inducible factor-1,HIF-1）信号通路等。结合文献分析发现,与骨质疏松症相关的上调基因Fosl1、Cxcl13、Clec11a、Phex、Fasl、Camk4、Stab1、Zbp1、Adora2a、Igfbp7、Cxcr2、Hp、MTI;与骨质疏松症相关的下调基因包括Nog, Zeb1, Esr1, Igf2bp2,Plxnb3,Rgs14。结论：Exendin-4 可能通过影响细胞免疫、细胞黏着和蛋白质水解相关基因的表达水平,促进前体细胞向成骨细胞的分化过程,改善骨质疏松。

关键词: 胰高血糖素样肽-1, 受体激动剂, 成骨细胞, 骨质疏松症, 转录组学

Abstract:

Objective: To observe the effects of Exendin-4, a glucagon-like peptide-1(GLP-1) receptor agonist, on proliferation and osteogenic differentiation of Mc3t3-E1(mouse embryonic osteoblast precursor cells) in vitro. Methods: Mc3t3-E1 cells were treated with Exendin-4 at a pre-experimental concentration of 30 nmol/L for 72 h. The cells were collected for sequencing, and the RNA-SEQ data were obtained. The differentially expressed genes greater than 2 times were screened and KEGG PATHWAY enrichment analysis and GO enrichment analysis were performed. Results: Mc3t3-E1 cells were proliferated after Exendin-4 treatment, and a total of 418 differentially expressed genes were screened, including 236 up-regulated genes and 182 down-regulated genes. GO enrichment analysis showed that the up-regulated differentially expressed genes were mainly enriched in the immune system, cellular adhesion and proteolytic pathways. KEGG PATHWAY enrichment analysis showed that The down-regulated differentially expressed genes were mainly enriched in PI3K-Akt signaling pathway, abnormal transcription in cancer, mammalian target of Rapamycin (mTOR) receptor signaling pathway, and hypoxia inducible factor 1 (HYPO xia inducible) Factor-1, HIF-1) signaling pathway. It revealed that up-regulated genes related to osteoporosis included Fosl1, Cxcl13, Clec11a, Phex, Fasl, Camk4, Stab1, Zbp1, Adora2a, Igfbp7, Cxcr2, Hp, MTI; while for Down-regulated genes included Nog, Zeb1, Esr1, Igf2bp2, Plxnb3, Rgs14. Conclusions: Exendin-4 can affect the differentiation of precursor cells into osteoblasts by influencing the expression levels of gene related to cell immunity, cell adhesion and proteolysis related genes, and ultimately affect the formation and development of osteoporosis.

Key words: Cell proliferation, Exendin-4, Osteoblast, Osteoporosis, RNA-Seq

中图分类号:

R541.7

刘欣, 綦才辉, 王振竞, 吕娜, 王少婷, 王淑萍. 胰高血糖素样肽-1激动剂Exendin-4 刺激小鼠胚胎成骨细胞前体细胞MC3T3-E1的转录组学体外研究[J]. 诊断学理论与实践, 2022, 21(03): 367-373.

LIU Xin, QI Caihui, WANG Zhenjing, LÜ Na, WANG Shaoting, WANG Shuping. Transcriptome study of glucagon like peptide-1 agonist Exendin-4 on mouse embryonic osteoblast precursor MC3T3-E1 in vitro[J]. Journal of Diagnostics Concepts & Practice, 2022, 21(03): 367-373.

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引物	序列
Id3-F	5′ACTTACCCTGAACTCAACGC3′
Id3-R	5′CTCCAAGGAAACCAGAAGAA3′
Rhbdl3-F	5′GCCTTGTGCTGTCCATACCC3′
Rhbdl3-R	5′CTCATCCTCCTGCCTCCCTC3′
Tbata-F	5′GGAACCAGCCCCTGTTTCTG3′
Tbata-R	5′TCATCCCTACGAGGTGCTAA3′
Col14a1-F	5′TCACGGGTGTTCAGAAATAA3′
Col14a1-R	5′AAAGCAATCCCTGTAAGTCG3′
Cxcr2-F	5′AGATGGCTCAGTGGGTAAGA3′
Cxcr2-R	5′ATGTGGTTGCTGGGATTTGA3′
Pag1-F	5′AACACCCGTGTCAGTTCCTT3′
Pag1-R	5′TCTGTGCCTCAGTTTCTCCA3′
Plxnb3-F	5′CCTTGGTGGAATACAAAGTG3′
Plxnb3-R	5′AATACAGTGGGAAAGAGGGT3′
Hp-F	5′GACCTTCTCAATCTCCACCAGC3′
Hp-R	5′ATCTCCCGCCACGGACTCAC3′
Igf2bp2-F	5′CGCTTGAGTAACCGAGTGTA3′
Igf2bp2-R	5′TCCTTATCTCCCTCCTAACC3′
Igfbp7-F	5′GAGGGCATCAACCACTGTAA3′
Igfbp7-R	5′TCTCCTCTAAGTAAGGAGGACG3′
Prss53-F	5′GCTCTGGCTCCTCAGCGAAGT3′
Prss53-R	5′ATGGTTCCTGGTTGGACTGC3′
Rgs14-F	5′GTTTGCCTACCCATTCCTCC3′
Rgs14-R	5′CCAGTACACCCAGGTCTTACTTAT3′
Hoxb8-F	5′CCGCTAGTGGTAGTATCTCGTAA3′
Hoxb8-R	5′TAGGCAGTTTCATGTTGTTGG3′
MTI-F	5′CGTGCTGTGCCTGATGTGAC3′
MT1-R	5′TAGGAAGACGCTGGGTTGGT3′
FXYD7-F	5′TGGAGAAGGAGACCAGGAGATG3′
FXYD7-R	5′CAGAGTGCAGGAACCAGAGGAC3′