猪C1QTNF3基因可变剪接体的鉴定及介导miR-101调控成脂分化的研究

doi:10.11843/j.issn.0366-6964.2021.011.006

摘要/Abstract

摘要： 旨在研究猪C1QTNF3基因可变剪接体的特性及miR-101通过C1QTNF3基因促进猪脂肪SV细胞成脂分化的机制。本研究采集3头30日龄健康马身猪仔公猪心、肝、脾、肺、肾、胃、股二头肌、腰大肌、皮下脂肪和背部脂肪组织样品，首先利用RT-PCR技术和生物信息学方法对C1QTNF3基因的不同转录本进行扩增和生物学特性分析，采用qRT-PCR技术检测C1QTNF3基因不同转录本在猪组织中的表达变化。随后，利用生物信息学预测发现，C1QTNF3上游的调控因子是miR-101，采用双荧光素酶报告试验验证miR-101对C1QTNF3的调控作用。最后，利用油红O染色和qRT-PCR技术检测过表达miR-101对猪脂肪SV细胞成脂分化的影响。基因克隆得到猪C1QTNF3存在两个转录本C1QTNF3和C1QTNF3-1，其中C1QTNF3-1为新鉴定的转录本；测序分析显示，与C1QTNF3相比，C1QTNF3-1的第1外显子区域缺失了219个碱基，少编码73个氨基酸。进化树分析显示，猪C1QTNF3和C1QTNF3-1蛋白序列与人和马来亚穿山甲等物种的亲缘关系较近。C1QTNF3和C1QTNF3-1在猪各组织中均有表达，且在各组织中C1QTNF3-1的表达量均极显著高于C1QTNF3（P<0.01）。过表达miR-101极显著下调C1QTNF3 3'UTR区域的荧光素酶活性（P<0.01）和C1QTNF3 mRNA的表达（P<0.01），同时增加了脂肪细胞成脂分化关键基因PPARγ、C/EBPβ、SREBP-1c和FABP4的mRNA表达量（P<0.01）。本试验成功克隆了猪C1QTNF3基因的两个可变剪接体，其中C1QTNF3-1在猪不同组织中均高表达，推测该转录本为基因发挥功能的主要亚型。并深入研究了C1QTNF3基因的上游调控因子，揭示了miR-101靶向C1QTNF3促进成脂分化的机制，丰富了C1QTNF3的生物学作用和调控网络。

关键词: 猪, C1QTNF3基因, 成脂分化, miR-101, 可变剪接

Abstract: The study aimed to explore the alternative splicing isoforms of pig C1QTNF3 gene and mechanism of miR-101 targeting C1QTNF3 expression in promoting porcine adipose-derived stromal vascular cells (ASVC) adipogenesis. The heart, liver, spleen, lung, kidney, stomach, biceps femoris, psoas magnus, abdominal fat and back fat were obtained from 3 30-day-old healthy Mashen male piglets. The full-length coding sequence (CDS) of C1QTNF3 transcripts were cloned by RT-PCR, the biological characteristics of C1QTNF3 protein were analyzed by bioinformatics methods. The expression patterns of C1QTNF3 transcripts in porcine tissues were detected by qRT-PCR. Bioinformatics prediction revealed that miR-101 was the upstream regulatory factor of C1QTNF3. The effect of miR-101 on C1QTNF3 was verified by dual luciferase reporter assay. The effect of overexpression of miR-101 on adipogenesis of porcine ASVC was detected by Oil Red O staining and qRT-PCR. Two transcripts of C1QTNF3 gene, C1QTNF3 and C1QTNF3-1, were successfully obtained in this study. C1QTNF3-1 was a newly identified transcript, which lacked 219 bp (73 amino acids) in the first exon compared with C1QTNF3. The phylogenetic tree analysis showed that the porcine C1QTNF3 and C1QTNF3-1 proteins sequence were highly related to Homo sapiens and Manis javanica. C1QTNF3 and C1QTNF3-1 were expressed in all porcine tissues, the expression level of C1QTNF3-1 was significantly higher than that of C1QTNF3 (P<0.01). Overexpression of miR-101 significantly reduced the relative luciferase activity of C1QTNF3 3'UTR(P<0.01) and mRNA expression of C1QTNF3(P<0.01); the mRNA expression of key genes for adipogenesis, PPARγ, C/EBPβ, SREBP-1c and FABP4 were significantly increased (P<0.01). In this study, two transcripts of pig C1QTNF3 gene were successfully cloned. The expression of C1QTNF3-1 was significantly higher than C1QTNF3 in different porcine tissues, suggesting it was the predominant transcript. In addition, miR-101 was identified as the upstream regulatory factor of C1QTNF3. By reducing C1QTNF3 gene expression, miR-101 promoted porcine ASVC adipogenesis, which enriched the biological role and regulatory network of C1QTNF3.

Key words: pig, C1QTNF3 gene, adipogenesis, miR-101, alternative splicing

中图分类号:

S828.2

杨阳, 张雪莲, 张万锋, 李文霞, 李文新, 蔡春波, 路畅, 高鹏飞, 郭晓红, 李步高, 曹果清. 猪C1QTNF3基因可变剪接体的鉴定及介导miR-101调控成脂分化的研究[J]. 畜牧兽医学报, 2021, 52(11): 3042-3052.

YANG Yang, ZHANG Xuelian, ZHANG Wanfeng, LI Wenxia, LI Wenxin, CAI Chunbo, LU Chang, GAO Pengfei, GUO Xiaohong, LI Bugao, CAO Guoqing. Study of Alternative Splicing of Porcine C1QTNF3 Gene and Its Regulation on Adipogenesis by Mediating miR-101[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(11): 3042-3052.

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