miR-92a对奶山羊乳腺上皮细胞增殖及凋亡的调控分析

doi:10.11843/j.issn.0366-6964.2020.01.016

摘要/Abstract

摘要： miRNAs是对哺乳动物乳腺组织发育及泌乳机能进行调控的重要因子。本研究依据已有的关中奶山羊乳腺组织miRNA表达谱，选择不同泌乳时期差异表达的miR-92a为研究对象，探究miR-92a对山羊乳腺上皮细胞（GMEC）增殖及凋亡的调控作用，并挖掘其潜在的调控基因。采用荧光定量PCR、MTT检测、EdU检测及流式细胞术，检测miR-92a在不同泌乳时期乳腺组织的表达情况，并在细胞水平检测miR-92a对乳腺上皮细胞增殖、凋亡及细胞周期的调控作用。利用RNA-seq技术，分析过表达miR-92a乳腺上皮细胞中的差异表达基因。qRT-PCR结果显示，miR-92a在奶山羊泌乳初期乳腺组织中表达量极显著高于泌乳中期（P<0.01），表明miR-92a可能对奶山羊泌乳性状有重要的调控作用。GMEC过表达miR-92a后，试验结果显示：与NC对照组比较，miR-92a过表达组的EdU阳性细胞数极显著减少（P<0.01），S期的细胞数显著下降、G1期的细胞数显著增加，同时miR-92a组的凋亡细胞数目显著增加（P<0.05）。采用RNA-seq，构建了miR-92a过表达mRNA文库，发现下调基因54个，上调基因160个。GO terms及KEGG通路分析显示差异表达基因调控乳腺上皮细胞多种生物学功能。以上结果表明miR-92a促进GMEC凋亡、抑制其增殖，测序结果进一步证明miR-92a对奶山羊乳腺发育及泌乳性能具有潜在的调控作用。

关键词: miR-92a, 奶山羊, 乳腺上皮细胞, 转录组测序

Abstract: miRNAs are important factors in the regulation of breast tissue development and lactation function. In this study, according to existing miRNA expression profiles of breast tissues in different lactation stages of guanzhong dairy goats, miR-92a expressed differently in different lactation periods was taken as the research object to study the regulatory effect of miR-92a on the proliferation and apoptosis of goat mammary epithelial cells(GMEC),and explore its pontential regulatory genes. The expression of miR-92a in breast tissues at different lactation stages was detected by fluorescence quantitative PCR, MTT assay, EdU assay and flow cytometry, and the regulatory effect of miR-92a on the proliferation and apoptosis of GMECs and cell cycle was detected at the cell level. RNA-seq was used to analyze the differentially expressed genes in GMECs overexpressing miR-92a. qRT-PCR results showed that the expression level of miR-92a in breast tissues of dairy goats in the early lactation stage was significantly higher than that in the middle lactation stage (P<0.01), suggesting that miR-92a may play an important role in regulating lactation traits of dairy goats. After miR-92a was overexpressed in GMEC, the test results showed that compared with the NC group, the number of EdU positive cells in the miR-92a overexpressed group was significantly reduced (P<0.01), the number of cells in the S phase was significantly decreased, and the number of cells in the G1 phase was significantly increased, while the number of apoptotic cells in the miR-92a group was significantly increased (P<0.05).Transcriptome sequencing technology constructed the mRNA library of miR-92a overexpression, and found 54 genes were down-regulated, meanwhile 160 genes were up-regulated. Analysis of GO terms and KEGG pathway showed that differentially expressed genes involved in regulating various biological functions of mammary epithelial cells. The above results indicate that miR-92a promotes the apoptosis of mammary epithelial cells and inhibits cells proliferation. The sequencing results further prove that miR-92a plays an important role in the regulation of breast development and lactation performance in dairy goats.

Key words: miR-92a, dairy goat, mammary epithelial cells, RNA-Seq

中图分类号:

包黎娟, 刘育含, 马毅, 安小鹏, 张月, 张梦, 王建刚, 堵斌, 李广, 曹斌云. miR-92a对奶山羊乳腺上皮细胞增殖及凋亡的调控分析[J]. 畜牧兽医学报, 2020, 51(1): 137-149.

BAO Lijuan, LIU Yuhan, MA Yi, AN Xiaopeng, ZHANG Yue, ZHANG Meng, WANG Jiangang, DU Bin, LI Guang, CAO Binyun. The Regulatory of MiR-92a on Proliferation and Apoptosis of Dairy Goat Mammary Epithelial Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(1): 137-149.

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