miR-31-5p对山羊毛囊干细胞增殖和凋亡的影响

doi:10.3864/j.issn.0578-1752.2021.23.017

摘要/Abstract

摘要：

【目的】长江三角洲白山羊是我国及世界上唯一能生产优质笔料毛的山羊品种,课题组前期转录组测序结果表明：在优质笔料毛与非优质笔料毛个体皮肤组织中,MAP3K1的表达水平存在显著差异。探究优质笔料毛性状形成过程中与MAP3K1相互作用的关键miRNAs及其对山羊毛囊干细胞增殖和凋亡的影响,为长江三角洲白山羊的分子选育提供理论依据。【方法】通过生物信息学网站（StrBase、miRDB、TargetScan、miRWalk、DAVID、KEGG、RNAhybrid）预测、筛选与MAP3K1具有靶向关系的miRNAs,利用在线网站Venny 2.1绘制韦恩图。通过构建miR-31-5p过表达载体,MAP3K1、RASA1野生型和突变型双荧光素酶报告基因载体,验证miR-31-5p与MAP3K1、RASA1之间的靶向关系,并结合qPCR和Western Blot技术检测过表达后miR-31-5p对MAP3K1、RASA1 mRNA和蛋白表达水平的影响。为探究过表达miR-31-5p后对细胞增殖、凋亡的影响,分析了转染miR-31-5p后毛囊干细胞内增殖相关基因（PCNA, CDK1, CCND2）、抗凋亡基因（Bcl-2）和促凋亡基因（Bax）的mRNA和蛋白表达水平;同时结合CCK-8,EdU,流式细胞术等方法验证过表达miR-31-5p对毛囊干细胞活力、细胞周期以及凋亡的影响。【结果】通过数据库共同预测到3个可能与MAP3K1相作用的miRNAs,结合现有miRNAs在皮肤和毛囊细胞上研究,最终选用评分相对较高的miR-31-5p作为研究对象。转染miR-31-5p后检测细胞内的miR-31-5p的相对表达量,发现miR-31-5p表达含量极显著高于对照组及空白载体组（P<0.01）;双荧光素酶报告基因结果显示过表达miR-31-5p可促使MAP3K1活性升高（P<0.01）,结合TargetScan、KEGG数据库预测发现miR-31-5p可靶向MAPK信号通路中位于MAP3K1的上游抑制因子RASA1。过表达miR-31-5p抑制了RASA1的活性（P<0.01）;同时,qPCR及Western Blot表明：过表达miR-31-5p后显著抑制RASA1的mRNA和蛋白表达,促进了MAP3K1的表达（P<0.01）。CCK-8结果显示过表达miR-31-5p后提高了细胞增殖能力（P<0.01）,通过EdU染色发现过表达miR-31-5p后,EdU阳性细胞率显著高于空白组（P<0.01）,促进细胞增殖;细胞周期数据说明过表达miR-31-5p后, G1/G0期细胞所占比例为52.23%,显著低于Control组（56.81%,P<0.01）,减缓了G1/G0期细胞阻滞,而S期和G2/M期差异不显著,但仍有上升趋势。通过细胞凋亡试验发现过表达miR-31-5p组活细胞率为93.8%,总凋亡率为4.9%,而空白组活细胞率仅为90.1%,总凋亡率为8.41%,说明在过表达miR-31-5p后细胞凋亡率明显下降（P<0.05）;最后检测miR-31-5p对增殖和凋亡相关基因的影响,发现过表达miR-31-5p后显著提高了增殖相关基因、抗凋亡基因（Bcl-2）的mRNA和蛋白表达水平（P<0.05）,降低了促凋亡基因（Bax）的mRNA和蛋白表达水平。最终根据所研究出的结果绘制miR-31-5p在毛囊干细胞中的分子作用机制图。【结论】 miR-31-5p通过靶向抑制MAPK信号通路中的RASA1,上调MAP3K1表达水平,进而促进毛囊干细胞增殖并抑制其凋亡,为进一步阐明调控长江三角洲白山羊优质笔料毛性状的分子形成机制提供理论依据。

关键词: 长江三角洲白山羊, 优质笔料毛性状, 毛囊干细胞, MAP3K1, miR-31-5p, RASA1, 增殖, 凋亡

Abstract:

【Objective】 The Yangtze River Delta White Goat is the only goat breed that can produce superior-quality brush hair in China and the world. The transcriptome sequencing results of the research team showed that there were significant differences in the expression level of MAP3K1 in the individual skin tissues of superior-quality brush hair and normal-quality brush hair. This study aimed to explore the key miRNAs that interacted with MAP3K1 during the formation of superior-quality brush hair and their effects on the proliferation and apoptosis of goat hair follicle stem cells. 【Method】 The bioinformatics websites (StrBase, miRDB, TargetScan, miRWalk, DAVID, KEGG, and RNAhybrid) were used to predict and select the miRNAs, with targeted relationship with MAP3K1, and use the online website Venny 2.1 to draw a Venn diagram. Through the construction of miR-31-5p overexpression vector, wild-MAP3K1, wild/Mut-RASA1 Luciferase Reporter assay vector, the relationship between miR-31-5p and MAP3K1, RASA1 was verified, and the effects of miR31-5p on MAP3K1, RASA1 mRNA and protein expression were detected by qPCR and Western Blot. In order to explore the effect of overexpression of miR-31-5p on cell proliferation and apoptosis, the mRNA and protein expression levels of proliferation marker gene (PCNA,CDK1,CCND2), anti-apoptotic gene (Bcl-2) and pro-apoptotic gene (Bax) in hair follicle stem cells transfected with miR-31-5p were detected, and the effects of overexpression of miR-31-5p on the viability, cell cycle and apoptosis of hair follicle stem cells were verified by CCK-8, EdU, flow cytometry. 【Result】 Through the database, the final score of the three miRNAs were predicted, which might relatively highly interact with MAP3K1. Then, combined with the known miRNAs studies in skin and hair follicle cells, miR-31-5p with highest score was selected as research object. After transfection of miR-31-5p, the relative expression of miR-31-5p in cells was detected, and it was found that the expression of miR-31-5p was significantly higher than that in the control group and blank vector group (P<0.01). The results of double luciferase reporter genes showed that overexpression of miR-31-5p could increase the activity of MAP3K1. Combined with Target Scan and KEGG database, it was predicted that miR-31-5p could target RASA1, the upstream inhibitor of MAP3K1 in MAPK signal pathway. In order to verify the relationship between miR-31-5p and RASA1, it was found that overexpression of miR31-5p inhibited the activity of RASA1 (P<0.01); qPCR and Western Blot assays showed that overexpression of miR-31-5p significantly inhibited the expression of mRNA and protein of RASA1 and promoted the expression of MAP3K1 (P<0.01). CCK-8 assays showed that overexpression of miR-31-5p increased the ability of cell proliferation. EdU staining showed that the rate of positive cells overexpressing miR-31-5p was significantly higher than that in the blank group (P<0.01), and promoted cell proliferation. Cell cycle data showed that after overexpression of miR-31-5p, the proportion of cells in G1/G0 phase was 52.23%, which was significantly lower than that in Control group (56.81% P<0.01). It slowed down the cell arrest in G1/G0 phase, but there was no significant difference between S phase and G2/M phase, however there was still an upward trend. Through apoptosis experiment, it was found that the survival rate of miR-31-5p group was 93.8%, and the total apoptosis rate was 4.9%; while that of control group was only 90.1%, and the total apoptosis rate was 8.41%, which indicated that the apoptosis rate decreased significantly after overexpression of miR-31-5p. Finally, the effects of miR-31-5p on proliferation and apoptosis-related genes were detected. It was found that overexpression of miR-31-5p significantly increased the mRNA and protein expression levels of proliferation marker genes and anti-apoptosis genes (Bcl-2), and decreased the mRNA and protein expression levels of pro-apoptosis gene (Bax). According to the results of the research, the molecular mechanism of miR-31-5p in hair follicle stem cells was revealed. 【Conclusion】 miR-31-5p targeted RASA1 and up-regulated the expression level of MAP3K1, thereby promoting the proliferation of hair follicle stem cells and inhibiting their apoptosis. It provided a theoretical basis for further investigating the molecular mechanism that regulates the characteristics of superior-quality brush hair of the Yangtze River Delta White Goats.

Key words: Yangtze River Delta White Goat, superior-quality brush hair trait, hair follicle stem cells, MAP3K1, miR-31-5p, RASA1, proliferation, apoptosis

冯云奎,王健,马金亮,张柳明,李拥军. miR-31-5p对山羊毛囊干细胞增殖和凋亡的影响[J]. 中国农业科学, 2021, 54(23): 5132-5143.

FENG YunKui,WANG Jian,MA JinLiang,ZHANG LiuMing,LI YongJun. Effects of miR-31-5p on the Proliferation and Apoptosis of Hair Follicle Stem Cells in Goat[J]. Scientia Agricultura Sinica, 2021, 54(23): 5132-5143.

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名称 Name	引物名称 Primer name	引物序列（5′-3′） Primer sequence (5′-3′)
miR-31-5p	Stem-loop RT-miR-31-5p	GTCGTATCCAGTGCAGGGTCCGAGGT ATTCGCACTGGATACGACAGCTATGC
	miR-31-5p-F	AGGCAAGATGCTGGCAT
	miR-31-5p-R	GTGCAGGGTCCGAGGT
18S-rRNA ID:493779	18S-F	GTGGTGTTGAGGAAAGCAGACA
18S-rRNA ID:493779	18S-R	TGATCACACGTTCCACCTCATC
PCNA ID:102172276	PCNA-F	ATCAGCTCAAGTGGCGTGAA
PCNA ID:102172276	PCNA-R	TGCCAAGGTGTCCGCATTAT
CDK1 ID:10086361	CDK1-F	AGATTTTGGCCTTGCCAGAG
CDK1 ID:10086361	CDK1-R	AGCTGACCCCAGCAATACTT
CCND2 ID:102180657	CCND2-F	GGGCAAGTTGAAATGGAA
CCND2 ID:102180657	CCND2-R	TCATCGACGGCGGGTAC
Bax ID:100846984	Bax-F	TTTCCGACGGCAACTTCAA
Bax ID:100846984	Bax-R	TGAGCACTCCAGCCACAAA
Bcl-2 ID:100861254	Bcl-2-F	ATGTGTGTGGAGAGCGTCAA
Bcl-2 ID:100861254	Bcl-2-R	CCTTCAGAGACAGCCAGGAG
MAP3K1 ID:102187530	MAP3K1-F	GAGAGTTGGCAGTTGGCAGAG
MAP3K1 ID:102187530	MAP3K1-R	CAGTTGTTTGATTCAGTTTGGTTTCC
RASA1 ID:102170782	RASA1-F RASA1-R	TGCCAGAGGAAGAGTACAGC TTCCATCAAGGTGCTCGCAA
GAPDH ID:100860872	GAPDH-F	AGGTCGGAGTGAACGGATTC
GAPDH ID:100860872	GAPDH-R	CCAGCATCACCCCACTTGAT

名称 Name	引物名称 Primer name	引物序列（5′-3′） Primer sequence (5′-3′)
miR-31-5p	miR-31-5p F	CCCAAGCTTGCCACAACCTTCCTATGCTTGA
miR-31-5p	miR-31-5p R	GCTCTAGAGGCCAGCAAGGCTAAAATGAA
MAP3K1	Wild-MAP3K1-F	CCGCTCGAGTTTCCAGGTCTCTCGTGTGC
MAP3K1	Wild-MAP3K1-R	ATAAGAATGCGGCCGCGTGGGCATGGTGGTCTACAA
RASA1	Wild-RASA1-F	CCGCTCGAGTAACGATGTCAGGTAGCAGCC
	Wild-RASA1-R	ATAAGAATGCGGCCGCTCACTGGAATGTGGAAAGGTGT
	Mut-RASA1-F	CCGCTCGAGGTGCACAACAGCATGTACTGA
	Mut-RASA1-F	ATAAGAATGCGGCCGCATACCTCGCAAAGGAGACATTAT