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

Abstract

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

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

Effects of miR-31-5p on the Proliferation and Apoptosis of Hair Follicle Stem Cells in Goat

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