Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (23): 5132-5143.doi: 10.3864/j.issn.0578-1752.2021.23.017

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

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

FENG YunKui(),WANG Jian,MA JinLiang,ZHANG LiuMing,LI YongJun()   

  1. College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, Jiangsu
  • Received:2020-11-02 Accepted:2021-03-11 Online:2021-12-01 Published:2021-12-06
  • Contact: YongJun LI E-mail:feng1105387084@163.com;yzliyongjun@163.com

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

Table 1

Primer sequence for qPCR"

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-R TGATCACACGTTCCACCTCATC
PCNA
ID:102172276
PCNA-F ATCAGCTCAAGTGGCGTGAA
PCNA-R TGCCAAGGTGTCCGCATTAT
CDK1
ID:10086361
CDK1-F AGATTTTGGCCTTGCCAGAG
CDK1-R AGCTGACCCCAGCAATACTT
CCND2
ID:102180657
CCND2-F GGGCAAGTTGAAATGGAA
CCND2-R TCATCGACGGCGGGTAC
Bax
ID:100846984
Bax-F TTTCCGACGGCAACTTCAA
Bax-R TGAGCACTCCAGCCACAAA
Bcl-2
ID:100861254
Bcl-2-F ATGTGTGTGGAGAGCGTCAA
Bcl-2-R CCTTCAGAGACAGCCAGGAG
MAP3K1
ID:102187530
MAP3K1-F GAGAGTTGGCAGTTGGCAGAG
MAP3K1-R CAGTTGTTTGATTCAGTTTGGTTTCC
RASA1
ID:102170782
RASA1-F
RASA1-R
TGCCAGAGGAAGAGTACAGC
TTCCATCAAGGTGCTCGCAA
GAPDH
ID:100860872
GAPDH-F AGGTCGGAGTGAACGGATTC
GAPDH-R CCAGCATCACCCCACTTGAT

Table 2

Primer sequences used in plasmid construction"

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 R GCTCTAGAGGCCAGCAAGGCTAAAATGAA
MAP3K1 Wild-MAP3K1-F CCGCTCGAGTTTCCAGGTCTCTCGTGTGC
Wild-MAP3K1-R ATAAGAATGCGGCCGCGTGGGCATGGTGGTCTACAA
RASA1 Wild-RASA1-F CCGCTCGAGTAACGATGTCAGGTAGCAGCC
Wild-RASA1-R ATAAGAATGCGGCCGCTCACTGGAATGTGGAAAGGTGT
Mut-RASA1-F CCGCTCGAGGTGCACAACAGCATGTACTGA
Mut-RASA1-F ATAAGAATGCGGCCGCATACCTCGCAAAGGAGACATTAT

Fig. 1

Predicted miRNAs targeting MAP3K1"

Fig. 2

The effects of overexpression of miR-31-5p on MAP3K1 and RASA1 A: Overexpression efficiency of miR-31-5p; B: The result of Double-Luciferase Reporter of MAP3K1; C: The result of Double-Luciferase Reporter of RASA1; D: Effects of MAP3K1 and RASA1 mRNA after overexpression of miR-31-5p; E, F: Effects of MAP3K1 and RASA1 protein after overexpression of miR-31-5p"

Fig. 3

The effects of overexpression of miR-31-5p on the proliferation and cell cycle of hair follicle stem cells A: Result of CCK-8结果; B: Result of EdU assay; C: Percentage of EdU-positive Cells; D: Periodic distribution diagram of Control; E: Periodic distribution diagram of pre-miR-31-5p; F: Comparison of cell number in G0/G1, S and G2/M phase between the Control and the pre-miR-31-5p"

Fig. 4

The effect of overexpression of miR-31-5p on the apoptosis of hair follicle stem cells A: Apoptosis profile of Control; B: Apoptosis profile of pre-miR-31-5p; C: Comparison of apoptosis rate between the Control and the pre-miR-31-5p"

Fig. 5

The effect of overexpression of miR-31-5p on proliferation and apoptosis related genes A: Effect of overexpression of miR-31-5p on the mRNA of proliferation and apoptosis related genes; B: Effect of overexpression of miR-31-5p on the proteins of proliferation and apoptosis related genes"

Fig. 6

The molecular mechanism of miR-31-5p"

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