中国农业科学 ›› 2021, Vol. 54 ›› Issue (23): 5132-5143.doi: 10.3864/j.issn.0578-1752.2021.23.017
收稿日期:
2020-11-02
接受日期:
2021-03-11
出版日期:
2021-12-01
发布日期:
2021-12-06
通讯作者:
李拥军
作者简介:
冯云奎,E-mail: 基金资助:
FENG YunKui(),WANG Jian,MA JinLiang,ZHANG LiuMing,LI YongJun(
)
Received:
2020-11-02
Accepted:
2021-03-11
Online:
2021-12-01
Published:
2021-12-06
Contact:
YongJun LI
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。
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.
表1
荧光定量PCR引物序列"
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 |
表2
构建载体的引物序列"
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 |
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