生物技术通报 ›› 2022, Vol. 38 ›› Issue (10): 254-261.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0015
张浩1,2(), 何长晟1,2, 李艳艳1,2, 王永1,2(), 朱江江2, 俄木曲者3, 林亚秋1,2()
收稿日期:
2022-01-05
出版日期:
2022-10-26
发布日期:
2022-11-11
作者简介:
张浩,男,硕士研究生,研究方向:动物遗传育种与繁殖;E-mail:基金资助:
ZHANG Hao1,2(), HE Chang-sheng1,2, LI Yan-yan1,2, WANG Yong1,2(), ZHU Jiang-jiang2, Emu Quzhe3, LIN Ya-qiu1,2()
Received:
2022-01-05
Published:
2022-10-26
Online:
2022-11-11
摘要:
旨在明确miR-301b对山羊肌内脂肪细胞分化的调控作用,探讨其发挥调控作用的可能机制。利用化学合成的miR-301b mimics和miR-301b siRNA,通过脂质体转染法在山羊肌内脂肪细胞过表达和干扰miR-301b,并通过油红O染色、qPCR和生物信息学分析等方法,从细胞形态学和mRNA水平明确miR-301b对山羊肌内脂肪细胞分化的调控作用,并初步探究其可能的作用机制。结果表明,过表达miR-301b效率约29 290倍,细胞形态学结果显示,过表达miR-301b后山羊肌内脂肪细胞脂滴积聚减少,甘油三酯合成降低。qPCR结果显示,过表达miR-301b后成脂标志基因CEBPα、PPARγ、SREBP1、LPL表达水平极显著下调(P<0.01)。miR-301b干扰效率约60%,干扰miR-301b表达后,细胞形态学结果显示,山羊肌内脂肪细胞脂滴积聚增加,甘油三酯合成升高。qPCR结果显示,干扰miR-301b后成脂标志基因AP2、CEBPα、CEBPβ、PPARγ、SREBP1、LPL表达水平极显著上调(P<0.01)。通过对miR-301b生物信息学分析,推测KLF3为miR-301b的靶标基因,qPCR验证发现过表达miR-301b后显著抑制KLF3的mRNA表达水平(P<0.05),干扰miR-301b后显著促进KLF3的表达水平(P<0.05)。过表达miR-301b抑制山羊肌内前体脂肪细胞分化,同时KLF3表达水平下调;干扰miR-301b促进山羊肌内前体脂肪细胞分化,KLF3表达水平上调,推测miR-301b可能通过KLF3抑制山羊肌内脂肪细胞分化。
张浩, 何长晟, 李艳艳, 王永, 朱江江, 俄木曲者, 林亚秋. miR-301b对山羊肌内脂肪细胞分化的调控作用[J]. 生物技术通报, 2022, 38(10): 254-261.
ZHANG Hao, HE Chang-sheng, LI Yan-yan, WANG Yong, ZHU Jiang-jiang, Emu Quzhe, LIN Ya-qiu. Regulation of miR-301b on Goat Intramuscular Adipocyte Differentiation[J]. Biotechnology Bulletin, 2022, 38(10): 254-261.
名称Name | 序列Sequence(5'-3') |
---|---|
miR-301b mimics | CAGUGCAAUGAUAUUGUCAAAGC UUUGACAAUAUCAUUGCACUGUU |
mimics NC | UUCUCCGAACGUGUCACGUTTACGUGACACG- UUCGGAGAATT |
miR-301b Inhibitor | GCUUUGACAAUAUCAUUGCACUG |
Inhibitor NC | CAGUACUUUUGUGUAGUACAA |
表1 miRNA mimic和 inhibitor序列
Table 1 Sequences of miRNA mimic and inhibitor
名称Name | 序列Sequence(5'-3') |
---|---|
miR-301b mimics | CAGUGCAAUGAUAUUGUCAAAGC UUUGACAAUAUCAUUGCACUGUU |
mimics NC | UUCUCCGAACGUGUCACGUTTACGUGACACG- UUCGGAGAATT |
miR-301b Inhibitor | GCUUUGACAAUAUCAUUGCACUG |
Inhibitor NC | CAGUACUUUUGUGUAGUACAA |
基因 Gene/miRNA | 序列 Sequence(5'-3') | Tm/℃ | 产物长度 Product length/ bp | 登录号 GenBank No. |
---|---|---|---|---|
AP2 | TGAAGTCACTCCAGATGACAGGTGACACATTCCAGCACCAGC | 58 | 143 | NM_001285623.1 |
CEBPα | CCGTGGACAAGAACAGCAACAGGCGGTCATTGTCACTGGT | 58 | 142 | XM_018062278.1 |
CEBPβ | CAAGAAGACGGTGGACAAGCAACAAGTTCCGCAGGGTG | 66 | 204 | XM_018058020.1 |
PPARγ | AAGCGTCAGGGTTCCACTATG GAACCTGATGGCGTTATGAGAC | 60 | 197 | NM_001285658.1 |
Pref1 | CCGGCTTCATGGATAAGACCTGCCTCGCACTTGTTGAGGAA | 65 | 184 | KP686197.1 |
SREBP1 | AAGTGGTGGGCCTCTCTGA GCAGGGGTTTCTCGGACT | 58 | 127 | NM_001285755.1 |
KLF3 | CTCGGTGTCATACCCGTCTAATCCATCTGCATCCCGTGAGAC | 60 | 128 | KU041753.1 |
UXT | GCAAGTGGATTTGGGCTGTAACATGGAGTCCTTGGTGAGGTTGT | 60 | 180 | XM_005700842.2 |
U6 | TGGAACGCTTCACGAATTTGCGGGAACGATACAGAGAAGATTAGC | 60 | ||
miR-301b | CTGGAGCAGTGCAATGATGAAAGTGCAGGGTCCGAGGT | 58 |
表2 特异性引物序列信息
Table 2 Sequences information of specific primers
基因 Gene/miRNA | 序列 Sequence(5'-3') | Tm/℃ | 产物长度 Product length/ bp | 登录号 GenBank No. |
---|---|---|---|---|
AP2 | TGAAGTCACTCCAGATGACAGGTGACACATTCCAGCACCAGC | 58 | 143 | NM_001285623.1 |
CEBPα | CCGTGGACAAGAACAGCAACAGGCGGTCATTGTCACTGGT | 58 | 142 | XM_018062278.1 |
CEBPβ | CAAGAAGACGGTGGACAAGCAACAAGTTCCGCAGGGTG | 66 | 204 | XM_018058020.1 |
PPARγ | AAGCGTCAGGGTTCCACTATG GAACCTGATGGCGTTATGAGAC | 60 | 197 | NM_001285658.1 |
Pref1 | CCGGCTTCATGGATAAGACCTGCCTCGCACTTGTTGAGGAA | 65 | 184 | KP686197.1 |
SREBP1 | AAGTGGTGGGCCTCTCTGA GCAGGGGTTTCTCGGACT | 58 | 127 | NM_001285755.1 |
KLF3 | CTCGGTGTCATACCCGTCTAATCCATCTGCATCCCGTGAGAC | 60 | 128 | KU041753.1 |
UXT | GCAAGTGGATTTGGGCTGTAACATGGAGTCCTTGGTGAGGTTGT | 60 | 180 | XM_005700842.2 |
U6 | TGGAACGCTTCACGAATTTGCGGGAACGATACAGAGAAGATTAGC | 60 | ||
miR-301b | CTGGAGCAGTGCAATGATGAAAGTGCAGGGTCCGAGGT | 58 |
图1 过表达miR-301b对山羊肌内脂肪细胞形态学影响 A:miR-301b 过表达效率;B:油红O 染色和Bodipy 染色(×400);C:油红O 染色OD值;D:甘油三酯测定。*代表P<0.05,**代表P<0.01,下同
Fig.1 Effect of overexpressing miR-301b on the morphol-ogy of goat intramuscular adipocytes A:The overexpressing efficiency of miR-301b. B:Oil red O staining and Bodipy staining(×400). C:OD value of oil red O staining. D:Triglyceride(TG)content assay. * indicates P<0.05, ** indicates P<0.01, the same below
图3 干扰miR-301b 对山羊肌内脂肪细胞形态学影响 A:miR-301b效率;B:油红O 染色和Bodipy 染色(×400);C:油红O 染色OD值;D:甘油三酯测定
Fig. 3 Effect of interfering miR-301b on the morphology of goat intramuscular adipocytes A:Efficiency of miR-301b. B:Oil red O staining and Bodipy staining(×400).C:OD value of oil red O staining. D:Triglyceride(TG)content assay
图4 干扰miR-301b 山羊对肌内脂肪细胞分化标志基因表达的影响
Fig.4 Effect of interferring mir-301b goat on the expression of differentiation marker genes of intramuscular adipocytes
图5 miR-301b靶基因预测及表达水平检测 A:miR-301b靶基因预测韦恩图;B:KLF3潜在miRNA结合位点预测;C:过表达miR-301b后KLF3表达水平;D:干扰miR-301b表达后KLF3表达水平
Fig. 5 Prediction and expression level detection of miR-301b target gene A:miR-301b target gene prediction Venn diagram;B:KLF3 potential miRNA binding site prediction;C:KLF3 expression level after simulating miR-301b expression;D:KLF3 expression level after interfering with miR-301b expression
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