miR-301b对山羊肌内脂肪细胞分化的调控作用

doi:10.13560/j.cnki.biotech.bull.1985.2022-0015

摘要/Abstract

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。

关键词: 山羊, miR-301b, KLF3, 肌内脂肪细胞, 分化

Abstract:

The purpose of this experiment is to clarify the regulatory effect of miR-301b on goat intramuscular adipocyte differentiation and to explore the possible mechanism of its regulatory effect. Chemically-synthesized miR-301b mimics and miR-301b siRNA were used to overexpress and interfere miR-301b in goat intramuscular adipocytes by liposome transfection，and the regulatory effect of miR-301b on goat intramuscular adipocyte differentiation was clarified from the cell morphology and mRNA level by oil red O staining，qPCR and bioinformatics analysis，and its possible mechanism was explored preliminary. The results showed that the overexpression efficiency of miR-301b was about 29 290 times. The results of cell morphology showed that the accumulation of lipid droplets and triglyceride synthesis of goat intramuscular adipocytes decreased after the overexpression of miR-301b. qPCR results showed that expressions of lipogenic marker gene CEBPα，PPARγ，SREBP1 and LPL in the overexpressed miR-301b significantly reduced（P<0.01）. The interference efficiency of miR-301b was about 60%. After the expression of miR-301b was interfered，the results of cell morphology showed that the accumulation of lipid droplets in goat intramuscular adipocytes increased and the synthesis of triglycerides increased. qPCR results showed that interfering miR-301b significantly promoted expressions of adipogenic marker gene AP2，CEBPα，CEBPβ，PPARγ，SREBP1 and LPL. Through bioinformatics analysis of miR-301b，it was speculated that KLF3 was the target gene of miR-301b. qPCR verification found that overexpression of miR-301b significantly inhibited the mRNA expression of KLF3，and interference to miR-301b significantly promoted the expression of KLF3. In conclusion，overexpression of miR-301b inhibited the differentiation of intramuscular preadipocytes in goats，and down-regulated the expression of KLF3；interfering miR-301b promoted the differentiation of intramuscular preadipocytes in goats，and the expression of KLF3 was up-regulated. Thus it is speculated that miR-301b may inhibit the differentiation of goat intramuscular adipocytes through KLF3.

Key words: goat, miR-301b, KLF3, intramuscular adipocytes, differentiation

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.

图/表 7

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

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2 miR-301b 山羊对肌内脂肪细胞分化标志基因表达的影响

Fig.2 Effects of miR-301b goat on the expressions of diffe-rentiation marker genes of intramuscular adipocytes

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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