Regulation of miR-301b on Goat Intramuscular Adipocyte Differentiation

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

Abstract

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

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.

Figures/Tables 7

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

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

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

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

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

Fig.4 Effect of interferring mir-301b goat on the expression of differentiation marker genes of intramuscular adipocytes

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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