干扰山羊KLF12促进皮下脂肪细胞分化

doi:10.3864/j.issn.0578-1752.2022.01.015

Abstract

Abstract:

【Background】Subcutaneous adipose tissue (SAT) under the skin is an important factor affecting the taste of meat. Exploring the molecular regulation mechanisms of SAT deposition is very important for breeding improvement and the development of animal husbandry. Krüppel-like factors 12 (KLF12) is a conserved transcription factor that evolutionarily conserved, and it was found that it could be expressed in a variety of cell types and control a wide range of cellular processes. 【Objective】 This study aimed to obtain the coding sequence (CDS) of goat KLF12 and to explore its molecular characteristics. Moreover, the study also intended to clarify the expression pattern of KLF12 in goat tissues and subcutaneous adipocytes, and to explore the role of KLF12 in goat subcutaneous preadipocytes differentiation via interference KLF12, so as to provide a theoretical basis for further research on the potential role of KLF12 in the process of fat deposition. 【Method】 In this study, the goat KLF12 CDS sequence was cloned by Reverse Transcription PCR ( RT-PCR) method, and the nucleotide sequence and amino acid sequence of goat KLF12 were analyzed on online bioinformatics analysis software. The Quantitative Real-time PCR (qRT-PCR) technology was used to detect the expression levels of KLF12 in goat heart, liver, abdominal fat, subcutaneous fat, triceps brachii, longissimus dorsi and other 14 tissues. Furthermore, the expression level of KLF12 in subcutaneous preadipocytes in different differentiation periods was investigated. Then, the goat KLF12 small interfering RNA (si-KLF12) was chemically synthesized and transfected into goat subcutaneous preadipocyte in vitro by using Lipofectamine RNAiMAX transfection reagent. Subsequently, 100 µmol·L^-1 oleic acid induced adipocyte differentiation. Oil red O and Bodipy staining methods and qRT-PCR techniques were used to clarify the effects of interference KLF12 on the accumulation of lipid droplets in subcutaneous preadipocytes and the mRNA expression levels of adipose differentiation marker genes from the perspectives of morphology and molecular biology. 【Result】 The goat KLF12 (1 315 bp) were successfully obtained, which contained an Open Reading Frame (ORF) (1 209 bp) and encoded 402 amino acids. The subcellular localization results showed that KLF12 was mainly located in the nucleus. In addition, KLF12 had no transmembrane domain and signal peptide but 3 typical zinc finger domains (ZnF_C2H2) at amino acids 317-341, 347-371 and 377-399. Tissue expression profile showed that the expression level of KLF12 in goats’ heart and spleen were significantly higher than that in other tissues (P<0.01). Moreover, during subcutaneous preadipocytes differentiation, the expression level of KLF12 was peaked at 60 h. After transfection of si-KLF12 into goat subcutaneous preadipocytes, the results of oil red O and Bodipy saining showed that accumulation of lipid droplets in adipocytes were significantly increased. At the same time, the results of qRT-PCR showed that the expression levels of key adipogenic regulatory genes like lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor γ (PPARγ) were significantly increased (P<0.05), while the expression level of preadipocyte growth factor (Pref-1) was extremely significantly reduced (P<0.01). Combined with the morphological observation results and the changes in the expression levels of key adipogenic regulatory genes, it was speculated that KLF12 played a negative regulatory role in the differentiation of subcutaneous adipocytes. 【Conclusion】 By investigating the basic molecular biological characteristics and its expression pattern between tissues and cells of goat KLF12 and analyzing the potential regulatory effects of KLF12 on differentiation process of goat subcutaneous adipocytes, the results suggested that KLF12 played a negative role in goats subcutaneous preadipocytes differentiation, and this effect achieved by regulating LPL, PPARγ and Pref-1, which laid a foundation for further exploring the molecular mechanism of KLF12 in regulating the differentiation of adipocytes.

Key words: goat, KLF12, molecular characteristics, knockdown, subcutaneous adipocytes

DU Yu,WANG Yong,MENG QingYong,ZHU JiangJiang,LIN YaQiu. Knockdown Goat KLF12 to Promote Subcutaneous Adipocytes Differentiation[J].Scientia Agricultura Sinica, 2022, 55(1): 184-196.

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References 44

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名称 Name	引物序列5°至3° Primer Sequence	退火温度 Tm (℃)	产物长度 Products length (bp)	用途 Purpose	登录号 miRbase ID
KLF12	S: TTAGCGCATCATGTGATCCG A: TGGGGTGCCGCTAAGAGAT	58	1315	RT-PCR	XM_005687692.2
KLF12	S: TCTAAGGTCACATTTGGCAGGTC A: CCAATCGGTGCCTGTTGTCTAC	61	183	qRT-PCR	KX247669.1
UXT	S: GCAAGTGGATTTGGGCTGTAAC A: ATGGAGTCCTTGGTGAGGTTGT	60	180	qRT-PCR	XP_005700899.1

名称 Name	引物序列5°至3° Primer sequence	退火温度 Tm (℃)	产物长度 Products length (bp)	用途 Purpose	登录号 miRbase ID
si-KLF12	UGGACAAGUCCACUGGCUCAGUUUG			siRNA
Negative Control	S: UUCUCCGAACGUGUCACGUTT A: ACGUGACACGUUCGGAGAATT			RNAi
LPL	S: TCCTGGAGTGACGGAATCTGT A: GACAGCCAGTCCACCACGAT	60	174	qPCR	NM_001285607.1
PPARγ	S: AAGCGTCAGGGTTCCACTATG A: GAACCTGATGGCGTTATGAGAC	60	197	qPCR	NM_001285658.1
AP2	S: TGAAGTCACTCCAGATGACAGG A: TGACACATTCCAGCACCAGC	58	143	qPCR	NM_001285623.1
C/EBPβ	S: CAAGAAGACGGTGGACAAGC A: AACAAGTTCCGCAGGGTG	66	204	qPCR	XM_018058020.1
Pref1	S: CCGGCTTCATGGATAAGACCT A: GCCTCGCACTTGTTGAGGAA	65	178	qPCR	KP686197.1
SREBP1	S: AAGTGGTGGGCCTCTCTGA A: GCAGGGGTTTCTCGGACT	58	127	qPCR	NM_001285755.1
UXT	S: GCAAGTGGATTTGGGCTGTAAC A: ATGGAGTCCTTGGTGAGGTTGT	60	180	qPCR	XP_005700899.1

Knockdown Goat KLF12 to Promote Subcutaneous Adipocytes Differentiation

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