小尾寒羊SP1基因克隆及其对前体脂肪细胞分化的影响

doi:10.16431/j.cnki.1671-7236.2021.05.005

Abstract

Abstract: The aim of this experiment was to clone the specificity protein 1 (SP1) gene,analyze its sequence,and investigate the effect of SP1 gene on the preadipocytes differentiation of Small-tailed Han sheep.The 1-month-old adipose tissue in the tail of Small-tailed Han sheep was selected for the isolation,culture and induction differentiation of preadipocytes.SP1 gene CDS was cloned by overlapping PCR.Bioinformatics softwares were used to analyze the SP1 gene CDS,and predict the structure,functional domain,subcellular localization and post-translational modification of SP1 protein.Overexpression and inhibition of SP1 gene in preadipocytes was performed by lentivirus infection.The overexpression and antisense vectors of SP1 gene and control plasmids packed with lentivirus were transfected into 293T cells,and the preadipocytes of Small-tailed Han sheep were infected with lentiviruses.Oil Red O staining was used to observe the ability of lipid accumulation in adipocytes.The mRNA expression of SP1 gene and adipogenic marker genes were detected by Real-time quantitative PCR.The results showed that the full-length of SP1 gene CDS was 2 340 bp,encoding 779 amino acids.Sequence similarity analysis showed that SP1 gene CDS of Small-tailed Han sheep had the highest similarity with Ovis aries and Capra hircas,followed by Bos taurus,Equus caballus,Sus scrofa,Homo sapiens,Mus musculus and Rattus norvegicus,and the lowest similarity with Gallus gallus,which was consistent with the result of phylogenetic analysis.The SP1 protein was located in the nucleus with 109 phosphorylation sites.Its secondary structure consisted of alpha helix,beta turn,radom coil and extended chain,and the proportion of them were 16.94%,8.60%,54.17% and 20.28%,respectively.Three zinc finger domains were found in both secondary and tertiary structures.The overexpression,interference and control plasmids were transfected into 293T cells,and the cells in each group presented strong green fluorescence,which indicated the vector was successfully transfected into 293T cells.After the lentiviruses infects the preadipocytes of the Small-tail Han sheep,the mRNA expression of SP1 gene in overexpression group was extremely significant higher than that in control group (P<0.01),while the expression of SP1 gene mRNA in inhibition group was extremely significant lower than that in control group (P<0.01).After SP1 gene was overexpressed,the mRNA expression of lipogenic marker genes were extremely significant up-regulated (P<0.01),and more lipid droplets were deposited.The results were opposite after SP1 gene was inhibited.Therefore,SP1 gene had a positive regulatory effect on the differentiation of preadipocyte in the caudal adipose tissue of Small-tailed Han sheep.

Key words: Small-tailed Han sheep; SP1 gene; cloning; preadipocyte; differentiation

CLC Number:

S811.3

WANG Feng, ZHAO Bishi, LIU Xuying, LIANG Yu, QIAO Liying, LIU Wenzhong, LIU Jianhua. Cloning of SP1 Gene and Its Effect on Preadipocytes Differentiation in Small-tailed Han Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(5): 1544-1557.

References

[1] BINGOL M,AYGUN T,GOKDAL O,et al.The effects of docking on fattening performance and carcass characteristics in fat-tailed Norduz male lambs[J].Small Ruminant Research,2006,64(1-2):101-106.
[2] TAO Z,LIU L,ZHENG L D,et al.Autophagy in adipocyte differentiation[J].Methods in Molecular Biology,2019,1854:45-53.
[3] MOTA DE SÁ P,RICHARD A J,HANG H,et al.Transcriptional regulation of adipogenesis[J].Compre-hensive Physiology,2017,7(2):635-674.
[4] POULOS S P,DODSON M V,CULVER M F,et al.The increasingly complex regulation of adipocyte differentiation[J].Experimental Biology and Medicine,2016,241(5):449-456.
[5] VAN VLIET J,CROFTS L A,QUINLAN K G R,et al.Human KLF17 is a new member of the SP/KLF family of transcription factors[J].Genomics,2006,87(4):474-482.
[6] PHILIPSEN S,SUSKE G.A tale of three fingers:The family of mammalian SP/XKLF transcription factors[J].Nucleic Acids Research,1999,27(15):2991-3000.
[7] KADONAGA J T,CARNER K R,MASIARZ F R,et al.Isolation of cDNA encoding transcription factor SP1 and functional analysis of the DNA binding domain[J].Cell,1987,51(6):1079-1090.
[8] BRIGGS M R,KADONAGA J T,BELL S P,et al.Purification and biochemical characterization of the promoter-specific transcription factor,SP1[J].Science,1986,234(4772):47-52.
[9] ZHOU B,HUANG W H,CHEN S,et al.GDF15 serves as a co-activator to enhance KISS-1 gene transcription through interacting with SP1[J].Carcinogenesis,2020,23:bgaa103.
[10] LEE S Y,YANG J,PARK J H,et al.The microRNA-92a/SP1/MyoD axis regulates hypoxic stimulation of myogenic lineage differentiation in mouse embryonic stem cells[J].Molecular Therapy,2020,28(1):142-156.
[11] CAI H,LIU B,WANG H,et al.SP1 governs primordial folliculogenesis by regulating pregranulosa cell development in mice[J].Journal of Molecular Cell Biology,2020,12(3):230-244.
[12] LOSHCHENOVA P S,SERGEEVA S V,LIMONOV D V,et al.SP1-independent downregulation of NHEJ in response to BER deficiency[J].DNA Repair,2020,86:102740.
[13] ZHU J J,LUO J,XU H F,et al.Short communication:Altered expression of specificity protein 1 impairs milk fat synthesis in goat mammary epithelial cells[J].Journal of Dairy Science,2016,99(6):4893-4898.
[14] LV X,SUN W,ZOU S,et al.Characteristics of the BMP7 promoter in Hu sheep[J].Animals,2019,9(11):874.
[15] TSENG Y H,KOKKOTOU E,SCHULZ T J,et al.New role of bone morphogenetic protein 7 in brown adipogenesis and energy expenditure[J].Nature,2008,454(7207):1000-1004.
[16] FANG Q,YANG W,LI H,et al.Negative regulation of DsbA-L gene expression by the transcription factor SP1[J].Diabetes,2014,63(12):4165-4171.
[17] RUIZ DE EGUINO G,INFANTE A,SCHLANGEN K,et al.SP1 transcription factor interaction with accumulated prelamin a impairs adipose lineage differentiation in human mesenchymal stem cells:Essential role of SP1 in the integrity of lipid vesicles[J].Stem Cells Translational Medicine,2012,1(4):309-321.
[18] LU S,ARCHER M C.SP1 coordinately regulates de novo lipogenesis and proliferation in cancer cells[J].International Journal of Cancer,2010,126(2):416-425.
[19] CHEN S,HU Z,HE H,et al.Fatty acid elongase7 is regulated via SP1 and is involved in lipid accumu-lation in bovine mammary epithelial cells[J].Journal of Cellular Physiology,2018,233(6):4715-4725.
[20] NAGANUMA T,SATO Y,SASSA T,et al.Biochemical characterization of the very long-chain fatty acid elongase ELOVL7[J].FEBS Letters,2011,585(20):3337-3341.
[21] 王强,潘洋洋,乔利英,等.miR-33a靶向Lipin1和IRS2调节绵羊前体脂肪细胞分化的研究[J].畜牧兽医学报,2020,51(4):701-712. WANG Q,PAN Y Y,QIAO L Y,et al.miR-33a regulates ovine preadipocyte differentiation by targeting Lipin1 and IRS2[J].Acta Veterinaria et Zootechnica Sinica,2020,51(4):701-712.(in Chinese)
[22] SHI T,YAN X,QIAO L,et al.miR-330-5p negatively regulates ovine preadipocyte differentiation by targeting branched-chain aminotransferase 2[J].Animal Science Journal,2018,89(6):858-867.
[23] 姚一龙,李隐侠,安外尔·热合曼,等.湖羊SP1基因CDS区克隆及其对颗粒细胞增殖和凋亡的影响[J].畜牧兽医学报,2017,48(11):2098-2106. YAO Y L,LI Y X,REHEMAN A,et al.Cloning of SP1 gene CDS region of Hu sheep and its effect on proliferation and apoptosis of granulosa cells[J].Acta Veterinaria et Zootechnica Sinica,2017,48(11):2098-2106.(in Chinese)
[24] 杨红,郑晓珂.生物化学[M].北京:中国医药科技出版社,2016. YANG H,ZHEN X H.Biochemistry[M].Beijing:China Medical Science Press,2016.(in Chinese)
[25] BRUNO M,MAHGOUB M,MACFARLAN T S.The arms race between KRAB-Zinc finger proteins and endogenous retroelements and its impact on mammals[J].Annual Review of Genetics,2019,53:393-416.
[26] 张广杰,崔悦悦,邱庆庆,等.广西巴马小型猪SP1基因克隆测序及其真核表达载体的构建[J].南方农业学报,2018,49(2):360-366. ZHANG G J,CUI Y Y,QIU Q Q,et al.Clone sequencing of gene SP1 in Guangxi Bama mini pig and construction of its eukaryotic expression vector[J].Journal of Southern Agriculture,2018,49(2):360-366.(in Chinese)
[27] 郝光.草鱼(Ctenopharyngodon idellus) SP1的克隆及其表达研究[D].新乡:河南师范大学,2014. HAO G.Cloning and expression of transcription factor SP1 from Ctenopharyngodon idellus[D].Xinxiang:Henan Normal University,2014.(in Chinese)
[28] LIN C J,HSIAO T H,CHUNG Y S,et al.Zebrafish SP1-like protein is structurally and functionally comparable to human SP1[J].Protein Expression and Purification,2011,76(1):36-43.
[29] GRAVES J D,KREBS E G.Protein phosphorylation and signal transduction[J].Pharmacology and Therapeutics,1999,82(2-3):111-121.
[30] HUNTER T.Signaling——2000 and beyond[J].Cell,2000,100(1):113-127.
[31] DUPUIS-MAURIN V,BRINZA L,BAGUET J,et al.Overexpression of the transcription factor SP1 activates the OAS-RNAse L-RIG-I pathway[J].PLoS One,2015,10(3):e0118551.
[32] LIU T,LI Y,SU H,et al.Nuclear localization of the tyrosine kinase BMX mediates VEGFR2 expression[J].Journal of Cellular and Molecular Medicine,2020,24(1):126-138.
[33] PRIETO-HONTORIA P L,PÉREZ-MATUTE P,FERNÁNDEZ-GALILEA M,et al.Lipoic acid inhibits leptin secretion and SP1 activity in adipocytes[J].Molecular Nutrition & Food Research,2011,55(7):1059-1069.
[34] CHUANG J Y,WANG S A,YANG W B,et al.SP1 phosphorylation by cyclin-dependent kinase 1/cyclin B1 represses its DNA-binding activity during mitosis in cancer cells[J].Oncogene,2012,31(47):4946-4959.
[35] WANG Q,PAN Y,ZHAO B,et al.miR-33a inhibits the adipogenic differentiation of ovine adipose-derived stromal vascular fraction cells by targeting SIRT6[J].Domestic Animal Endocrinology,2020,74:106513.
[36] WANG X,LIU Y,NI Y,et al.Lentivirus vector-mediated knockdown of SOX9 shows neuroprotective effects on light damage in rat retinas[J].Molecular Vision,2019,25:703-713.
[37] 范少鹏,李晓辉,时彩霞,等.慢病毒介导BMP-2过表达质粒转染骨髓间充质干细胞联合丝素蛋白支架向成骨细胞转化的实验研究[J].中国骨伤,2019,32(9):853-860. FAN S P,LI X H,SHI C X,et al.Lentivirus mediated BMP-2 overexpression plasmid transfection into bone marrow mesenchymal stem cells combined with silk fibroin scaffold for osteoblast transformation[J].China Journal of Orthopaedics and Traumatology,2019,32(9):853-860.(in Chinese)
[38] 蔡晗.转录因子SP1调控小鼠原始卵泡形成的机制研究[D].北京:中国农业大学,2019. CAI H.The role and the mechanism of transcription factor SP1 in the formation of primordial follicles in mice[D].Beijing:China Agricultural University,2019.(in Chinese)
[39] 邓渊润.SP1对宫颈癌放疗敏感性的影响及其机制研究[D].广州:南方医科大学,2017. Deng Y R.The role and mechanism of SP1 on the radiosensitivity of cervical cancer cell lines[D].Guangzhou:Southern Medical University,2017.(in Chinese)
[40] 宋慧胜,邱惠思,吴华振,等.特异性蛋白1(SP1)上调FOXP4-AS1促进结直肠癌细胞增殖[J].中国生物化学与分子生物学报,2019,35(3):304-309. SONG H S,QIU H S,WU H Z,et al.Specificity protein 1(SP1) upregulated FOXP4-AS1 to promote the proliferation of colorectal cancer cells[J].Chinese Journal of Biochemistry and Molecular Biology,2019,35(3):304-309.(in Chinese)
[41] 董庆生,张瑞,刘俊,等.转录因子SP1-siRNA对胶质瘤细胞系U251凋亡与增殖的影响[J].江苏医药,2014,40(4):373-376. DONG Q S,ZHANG R,LIU J,et al.Effects of SP1-siRNA on apoptosis and proliferation of glioblastoma U251 cells[J].Jiangsu Medical Journal,2014,40(4):373-376.(in Chinese)
[42] YUAN J,TANG Z,YANG S,et al.CRABP2 promotes myoblast differentiation and is modulated by the transcription factors MyoD and SP1 in C2C12 cells[J].PLoS One,2013,8(1):e55479.
[43] 汤峰林,丁涛,朱浩明,等.miR-375靶向SP1调控骨肉瘤细胞增殖凋亡[J].中国老年学杂志,2018,38(21):5308-5311. TANG F L,DING T,ZHU H M,et al.miR-375 targets SP1 to regulate the proliferation and apoptosis of osteosarcoma cells[J].Chinese Journal of Gerontology,2018,38(21):5308-5311.(in Chinese)
[44] 郭剑津.人肥胖基因(FTO)的表达调控及其参与氧化应激对脂肪肝形成的影响[D].南京:南京医科大学,2013. GUO J J.Expression,transcriptional regulation and effect on NFALD of human FTO gene[D].Nanjing:Nanjing Medical University,2013.(in Chinese)
[45] FONSECA A C P D,MARCHESINI B,ZEMBRZUSKI V M,et al.Genetic variants in the fat mass and obesity-associated (FTO) gene confer risk for extreme obesity and modulate adiposity in a Brazilian population[J].Genetics and Molecular Biology,2020,43(1):e20180264.
[46] ROY D,FARABAUGH K T,WU J,et al.Coordinated transcriptional control of adipocyte triglyceride lipase (Atgl) by transcription factors SP1 and peroxisome proliferator-activated receptor γ (PPARγ) during adipocyte differentiation[J].Journal of Biological Chemistry,2017,292(36):14827-14835.
[47] SCHREIBER R,XIE H,SCHWEIGER M.Of mice and men:The physiological role of adipose triglyceride lipase (ATGL)[J].Biochimica et Biophysica Acta Molecular and Cell Biology of Lipids,2019,1864(6):880-899.
[48] ZHANG X M,WANG L H,SU D J,et al.microRNA-29b promotes the adipogenic differentia-tion of human adipose tissue-derived stromal cells[J].Obesity (Silver Spring,Md.),2016,24(5):1097-1105.

Cloning of SP1 Gene and Its Effect on Preadipocytes Differentiation in Small-tailed Han Sheep

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