皱皮香猪泛素化连接酶2基因结构变异的差异表达研究

doi:10.11843/j.issn.0366-6964.2025.01.013

Abstract

Abstract:

The present study aimed to explore whether the structural variation in CUL2 affected the gene expression and wrinkle severity on skin of Xiang pigs. The 200 skins and blood samples were collected from Xiang pigs with wrinkled skin and common Xiang pigs in 6 months old. The polymorphic distribution of the CUL2-I3-sv300 site was examined using PCR method. Based on the prediction of potential functional elements within the structural variation region, and the interaction between CUL2 gene and other related genes by softwares online such as UCSC, the expression levels of CUL2 and those collagen formation-related genes were determined using RT-qPCR and Western blot methods. The results showed that the structural variation CUL2-I3-sv300 exhibited abundant polymorphism within the pig populations. Genotypes of the mutant (MM), the heterozygous (WM) and the wild (WW) were detected from skins of the common Xiang pigs, while type WW was not detected from the Xiang pig with wrinkled skin. Furthermore, the allele M was significantly more abundant in Xiang pigs with wrinkled skin compared with that in the common Xiang pigs (P < 0.05). Based on the bioinformatics analysis, several elements were screened from the structural variation region including a short interspersed nuclear element (SINE), three intronic splicing silencers (ISS) and four RNA binding protein sites (RBPs). And CUL2 might regulate HIF-1α, indirectly change VEGFA, MMP-1, and MMP-9, and eventually affect the formation of skin collagen. After detection by using both of the wrinkled skin and common skin, the mRNA and protein levels of CUL2 expression were higher in the wrinkled skin with MM genotypes than that with other genotypes in wrinkled and common skins. Moreover, the expression of CUL2, MMP-1 and MMP-9 were up-regulated, while mRNA levels of HIF-1α, VEGFA, COL1A1, COL3A1 and COL6A3 genes were down-regulated in Xiang pig with wrinkled skin compared with samples from the common Xiang pig.It is suggested that the loss of those functional elements in the structural variation region might promote the expression of CUL2 while inhibit collagen formation, thereby aggravating the severity of skin wrinkles on Xiang pig body.

Key words: Xiang pigs with wrinkled skin, CUL2 gene, collagen, wrinkled skin, bioinformatics analysis

CLC Number:

S828.2

ZHOU Xianshan, HUANG Shihui, NIU Xi, RAN Xueqin, WANG Jiafu. Differential Expression Study of Structural Variation in the Ubiquitin Ligase 2 Gene of Xiang Pigs with Wrinkled Skin[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 136-146.

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References 48

1	ZHANGS B,DUANE K.Fighting against skin aging: the way from bench to bedside[J].Cell Transplant,2018,27(5):729-738. doi: 10.1177/0963689717725755
2	KIMJ,LEES G,LEEJ,et al.Oral supplementation of low-molecular-weight collagen peptides reduces skin wrinkles and improves biophysical properties of skin: a randomized, double-blinded, placebo-controlled study[J].J Med Food,2022,25(12):1146-1154. doi: 10.1089/jmf.2022.K.0097
3	ADDORF A S.Beyond photoaging: additional factors involved in the process of skin aging[J].Clin Cosmet Investig Dermatol,2018,11,437-443. doi: 10.2147/CCID.S177448
4	POONF,KANGS,CHIENA L.Mechanisms and treatments of photoaging[J].Photodermatol Photoimmunol Photomed,2015,31(2):65-74. doi: 10.1111/phpp.12145
5	GONZALOS,KREIENKAMPR,ASKJAERP.Hutchinson-Gilford Progeria Syndrome: a premature aging disease caused by LMNA gene mutations[J].Ageing Res Rev,2017,33,18-29. doi: 10.1016/j.arr.2016.06.007
6	LAWM H,MEDLANDS E,ZHUG,et al.Genome-wide association shows that pigmentation genes play a role in skin aging[J].J Invest Dermatol,2017,137(9):1887-1894. doi: 10.1016/j.jid.2017.04.026
7	ABITBOLM,BOSSÉP,THOMASA,et al.A deletion in FOXN1 is associated with a syndrome characterized by congenital hypotrichosis and short life expectancy in Birman cats[J].PLoS One,2015,10(3):e0120668. doi: 10.1371/journal.pone.0120668
8	DUQUE LASIOM L,KOZELB A.Elastin-driven genetic diseases[J].Matrix Biol,2018,71-72,144-160. doi: 10.1016/j.matbio.2018.02.021
9	徐婷婷,齐芬芳,黄世会,等.香猪MAP3K4基因结构变异多态性和基因表达研究[J].畜牧兽医学报,2023,54(12):5046-5055.
	XUT T,QIF F,HUANGS H,et al.Study of the polymorphisms of structural variation and the expression of MAP3K4 gene in Xiang pig[J].Acta Veterinaria et Zootechnica Sinica,2023,54(12):5046-5055.
10	FLOODK S,HOUSTONN A,SAVAGEK T,et al.Genetic basis for skin youthfulness[J].Clin Dermatol,2019,37(4):312-319. doi: 10.1016/j.clindermatol.2019.04.007
11	CSEKESE,RAČOVÁL.Skin aging, cellular senescence and natural polyphenols[J].Int J Mol Sci,2021,22(23):12641. doi: 10.3390/ijms222312641
12	ORIOLID,DELLAMBRAE.Epigenetic regulation of skin cells in natural aging and premature aging diseases[J].Cells,2018,7(12):268. doi: 10.3390/cells7120268
13	LIUX L,HUF B,HUANGS H,et al.Detection of genomic structure variants associated with wrinkled skin in Xiang pig by next generation sequencing[J].Aging (Albany NY),2021,13(22):24710-24739.
14	HUANGT,ZHANGM P,YANG R,et al.Genome-wide association and evolutionary analyses reveal the formation of swine facial wrinkles in Chinese Erhualian pigs[J].Aging (Albany NY),2019,11(13):4672-4687.
15	ZHAOP J,YUY,FENGW,et al.Evidence of evolutionary history and selective sweeps in the genome of Meishan pig reveals its genetic and phenotypic characterization[J].Gigascience,2018,7(5):giy058. doi: 10.1093/gigascience/giy058
16	胡冯彬. 香猪皱皮性状的转录组学研究[D]. 贵阳: 贵州大学, 2020.
	HU F B. Transcriptomic research on the wrinkled skin of Xiang Pigs[D]. Guiyang: Guizhou University, 2020. (in Chinese)
17	张宏蕾,赵卫红.E3泛素连接酶CUL2在宫颈癌中的研究进展[J].国际妇产科学杂志,2021,48(2):121-125.
	ZHANGH L,ZHAOW H.Research progress of E3 ubiquitin ligase CUL2 in cervical cancer[J].Journal of International Obstetrics and Gynecology,2021,48(2):121-125.
18	FRÖHLICHL F.Micrornas at the interface between osteogenesis and angiogenesis as targets for bone regeneration[J].Cells,2019,8(2):121. doi: 10.3390/cells8020121
19	GAOS,LIUL F,LIZ L,et al.Seven novel genes related to cell proliferation and migration of VHL-mutated pheochromocytoma[J].Front Endocrinol (Lausanne),2021,12,598656. doi: 10.3389/fendo.2021.598656
20	ZIERENR C,DONGL,CLARKD J,et al.Defining candidate mRNA and protein EV biomarkers to discriminate ccRCC and pRCC from non-malignant renal cells in vitro[J].Med Oncol,2021,38(9):105. doi: 10.1007/s12032-021-01554-2
21	张蕊蕊. 猪基因组中SINE多态性与肌肉组织eQTL信号的联合分析及验证[D]. 武汉: 华中农业大学, 2021.
	ZHANG R R. Joint analysis and validation of sine polymorphism with muscle tissue eQTL signaling in the porcine genome[D]. Wuhan: Huazhong Agricultural University, 2021. (in Chinese)
22	SONGZ X,SHAHS,LVB X,et al.Anti-aging and anti-oxidant activities of murine short interspersed nuclear element antisense RNA[J].Eur J Pharmacol,2021,912,174577. doi: 10.1016/j.ejphar.2021.174577
23	李兰会,杜小龙,王麒,等.601 bp SINE插入突变导致水貂Agouti基因沉默[J].农业生物技术学报,2019,27(2):297-306.
	LIL H,DUX L,WANGQ,et al.Gene silencing of mink (Neovison vison) Agouti induced by 601 bp SINE insertion mutation[J].Journal of Agricultural Biotechnology,2019,27(2):297-306.
24	WANGX Y,CHIC L,HEJ,et al.SINE insertion may act as a repressor to affect the expression of pig LEPROT and growth traits[J].Genes (Basel),2022,13(8):1422. doi: 10.3390/genes13081422
25	ZHAOC,XIEW,ZHUH C,et al.LncRNAs and their RBPs: how to influence the fate of stem cells?[J].Stem Cell Res Ther,2022,13(1):175. doi: 10.1186/s13287-022-02851-x
26	JUNGH,LEEK S,CHOIJ K.Comprehensive characterisation of intronic mis-splicing mutations in human cancers[J].Oncogene,2021,40(7):1347-1361. doi: 10.1038/s41388-020-01614-3
27	JACOBA G,SMITHC W J.Intron retention as a component of regulated gene expression programs[J].Hum Genet,2017,136(9):1043-1057. doi: 10.1007/s00439-017-1791-x
28	HUAY M,VICKERST A,OKUNOLAH L,et al.Antisense masking of an hnRNP A1/A2 intronic splicing silencer corrects SMN2 splicing in transgenic mice[J].Am J Hum Genet,2008,82(4):834-848. doi: 10.1016/j.ajhg.2008.01.014
29	STATELLOL,MAUGERIM,GARREE,et al.Identification of RNA-binding proteins in exosomes capable of interacting with different types of RNA: RBP-facilitated transport of RNAs into exosomes[J].PLoS One,2018,13(4):e0195969. doi: 10.1371/journal.pone.0195969
30	CHABOTB,SHKRETAL.Defective control of pre-messenger RNA splicing in human disease[J].J Cell Biol,2016,212(1):13-27. doi: 10.1083/jcb.201510032
31	李爱民. 中国地方黄牛ANGPTL6基因遗传变异, 可变剪切及克隆表达研究[D]. 杨凌: 西北农林科技大学, 2012.
	LI A M. Study of genetic variation, alternative splicing, cloning, and expression of the ANGPTL6 gene in Chinese native cattle[D]. Yangling: Northwest A & F University, 2012. (in Chinese)
32	ANNAA,MONIKAG.Splicing mutations in human genetic disorders: examples, detection, and confirmation[J].J Appl Genet,2018,60(2):231.
33	XUB Q,WANGR H,ZHANGJ S,et al.Bioinformatics analysis of CUL2/4A/9 and its function in head and neck squamous cell carcinoma[J].Endokrynol Pol,2023,74(3):315-330. doi: 10.5603/EP.a2023.0029
34	CHIP L,CHENGC C,WANGM T,et al.Induced pluripotent stem cell-derived exosomes attenuate vascular remodelling in pulmonary arterial hypertension by targeting HIF-1α and Runx2[J].Cardiovasc Res,2024,120(2):203-214. doi: 10.1093/cvr/cvad185
35	张虔,崔燕,余四九,等.幼年牦牛不同部位皮肤毛囊的组织学结构及TGF-β2与HIF-1α差异性表达分析[J].畜牧兽医学报,2021,52(11):3224-3233. doi: 10.11843/j.issn.0366-6964.2021.011.024
	ZHANGQ,CUIY,YUS J,et al.Histological of hair follicles from different parts of skin and expression of TGF-β2 and HlF-1α in Young Yak (Bos grunniens)[J].Acta Veterinaria et Zootechnica Sinica,2021,52(11):3224-3233. doi: 10.11843/j.issn.0366-6964.2021.011.024
36	RAOV H,KANSALV,STOUPAS,et al.MMP-1 and MMP-9 regulate epidermal growth factor-dependent collagen loss in human carotid plaque smooth muscle cells[J].Physiol Rep,2014,2(2):e00224. doi: 10.1002/phy2.224
37	高建萍,张扬,邢芳毓,等.小鼠生长过程中肝, 肺, 肾胶原蛋白与MMP-1含量变化[J].生物工程学报,2021,37(2):646-654.
	GAOJ P,ZHANGY,XINGF Y,et al.Changes of collagen and MMP-1 in liver, lung and kidney during growth of mice[J].Chinese Journal of Biotechnology,2021,37(2):646-654.
38	JIR C.Hypoxia and lymphangiogenesis in tumor microenvironment and metastasis[J].Cancer Lett,2014,346(1):6-16. doi: 10.1016/j.canlet.2013.12.001
39	KASRAVIM,YAGHOOBIA,TAYEBIT,et al.MMP inhibition as a novel strategy for extracellular matrix preservation during whole liver decellularization[J].Biomater Adv,2024,156,213710. doi: 10.1016/j.bioadv.2023.213710
40	王波,刁志成,马勇,等.日粮添加油脂和出生类型对羔羊肌肉胶原蛋白特性及相关基因表达的影响[J].畜牧兽医学报,2019,50(4):771-780.
	WANGB,DIAOZ C,MAY,et al.Effects of dietary oil supplementation and birth types on muscle collagen characteristics and related genes expression in muscle of lamb[J].Acta Veterinaria et Zootechnica Sinica,2019,50(4):771-780.
41	ZHANGQ,DA CUNHAA P,LIS,et al.IL-27 regulates HIF-1α-mediated VEGFA response in macrophages of diabetic retinopathy patients and healthy individuals[J].Cytokine,2019,113,238-247. doi: 10.1016/j.cyto.2018.07.011
42	THEOCHARISA D,SKANDALISS S,GIALELIC,et al.Extracellular matrix structure[J].Adv Drug Deliv Rev,2016,97,4-27. doi: 10.1016/j.addr.2015.11.001
43	JIZ W,WANGX Q,LIUY L,et al.MicroRNA-574-3p regulates HIF-α isoforms promoting gastric cancer epithelial-mesenchymal transition via targeting CUL2[J].Dig Dis Sci,2022,67(8):3714-3724. doi: 10.1007/s10620-021-07263-0
44	NEGARII P,KESHARIS,HUANGC M.Probiotic activity of Staphylococcus epidermidis induces collagen type I production through FFaR2/p-ERK signaling[J].Int J Mol Sci,2021,22(3):1414. doi: 10.3390/ijms22031414
45	SCHÖNBORNK,WILLENBORGS,SCHULZJ N,et al.Role of collagen XⅡ in skin homeostasis and repair[J].Matrix Biol,2020,94,57-76. doi: 10.1016/j.matbio.2020.08.002
46	SUNS,HENRIKSENK,KARSDALM A,et al.Collagen type Ⅲ and VI turnover in response to long-term immobilization[J].PLoS One,2015,10(12):e0144525. doi: 10.1371/journal.pone.0144525
47	GAOJ P,GUOZ H,ZHANGY,et al.Age-related changes in the ratio of Type I/Ⅲ collagen and fibril diameter in mouse skin[J].Regen Biomater,2023,10,rbac110. doi: 10.1093/rb/rbac110
48	PROKSCHE,SCHUNCKM,ZAGUEV,et al.Oral intake of specific bioactive collagen peptides reduces skin wrinkles and increases dermal matrix synthesis[J].Skin Pharmacol Physiol,2014,27(3):113-119. doi: 10.1159/000355523

引物名称 Primer	引物序列(5′→3′) Primers sequence	预期长度/bp Fragments size
PCR-CUL2	F: CTTTTACAACCCTGAACTCGATGA R: TGGCGCTGCTACAGAAGACAC	589
q-PCR-CUL2	F: ACAGACGAGAAGTGCTGTCG R: AAACCGAGCTCTCGATTGGC	136
q-PCR- HIF-1α	F: TGAAAGCCTTGGATGGTTTTGT R: GCTCCGCTGTGTATTTTGCT	221
q-PCR-MMP-1	F: CAAGGTCTCCGAGGGTCAAG R: TGGGACAGCTGAACATCACC	300
q-PCR-MMP-9	F: TTCTGCCCTAGCGAGAGACT R: TTGTCCTGGTCGTAGTTGGC	164
q-PCR-VEGFA	F: GTGCCCACTGAGGAGTTCAA R: GCAACGCGAGTCTGTGTTTT	234
q-PCR- COL1A1	F: AGCCCTGGTGAAAATGGAGC R: CACCCTTAGCACCAACAGCA	190
q-PCR- COL3A1	F: TGGAACATGCCAGTGGGAAT R: TAGTCTCACAGCCTTGCGTG	182
q-PCR- COL6A3	F: GATCACGGAGAACAGTGCCA R: GACCGTGAGGTTCTGCTTCA	124
GAPDH	F: ACGACCACTTCGTCAAGCTC R: CAGGAGATGCTCGGTGTGTT	208

类别 Type	软件 Software	网址 Website
短散在元件Short interspersed nuclear element	UCSC	https://genome.ucsc.edu/
内含子剪切抑制子Intronic splicing silencer	SpliceAid	http://www.introni.it/splicing.html
RNA结合蛋白结合位点RBP binding site	RBP suite	http://www.csbio.sjtu.edu.cn/bioinf/RBPsuite/
RNA类型RNA type	Web Circ RNA	https://rth.dk/resources/webcircrna/submit
蛋白互作网络Protein interaction network	STRING	https://string-db.org/

猪群 Population	样本数 Number of sample	样本数Number of sample			等位基因频率Allele frequency
猪群 Population	样本数 Number of sample	MM	WM	WW	W	M
皱皮香猪ZXP	50	47	3	0	0.03^a	0.97^b
普通香猪XP	150	97	42	11	0.213^c	0.787^d

元件Element	位置/bp^* Location^*
短散在元件Short interspersed nuclear element(SINE)	42~300
内含子剪切抑制子Intronic splicing silencer(ISS)	81~85、236~241、281~286

RNA结合蛋白RBPs	评分Score	RBPs结合的碱基序列Sequence of RBP binding
FMRP	0.689	CGACUAG
IGF2BP2	0.638	AACCAUGAGGU
LIN28B	0.511	UGGUGUA
LIN28A	0.665	UGGUGUA

Differential Expression Study of Structural Variation in the Ubiquitin Ligase 2 Gene of Xiang Pigs with Wrinkled Skin

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