基于转录组测序分析SOX18在湖羊毛囊毛乳头细胞中的功能

doi:10.11843/j.issn.0366-6964.2024.06.013

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (6): 2409-2420.doi: 10.11843/j.issn.0366-6964.2024.06.013

基于转录组测序分析SOX18在湖羊毛囊毛乳头细胞中的功能

何明亮¹(), 吕晓阳²^,³, 蒋永清⁴, 宋正海⁵, 王叶青⁶, 杨会国⁷, 王善禾¹^,²^,³^,*(), 孙伟¹^,²^,³^,*()

1. 扬州大学动物科学技术学院, 扬州 225009
2. 教育部农业与农产品安全国际联合实验室, 扬州 225009
3. 江苏省普通高校家畜种质资源与遗传改良国际联合研究实验室, 扬州 225009
4. 浙江省农业科学院, 杭州 310021
5. 苏州市吴中区东山动物防疫站, 苏州 215107
6. 苏州太湖东山湖羊产业发展有限公司, 苏州 215107
7. 新疆畜牧科学院, 乌鲁木齐 830011

收稿日期:2023-09-25 出版日期:2024-06-23 发布日期:2024-06-28
通讯作者: 王善禾,孙伟 E-mail:13013706620@163.com;shanhe12315@163.com;dkxmsunwei@163.com
作者简介:何明亮(1995-)，男，河南信阳人，博士，主要从事动物遗传与育种研究，E-mail: 13013706620@163.com
基金资助:
江苏省自然科学基金(BK20210810);国家自然科学基金(32172689);江苏省农业重大新品种创制项目(PZCZ201739);江苏省“333工程”计划项目((2022)2-323);江苏省高等学校自然科学研究项目(20KJB230003);江苏省高等学校自然科学研究项目(22KJA230001);江苏省农业科技自主创新资金项目(CX(23)1036);扬州大学“高端人才计划-领军人才”培养项目;教育部农业与农产品安全国际合作联合实验室开放课题(JILAR-KF202103);江苏省研究生科研与实践创新计划(KYCX23_3593)

Function Analysis of SOX18 in Hu Sheep Hair Follicle Dermal Papilla Cells Based on Transcriptome Sequencing

Mingliang HE¹(), Xiaoyang LÜ²^,³, Yongqing JIANG⁴, Zhenghai SONG⁵, Yeqing WANG⁶, Huiguo YANG⁷, Shanhe WANG¹^,²^,³^,*(), Wei SUN¹^,²^,³^,*()

1. College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China
2. Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou 225009, China
3. International Joint Research Laboratory in Universities of Jiangsu Province of China for Domestic Animal Germplasm Resources and Genetic Improvement, Yangzhou 225009, China
4. Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
5. Suzhou Wuzhong District Dongshan Animal Epidemic Prevention Station, Suzhou 215107, China
6. Suzhou Taihu Dongshan Hu Sheep Industry Development Co., Ltd, Suzhou 215107, China
7. Xinjiang Academy of Animal Science, Urumqi 830011, China

Received:2023-09-25 Online:2024-06-23 Published:2024-06-28
Contact: Shanhe WANG, Wei SUN E-mail:13013706620@163.com;shanhe12315@163.com;dkxmsunwei@163.com

摘要/Abstract

摘要：

旨在初步探究SOX18基因在湖羊毛囊毛乳头细胞中的功能。本研究首先采用免疫荧光染色试验对从湖羊毛囊中分离的细胞进行鉴定，然后构建SOX18基因过表达载体并通过qRT-PCR和Western blot检测其过表达效率。将经过鉴定且生长状态良好的毛乳头细胞分为两组，分别转染SOX18过表达载体和空载质粒，每组3个重复。采用Trizol法提取6个样本的总RNA并构建cDNA文库, 利用Illumina Novaseq6000平台进行测序，然后对样本相关性和差异表达基因进行分析，并对差异表达基因进行GO功能分类和KEGG通路注释, 寻找SOX18基因调控的相关候选基因和通路。为确认转录组数据的准确性, 随机选取9个差异表达基因进行qRT-PCR验证。免疫荧光结果显示，毛乳头相关基因在所分离的细胞中高表达，表明所分离的毛乳头细胞纯度高。qRT-PCR和Western blot检测发现，SOX18基因过表达能够增加毛乳头细胞中SOX18的mRNA和蛋白水平，表明构建的SOX18基因过表达载体可用于后续试验。转录组分析结果表明样本间相关性好，SOX18过表达组和对照组相比共发现157个基因差异表达，其中103个基因在SOX18过表达后上调，54个基因下调，qRT-PCR分析表明转录组数据准确性高。GO功能分析显示，一些差异基因富集于细胞进展和毛囊发育过程。KEGG富集分析表明，一些差异表达基因富集于细胞增殖和毛囊发育相关信号通路。GO功能分析和KEGG富集分析表明SOX18在毛乳头细胞的增殖和毛囊发育过程中起作用。本研究通过转录组测序分析发现SOX18可能通过影响细胞增殖和毛囊发育相关的基因和信号通路来影响毛乳头细胞增殖和毛囊生长发育，研究结果为解析SOX18基因在湖羊毛乳头细胞和毛囊中的分子调控机制提供了理论基础。

关键词: 转录组测序, SOX18, 毛囊, 毛乳头细胞, 湖羊

Abstract:

The purpose of this study was to preliminary investigate the function of the SOX18 gene in Hu sheep hair follicle dermal papilla cells. In this study, immunofluorescence staining was used to identify cells isolated from the hair follicles of Hu sheep. Subsequently, an overexpression vector of the SOX18 gene was conducted, and its overexpression efficacy was assessed using qRT-PCR and Western blot. The dermal papilla cells successfully identified and well-grown were divided into two groups. One group was transfected with SOX18 overexpression vector, while the other was transfected with empty plasmid. Each group had 3 replicates. The total RNA was extracted from 6 samples using the Trizol method, followed by the construction of a cDNA library. The Illumina Novaseq6000 platform was used for sequencing. Subsequently, sample correlation and differentially expressed genes were analyzed. The differentially expressed genes were categorized based on GO and annotated using the KEGG pathway analysis to identify candidate genes and pathways associated with the regulation of the SOX18 gene. To confirm the accuracy of the transcriptome data, 9 differentially expressed genes were randomly chosen for qRT-PCR analysis. The immunofluorescence results revealed dermal papilla-related genes had high expression in isolated cells, indicating a high purity of the isolated dermal papilla cells. qRT-PCR and Western blot results showed that overexpression of the SOX18 gene could increase the mRNA and protein levels of SOX18 in dermal papilla cells, indicating that the SOX18 overexpression vector could be used for subsequent experiments. Transcriptome analysis results showed a good correlation between samples. A total of 157 genes were differentially expressed between the SOX18 overexpression group and the control group, of which 103 genes were up-regulated after SOX18 overexpression and 54 genes were down-regulated. qRT-PCR analysis showed high accuracy of transcriptome data. GO functional analysis showed that some differential genes were enriched in cell progression and hair follicle development. KEGG enrichment analysis showed that some differentially expressed genes were enriched in signaling pathways related to cell proliferation and hair follicle development. GO functional analysis and KEGG enrichment analysis indicated that SOX18 played a role in the proliferation of dermal papilla cells and the development of hair follicles. In this study, transcriptome sequencing analysis found that SOX18 may affect the proliferation of dermal papilla cells and the development of hair follicles by affecting genes and signaling pathways related to cell proliferation and hair follicle development. The study results provide a theoretical basis for understanding the molecular regulatory mechanism of the SOX18 gene in Hu sheep dermal papilla cells and hair follicles.

Key words: transcriptome sequencing, SOX18, hair follicles, dermal papilla cells, Hu sheep

中图分类号:

S826.2

何明亮, 吕晓阳, 蒋永清, 宋正海, 王叶青, 杨会国, 王善禾, 孙伟. 基于转录组测序分析SOX18在湖羊毛囊毛乳头细胞中的功能[J]. 畜牧兽医学报, 2024, 55(6): 2409-2420.

Mingliang HE, Xiaoyang LÜ, Yongqing JIANG, Zhenghai SONG, Yeqing WANG, Huiguo YANG, Shanhe WANG, Wei SUN. Function Analysis of SOX18 in Hu Sheep Hair Follicle Dermal Papilla Cells Based on Transcriptome Sequencing[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6): 2409-2420.

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基因 Gene	登录号 Accession number	引物序列(5′→3′) Primer sequence	产物大小/bp Product size	退火温度/℃ Annealing temperature
AHR	XM_060414533.1	F: CGAATCCTTCAAAGAGGCATAG	403	60
		R: GCTCGGTCTTCGGTATGG
ANKRD1	NM_001252178.1	F: TACAGGGAAGAAGAACGG	338	60
		R: TAGGCACATCCACAGGTT
BMP4	XM_060418703.1	F: CCGTGAGGAGCTTCCACCA	191	60
		R: GTTTATACGATGAAAGCCCTGCTCC
CFB	XM_004018929.6	F: TGTCCTTCTGGCTTCTACCC	466	60
		R: AAGGCTCTGTTCCACTCCAA
GANC	XM_060419122.1	F: AGTCTACCTATCGGGCATTA	269	60
		R: ATCTCCTTTCCCACTTGC
HUNK	XM_004003742.6	F: TTGCCAGGAAGAAATACGG	189	60
		R: GCAGGAAGTTCACGGCTC
PTGFR	NM_001009789.2	F: TTTAGAAGTCAGCAGCAC	354	60
		R: ATCAGAAATAGCAGCAAC
PTGS2	NM_001009432.1	F: CCCGAACAGGATTCTATG	481	60
		R: GCTGGTCCTCGTTCAATA
SOX9	XM_027974011.3	F: TCAAGGGCTACGACTGGACG	366	60
		R: TGGCGTTGGGCGAGATGTGC
SOX18	XM_027976914.2	F: TGTGGGCGAAGGACGAGC	253	60
		R: GCCAAGCCTGGGAGGAGGAG
GAPDH	NM_001190390.1	F: TCTCAAGGGCATTCTAGGCTAC	151	60
		R: GCCGAATTCATTGTCGTACCAG

引物名称 Primer name	引物序列(5′→3′) Primer sequence	产物大小/bp Product size	退火温度/℃ Annealing temperature
OE-SOX18	F: CTAGCGTTTAAACTTAAGCTTATGCAGAGATCGCCGCTCG	1 176	62
	R: CCACACTGGACTAGTGGATCCCTATCCAGAGATGCAGGCGCTG

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基于转录组测序分析SOX18在湖羊毛囊毛乳头细胞中的功能

Function Analysis of SOX18 in Hu Sheep Hair Follicle Dermal Papilla Cells Based on Transcriptome Sequencing

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