ATG14调控家兔毛囊毛乳头细胞自噬进程的功能探究

doi:10.11843/j.issn.0366-6964.2024.08.020

摘要/Abstract

摘要：

旨在探究自噬相关蛋白14(autophagy-related protein 14，ATG14)调控家兔毛囊毛乳头细胞(dermal papilla cells，DPCs)自噬进程对毛囊发育生长的影响。本试验选取健康6月龄长毛兔，采集背部皮肤分离培养DPCs，通过克隆ATG14基因的编码序列(coding sequence，CDS)，利用生物信息学对ATG14的生物学特性进行初步分析，在家兔DPCs中过表达或敲减ATG14对自噬相关蛋白和毛囊生长发育相关基因的表达及DPCs增殖水平的影响进行探究。结果表明，ATG14基因CDS长度为1 479 bp，共编码492个氨基酸，不存在潜在信号肽及跨膜区，属于定位于细胞核的不稳定蛋白，在不同哺乳动物中存在同源性。家兔DPCs中过表达或敲减ATG14后，WB结果显示ATG14能够上调自噬标志蛋白LC3和Beclin1的蛋白表达，抑制自噬抑制蛋白P62表达水平。ATG14能够增加细胞中pEGFP-LC3B荧光表达，表明ATG14能够激活细胞中LC3B的表达。同时，在DPCs中过表达ATG14能够显著上调CCND1、FGF2、LEF1、BCL2和WNT2的mRNA表达水平，显著下调SFRP2和TGFβ-1的基因表达水平(P < 0.05)，敲减ATG14能够显著下调CCND1、FGF2、LEF1、BCL2和WNT2的基因表达水平，显著上调SFRP2和TGFβ-1的mRNA水平(P < 0.05)。ATG14能够上调LEF1和CCND1的蛋白表达水平。此外，DPC中过表达ATG14能够显著促进DPCs细胞增殖(P < 0.01)。本研究通过对家兔ATG14基因调控DPCs自噬进程的功能进行分析，为阐明家兔毛囊生长发育的调控机制提供理论依据。

关键词: ATG14, 自噬, 毛囊, 家兔, 毛乳头细胞

Abstract:

The study aimed to investigate the effect of autophagy-related protein 14 (ATG14) regulate hair follicles growth and development by autophagy progress in rabbit dermal papilla cells (DPCs). In this study, healthy 6-month-old Angora rabbits were selected to collect dorsal skin to isolate and culture DPCs. The coding sequence (CDS) of ATG14 was cloned, and biological characteristics of ATG14 was analyzed by bioinformatics. After the overexpressed and knockdown of ATG14 in DPCs, and the expression of autophagy related protein and hair follicle growth and development related genes was detected, and the effect of ATG14 on the cell proliferation was investigated. The results showed that the length of CDS was 1 479 bp for the ATG14 gene, which could code 492 amino acids. Bioinformatics analyses indicated that the ATG14 protein didn't have potential signal peptides and transmembrane regions, which belong to the unstable proteins localized in the nucleus, and exhibited high homology in different mammals. After the overexpression and knockdown of ATG14 in DPCs, the WB results showed that ATG14 could upregulate the protein expression of autophagy related protein LC3 and Beclin1, but downregulate the autophagy inhibitor P62 protein expression. ATG14 could increase the fluorescent expression of pEGFP-LC3B in DPCs, indicating that ATG14 could activate the expression of LC3B. What's more, the overexpression of ATG14 could upregulate the mRNA expression of BCL2, CCND1, FGF2, WNT2 and LEF1, downregulate the gene expression of SFRP2 and TGFβ-1 (P < 0.05), the knockdown of ATG14 could downregulate the gene expression of BCL2, CCND1, FGF2, WNT2 and LEF1, upregulate the mRNA expression of SFRP2 and TGFβ-1 (P < 0.05). ATG14 could upregulate the expression of LEF1 and CCND1. The overexpression of ATG14 could promote the cell proliferation in DPCs (P < 0.01). In this study, the rabbit ATG14 gene regulating the autophagy process in DPCs was analyzed, which provided the theoretical basis for elucidating the regulatory mechanism of hair follicle growth and development in rabbit.

Key words: ATG14, autophagy, hair follicle, rabbit, dermal papilla cells

中图分类号:

S829.1

曹馨予, 蔡佳炜, 鲍志远, 姚漱玉, 李云鹏, 陈阳, 吴信生, 赵博昊. ATG14调控家兔毛囊毛乳头细胞自噬进程的功能探究[J]. 畜牧兽医学报, 2024, 55(8): 3472-3481.

Xinyu CAO, Jiawei CAI, Zhiyuan BAO, Shuyu YAO, Yunpeng LI, Yang CHEN, Xinsheng WU, Bohao ZHAO. The Function Analysis of ATG14 Regulates the Autophagy Process in Rabbit Hair Follicle Dermal Papilla Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3472-3481.

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引物名称 Primer	引物序列(5′→3′) Sequence
pcDNA3.1-ATG14	F：tttaaacttaagctt$\underline{ggtacc}$ATGGCGTCTCCCAGTGGG
	R：gccgccactgtgctg$\underline{gatatc}$CTAGCGGTGGCCAGTGTAAGC
siRNA-ATG14	F：GCUGGUCAACAUUCUGUCUTT
	R：AGACAGAAUGUUGACCAGCTT
siRNA-NC	F：UUCUCCGAACGUGUCACGUTT
	R：ACGUGACACGUUCGGAGAATT

引物名称 Primer	引物序列(5′→3′) Sequence	产物长度/bp Product length	退火温度/℃ Tm
CCND1	F：GAACGCTACCTTCCCCAGTGCTC	103	57.2
	R：CCTCACAGACCTCCAGCATCCAG
FGF2	F：GTGTGTGCAAACCGTTACCTT	159	50.9
	R：TCGTTTCAGTGCCACATACCAG
LEF1	F：CATCTCGGGTGGATTCAGG	121	53.2
	R：ATGAGGGATGCCAGTTGTG
BCL2	F：ACATCGCCCTGTGGATGACTG	183	57.6
	R：CGAGGGTGATGCAAGCTCCTAT
WNT2	F：AGCCATCCAGGTCGTCATGAACCAG	164	56.3
	R：TGCACACACGACCTGCTGTACCC
SFRP2	F：CCAGCCCGACTTCTCCTACAAGC	135	57.5
	R：TCCAGCACCTCTTTCATGGTCT
TGFβ-1	F：CAGGTCCTTGCGGAAGTCAA	126	60.0
	R：CTGGAACGGGCTCAACATCTA
GAPDH	F：CACCAGGGCTGCTTTTAACTCT	145	53.9
	R：CTTCCCGTTCTCAGCCTTGACC

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