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

Abstract

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

CLC Number:

S829.1

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.

Figures/Tables 6

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Table 2

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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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The Function Analysis of ATG14 Regulates the Autophagy Process in Rabbit Hair Follicle Dermal Papilla Cells

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