KRT16在长毛兔毛囊发育过程中的表达规律及功能探究

doi:10.11843/j.issn.0366-6964.2023.01.015

摘要/Abstract

摘要： 旨在探究KRT16在长毛兔毛囊发育过程中的表达规律及功能。本试验选取12只健康的6月龄皖系长毛兔，于剪毛后在生长期、退行期和休止期选取背部皮肤采样。通过克隆得到兔KRT16基因的编码序列，利用生物信息学软件对KRT16编码序列(CDS)的生物学特性进行初步分析。实时荧光定量PCR(quantitative real-time PCR，qRT-PCR)分析KRT16在毛囊不同时期表达量。在毛囊毛乳头细胞(dermal papilla cell，DPC)中过表达和敲低KRT16，探究KRT16对毛囊生长发育相关基因的调控作用，以及对DPC增殖的影响。结果显示，KRT16基因的编码序列全长1431bp，可编码476个氨基酸，在不同哺乳动物中表现出高度同源性。实时荧光定量PCR结果显示，在毛囊发育周期中，KRT16在毛囊发育周期呈现出不同的表达水平，在生长期高表达。通过在兔毛乳头细胞中过表达与敲减KRT16，检测毛囊发育相关基因的表达水平变化，过表达KRT16后，SFRP2和TGFβ1基因的mRNA表达量极显著下降(P<0.01)，BCL2、CCND1、EGF、LEF1和CTNNB1基因的mRNA表达量极显著提高(P<0.01)；而敲降KRT16后，SFRP2和TGFβ1基因的表达量极显著提高(P<0.01)，BCL2、CCND1、EGF、LEF1和CTNNB1基因的表达量极显著下降(P<0.01)。同时，KRT16能够显著促进毛乳头细胞增殖。综上表明，KRT16参与毛囊周期性发育过程，并能够调控毛囊生长发育相关基因，促进毛乳头细胞增殖。该研究通过对KRT16基因功能的初步研究，为长毛兔毛囊发育的基础研究提供参考，同时为毛囊发育相关基因的功能研究提供依据。

关键词: 长毛兔, KRT16, 毛乳头细胞, 毛囊发育

Abstract: The study aimed to investigate the expression pattern and function of KRT16 during hair follicle development in Angora rabbit. In this experiment, 12 healthy 6-month-old Wan Strain Angora rabbits were selected for dorsal skin sampling during the anagen, catagen, and telogen after hair shearing. The coding sequence of the rabbit KRT16 gene was obtained by cloning, and the biological properties of the KRT16 coding sequence (CDS) were initially analyzed using bioinformatics softwares. Quantitative real-time PCR (qRT-PCR) was performed to analyze the expression of KRT16 in hair follicles at different times. Overexpression and knockdown of KRT16 in dermal papilla cells (DPC) were performed to investigate the regulatory role of KRT16 on genes related to hair follicle growth and development, and the effect on DPC proliferation. The results showed that the coding sequence of the KRT16 gene was 1 431 bp, could encode 476 amino acids, and showed high homology in different mammals. qRT-PCR results showed that KRT16 showed different expression levels during the period of hair follicle development, with high expression during the anagen phase. The changes in the expression levels of hair follicle development-related genes were examined by overexpressing and knocking down KRT16 in rabbit hair papilla cells. The mRNA expression of SFRP2 and TGFβ1 genes was highly significantly decreased (P<0.01), and the mRNA expression of BCL2, CCND1, EGF, LEF1, and CTNNB1 genes was highly significantly increased after overexpression of KRT16 (P<0.01); while the expression of SFRP2 and TGFβ1 genes was highly significantly increased (P<0.01), and the expression of BCL2, CCND1, EGF, LEF1, and CTNNB1 genes was highly significantly decreased (P<0.01) after knockdown of KRT16. Meanwhile, KRT16 was able to significantly promote the proliferation of hair papilla cells. In summary, it is showed that KRT16 is involved in the cyclic development of hair follicles and can regulate hair follicle growth-and development-related genes to promote the proliferation of hair papilla cells. This study provides a reference for the basic research of hair follicle development through the preliminary study of KRT16 gene function and also provides a basis for the functional study of hair follicle development-related genes in Angora rabbits.

Key words: wool rabbit, KRT16, dermal papilla cell, hair follicle development

中图分类号:

S852.2

张希宇, 翟频, 王璠, 戴莹莹, 赵博昊, 陈阳, 吴信生. KRT16在长毛兔毛囊发育过程中的表达规律及功能探究[J]. 畜牧兽医学报, 2023, 54(1): 157-167.

ZHANG Xiyu, ZHAI Pin, WANG Fan, DAI Yingying, ZHAO Bohao, CHEN Yang, WU Xinsheng. Expression and Function of KRT 16 in Hair Follicle Development of Angora Rabbit[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 157-167.

参考文献 41

[1]	ZHAO B H,LI J L,LIU M,et al.DNA methylation mediates lncRNA2919 regulation of hair follicle regeneration[J].Int J Mol Sci,2022,23(16):9481.
[2]	SARMA A.Biological importance and pharmaceutical significance of keratin:a review[J].Int J Biol Macromol,2022, 219:395-413.
[3]	GU L H,COULOMBE P A.Keratin function in skin epithelia:a broadening palette with surprising shades[J].Curr Opin Cell Biol,2007,19(1):13-23.
[4]	CHEN Y,BAO Z Y,LIU M,et al.Promoter methylation changes in KRT17:a novel epigenetic marker for wool production in angora rabbit[J].Int J Mol Sci,2022,23(11):6077.
[5]	张静.角蛋白24在脱毛中的作用研究[D].杭州:浙江大学,2020.ZHANG J.The role of keratin 24 in hair loss[D].Hangzhou:Zhejiang University,2020.(in Chinese)
[6]	FITCH K R,MCGOWAN K A,VAN RAAMSDONK C D,et al.Genetics of dark skin in mice[J].Genes Dev,2003,17(2):214-228.
[7]	MCGOWAN K A,ARADHYA S,FUCHS H,et al.A mouse Keratin 1 mutation causes dark skin and epidermolytic hyperkeratosis[J]. J Invest Dermatol,2006,126(5):1013-1016.
[8]	MCGOWAN K A,FUCHS H,DE ANGELIS M H,et al.Identification of a Keratin 4 mutation in a chemically induced mouse mutant that models white sponge nevus[J].J Invest Dermatol,2007,127(1):60-64.
[9]	LESSARD J C,COULOMBE P A.Keratin 16-null mice develop palmoplantar keratoderma,a hallmark feature of pachyonychia congenita and related disorders[J].J Invest Dermatol,2012,132(5):1384-1391.
[10]	ZHANG X W,YIN M M,ZHANG L J.Keratin 6,16 and 17-critical barrier Alarmin molecules in skin wounds and psoriasis[J]. Cells,2019,8(8):807.
[11]	CHEN J G,FAN H Y,WANG T,et al.Silencing KRT16 inhibits keratinocyte proliferation and VEGF secretion in psoriasis via inhibition of ERK signaling pathway[J].Kaohsiung J Med Sci,2019,35(5):284-296.
[12]	ZHAO B H,CHEN Y,HU S S,et al.Characterization of HTATIP2 and its role during hair follicle cycles in Angora rabbit[J].Genome,2020,63(3):179-187.
[13]	赵采芹,王燕碧,朱杰,等.鸡TRAF6和TIFA蛋白的序列分析与相互作用验证[J].畜牧兽医学报,2021,52(6):1511-1522.ZHAO C Q,WANG Y B,ZHU J,et al.Sequence analysis and interaction verification of chicken TRAF6 and TIFA proteins[J]. Acta Veterinaria et Zootechnica Sinica,2021,52(6):1511-1522.(in Chinese)
[14]	ZHOU T,CHEN Y,ZHAO B H,et al.Characterization and functional analysis of SIAH1 during skin and hair follicle development in the angora rabbit (Oryctolagus cuniculus)[J].Hereditas,2020,157(1):10.
[15]	白少成,周娟,靳荣帅,等.新西兰白兔CYP11A1基因的克隆、生物信息学分析及其对繁殖相关基因的影响[J].中国畜牧兽医,2022,49(7):2484-2496.BAI S C,ZHOU J,JIN R S,et al.Cloning and bioinformatics analysis of CYP11A1 gene in New Zealand white rabbits and its effects on reproduction-related genes[J].China Animal Husbandry & Veterinary Medicine,2022,49(7):2484-2496.(in Chinese)
[16]	BAO Z Y,ZHAO B H,HU S S,et al.Characterization and functional analysis of SMAD2 regulation in hair follicle cycle in Angora rabbits[J].Gene,2021,770:145339.
[17]	张晖,潘阳阳,王靖雷,等.牦牛RBM3的基因克隆及其在不同繁殖时期卵巢、输卵管、子宫中的表达定位[J].畜牧兽医学报, 2022,53(7):2202-2214.ZHANG H,PAN Y Y,WANG J L,et al.RBM3 gene cloning and its expression,localization in ovary,fallopian tube and uterus at different reproductive stages of yak (Bos grunniens)[J].Acta Veterinaria et Zootechnica Sinica,2022,53(7):2202-2214.(in Chinese)
[18]	黄正洋,王钱保,李春苗,等.鸡Smad4基因克隆生物信息学及组织表达分析[J].中国畜牧兽医,2021,48(10):3522-3532.HUANG Z Y,WANG Q B,LI C M,et al.Cloning,bioinformatics and tissue expression analysis of Smad4 gene in chicken (Gallus gallus)[J].China Animal Husbandry & Veterinary Medicine,2021,48(10):3522-3532.(in Chinese)
[19]	陈红梅,程龙飞,傅光华,等.6种血清型鸭疫里默氏菌SSPA基因的克隆及序列分析[J].福建农业学报,2015,30(9):831-835.CHEN H M,CHENG L F,FU G H,et al.Cloning and sequence analysis of SSPA genes of Riemerella anatipestifer with different serotypes[J].Fujian Journal of Agricultural Sciences,2015,30(9):831-835.(in Chinese)
[20]	闵星宇,杨丽雪,于海玲,等.犏牛PPP1R11基因的克隆及在睾丸中的表达规律研究[J].畜牧兽医学报,2022,53(2):481-492.MIN X Y,YANG L X,YU H L,et al.Cloning and expression analysis of cattle-yak PPP1R11 gene in testis[J].Acta Veterinaria et Zootechnica Sinica,2022,53(2):481-492.(in Chinese)
[21]	KUMAR S,STECHER G,LI M,et al.MEGA X:molecular evolutionary genetics analysis across computing platforms[J].Mol Biol Evol,2018,35(6):1547-1549.
[22]	KIM S,COULOMBE P A.Intermediate filament scaffolds fulfill mechanical,organizational,and signaling functions in the cytoplasm[J].Genes Dev,2007,21(13):1581-1597.
[23]	GONG L,GUO S P,WANG D T,et al.A KRT6A and a novel KRT16 gene mutations in Chinese patients with pachyonychia congenita[J].Int J Gen Med,2021,14:903-907.
[24]	LESSARD J C,PIÑA-PAZ S,ROTTY J D,et al.Keratin 16 regulates innate immunity in response to epidermal barrier breach[J].Proc Natl Acad Sci U S A,2013,110(48):19537-19542.
[25]	MARDARYEV A N,AHMED M I,VLAHOV N V,et al.Micro-RNA-31 controls hair cycle-associated changes in gene expression programs of the skin and hair follicle[J].FASEB J,2010,24(10):3869-3881.
[26]	WANG Y N,CHANG W C.Induction of disease-associated Keratin 16 gene expression by epidermal growth factor is regulated through cooperation of transcription factors Sp1 and c-Jun[J].J Biol Chem,2003,278(46):45848-45857.
[27]	WU J H,ZHANG W G,LI J Q,et al.Hoxc13 expression pattern in cashmere goat skin during hair follicle development[J]. Agric Sci China,2009,8(4):491-496.
[28]	高丽霞,张燕军,张文广,等.内蒙古白绒山羊毛囊发育周期蛋白质表达谱分析[J].农业生物技术学报,2014,22(6):727-735.GAO L X,ZHANG Y J,ZHANG W G,et al.Analysis of protein expression profile of hair follicle cycle of inner Mongolia cashmere goat (Capra cashmere)[J].Journal of Agricultural Biotechnology,2014,22(6):727-735.(in Chinese)
[29]	TONG X M,COULOMBE P A.Keratin 17 modulates hair follicle cycling in a TNFα-dependent fashion[J].Genes Dev, 2006,20(10):1353-1364.
[30]	KIM B K,YOON S K.Expression of Sfrp2 is increased in Catagen of hair follicles and inhibits keratinocyte proliferation[J]. Ann Dermatol,2014,26(1):79-87.
[31]	FOITZIK K,LINDNER G,MUELLER-ROEVER S,et al.Control of murine hair follicle regression (catagen) by TGF-β1 in vivo[J].FASEB J,2000,14(5):752-760.
[32]	吴振宇.生长獭兔毛囊发育规律及蛋氨酸调控机制研究[D].泰安:山东农业大学,2016.WU Z Y.The hair follicle development regularity and the mechanism research of Met in growing Rex rabbit[D].Tai'an:Shandong Agricultural University,2016.(in Chinese)
[33]	SUN Y,ZHOU Y X,MSUTHWANA P,et al.The role of CTNNB1 and LEF1 in feather follicles development of Anser cygnoides and Anser anser[J].Genes Genomics,2020,42(7):761-771.
[34]	陈秋燃.IGF-1与EGF因子联合使用对毛兔毛囊生长的影响[D].扬州:扬州大学,2020.CHEN Q R.The effect of IGF-1 combined with EGF on the hair follicle growth of Angora rabbit[D].Yangzhou:Yangzhou University,2020.(in Chinese)
[35]	NANASHIMA N,AKITA M,YAMADA T,et al.The hairless phenotype of the Hirosaki hairless rat is due to the deletion of an 80-kb genomic DNA containing five basic keratin genes[J].J Biol Chem,2008,283(24):16868-16875.
[36]	CAI C K,ZHAO G Y,TIAN L Y,et al.miR-15a and miR-16-1 downregulate CCND1 and induce apoptosis and cell cycle arrest in osteosarcoma[J].Oncol Rep,2012,28(5):1764-1770.
[37]	XU X W,LYLE S,LIU Y P,et al.Differential expression of Cyclin D1 in the human hair follicle[J].Am J Pathol,2003,163(3):969-978.
[38]	MVLLER-RÖVER S,ROSSITER H,LINDNER G,et al.Hair follicle apoptosis and Bcl-2[J].J Investig Dermatol Symp Proc, 1999,4(3):272-277.
[39]	STENN K S,LAWRENCE L,VEIS D,et al.Expression of the bcl-2 protooncogene in the cycling adult mouse hair follicle[J]. J Invest Dermatol,1994,103(1):107-111.
[40]	BOTCHKAREVA N V,AHLUWALIA G,SHANDER D.Apoptosis in the hair follicle[J].J Invest Dermatol,2006,126(2):258-264.
[41]	LIU Y H,QIAN P D,ZHU W,et al.TFAP2A induced KRT16 as an oncogene in lung adenocarcinoma via EMT[J].Int J Biol Sci,2019,15(7):1419-1428.