KRT16在长毛兔毛囊发育过程中的表达规律及功能探究

Expression and Function of KRT16 in Hair Follicle Development of Angora Rabbit

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1 431 bp,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。

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.