Preliminary Study On Hr Gene Function And Differential Proteomics Of Yuyi Hairless Mouse Skin

Backgrounds: The Hairless gene(Hr)is a member of the Jumonji family located in the D2 region of chromosome 14,which spans 14 kb and contains 20 exons and 18 introns.The Hr gene is 19486 bp in length and encodes 1182 amino acids whose translation product is hairless protein(HR)of about 127 k Da.Loss of HR function causes the hair follicles to disintegrate,and the number of apoptotic cells in the outer root sheath cells increases significantly,leading to structure destruction of most outer root sheath,disrupting the normal growth cycle of the hair,and impeding the formation of new hair.At present,there is relatively fewer researches on the Hr gene,and we can not determine its exact function.However,the Hr gene mutation causes the Kunming mice to lose their hair and keep in the station of life long hairless.Therefore,the Yuyi hairless mouse can be used as a model of hair growth regulation.Objective The objective of our study is to explore the gene function of the Hr gene.And the further aim is to study the effect of Hr gene mutation on the molecular mechanism of skin in Kunming mice and to explore the causes of the changes in the phenotypes of Yuyi hairless mice.Methods(1)Construction of Hr gene over-expression vector,transfection into mouse fibroblasts cells(NIH3T3)by lipofection,Western Blot was used to investigate the expression of HR protein in the transfected cells,cells proliferation and apoptosis were determined by flow cytometry.(2)RNA interference experiments: transfection of fourgroups of synthesized mature si RNA into mouse fibroblasts cells(NIH3T3)by lipofection,and the expression of HR protein was deteced by Western Blot analysis,cells proliferation and apoptosis were determined by flow cytometry.(3)Differential proteomics: Differentially expressed proteins in the skin of wild Kunming mice and Yuyi hairless mice were screened by two-dimensional electrophoresis and biological mass spectrometry techniques.GO and KEGG analyses were performed on these differentially expressed proteins,and the results were validated by Western Blot.Results(1)The construction of p EGFP-Hr-M and p EGFP-Hr-N over-expression vectors were successful.After transfection into mouse fibroblasts cells(NIH3T3)by liposome,the expression of HR protein was significantly increased,but there was no significantly differencein cell proliferation and apoptosis between these two groups.(2)After the four groups of si RNA targeting Hr gene were introduced into NIH3T3 cells,the expression of HR protein decreased.The second group of si RNA had the highest efficiency in knocking down HR protein,but it had no significant effect on cell proliferation and apoptosis.(3)There were 850 differentially expressed proteins screened by two-dimensional electrophoresis.Some of them were selected for mass spectrometry identification and 26 protein spots were successfully identified.(4)GO analysis of the identified 26 spots: Analysis of the biological processes of these proteins and found that they are mainly involved in cellular process: 15%;single-organism process: 14%;response to stimulus: 12%;metabolic process: 12% cellular component organization or biogenesis: 9%;multicellular biological process: 8%;development process: 8%.Based on the analysis of cellular components: 24% of differential proteins were localized in cells,22% were localized in organelles,19% were located in extracellular region,11% were localized in macromolecular complexes,theothers were located on membranes,membrane-enclosed lumen,etc.According to the classification of molecular functions,50% of these proteins are function in binding,22% have catalytical activity,10% have structural molecular activity,and the remaining parts have molecular regulation function,molecular transducer activity,signal transducer activity and electron carrier activity.(5)The KEGG analysis of these differences revealed that the differential genes were mainly distributed in estrogen signaling pathways,antigen processing and expression,endoplasmic reticulum protein processing and procedural necrosis pathways.(6)Two of the skinand hairrelated differential proteins,keratin 17(KRT17)and type III collagen(COL3A1)were tested and the results were consistent with the twodimensional electrophoresis results.The expression of KRT17 protein in Yuyi hairless mice was higher than that in normal wild Kunming mice of both 2 months and 6 months mice.The expression level of type III collagen fibrin in Yuyi hairless mice of 2 months was higher than that in wild Kunming mice,but it was lower inYuyi hairless mice of 6 months than that in wild Kunming mice.Conclusions(1)Neither Hr gene over-expression nor knock-down of Hr gene had significant effect on mouse fibroblasts cells(NIH3T3)proliferation and apoptosis.(2)There were 850 differential proteins detected by two-dimensional electrophoresis,and then selected points were analyzed by biological mass spectrometry and bioinformatics to find 26 differential proteins.These differential proteins were mainly concentrated in Estrogen signal transduction pathways,antigen processing and expression,endoplasmic reticulum protein processing and programmed necrosis pathways.Hr gene mutation affects the relevant pathways in mice.(3)Hr mutation up-regulated the expression of KRT17,and increased expression of KRT17 resulted in changes in skin status in hairless mice.Mutation in the Hr gene have also affected the expression of collagen,which has led to increased expression of COL3A1 in 2 months and decreased expression in 6 monthes of Yuyi hairless mice,and a series of changes such as skin aging.