Study On The Molecular Regulatory Mechanisms Underlying The Unique Skin Thickness Trait In Chenghua Pigs

Skin is the organism’s outer covering in mammalian,which forms the largest protective tissue.The skin thickness is mainly depended on the content of collagen in the dermal matrix,and the ability of dermal fibroblasts to synthesize collagen is the key factor for skin thickness.Changes of the human skin thickness are closely related to physiological and pathological biological phenomena such as skin aging,wrinkles,and diseases.Domestic pig skin is more similar to human skin in terms of morphological structure and physiology,making them an ideal model animal for scientific research on human skin.Additionally,pig skin is rich in collagen,and its dietary value has received much attention,as seen in traditional Sichuan cuisine,which requires high-quality pork with thick skin from Cheng Hua(CH)pigs for dishes such as"twice-cooked pork".The modern pig breeding programs in China,skin thickness is becoming an important economically valuable trait for the selection and improvement of characteristic breeds(such as the local Sichuan breed,CH pigs),in order to meet the demand for high-quality and distinctive pork from local residents.Therefore,analyzing the molecular regulatory mechanisms underlying the formation of skin thickness in domestic pigs not only has important practical applications for the breeding of thick-skinned pigs,but also has theoretical significance for related research on delaying skin aging and promoting skin physiological health in humans.Therefore,this thesis takes the extremely thick skin of CH pigs as a unique biological phenomenon and uses a comprehensive biological research strategy.With the use of experimental techniques such as whole transcriptome and single-cell transcriptome sequencing,proteomics analysis,immunofluorescence staining,WB,and RIP,skin tissues from different dimensions(species,breeds,anatomical sites)and cells are studied at molecular,cellular,individual,and population levels.The following research is conducted:(1)the key biological pathways regulating the formation of the unique skin thickness in CH pigs are screened and identified at the whole transcriptome level;(2)the heterogeneity of fibroblasts and gene expression patterns in different skin anatomical sites of CH pigs are revealed at the single-cell transcriptome level;(3)small extracellular vesicles derived from dermal fibroblasts in CH pigs efficiently promote the activity of fibroblasts and the biosynthesis of type I collagen.Through the research,the following main research results are obtained:1.Morphological and biochemical analyses revealed the unique characteristics of the thick skin trait in CH pigs.Results showed that the dermal thickness in the back of CH pigs reached 8.5 mm,while the control group of Large White(LW)pigs only was 3.0 mm.CH pigs have a high content of type I collagen in skin(58.6 g/cm~2 in CH pigs versus 21.8 g/cm~2 in LW pigs).Moreover,there were significant differences in dermal thickness among different anatomical regions of CH pigs(P<0.05),with decreasing thickness in skin of the back,head,shoulder,leg,abdomen,and ear,in that order.2.Identification of key biological pathways regulating the formation of specific skin thickness traits in CH pigs through transcriptome-wide screeningThrough transcriptome-wide sequencing analysis of skin tissue samples from CH pigs and LW pigs,we found that there were 454 differentially expressed m RNAs,612differentially expressed circ RNAs,188 differentially expressed mi RNAs,and 19differentially expressed lnc RNAs between the two breeds,revealing breed-specific differences from gene transcription levels in skin tissue.In the differentially expressed genes,many circ RNA-mi RNA-m RNA regulatory networks were constructed,including 67circ RNAs,47 mi RNAs,and 48 m RNAs.Among them,the key candidate circ004463-mi R-23b-CADM3/MAP4K4 regulatory axis was identified,of which circ004463 is highly expressed in skin tissue of CH pigs.Gene functional validation experiments were performed at the cellular in vitro and mouse in vivo.The luciferase reporter and RIP analyses showed that circ004463 targets mi R-23b and that mi R-23b targets CADM3/MAP4K4.The RT-q PCR and ELISA analyses showed that overexpression of circ004463/CADM3/MAP4K4 and mi R-23b inhibitor promotes the biosynthesis of type I collagen.The CCK-8 and Ed U analyses showed that overexpression of circ004463 and mi R-23b inhibitor promotes fibroblast proliferation.Theα-SMA fluorescence staining analysis showed that overexpression of circ004463 promotes fibroblast-to-myofibroblast transformation.The P21 staining and WB analysis showed that the mi R-23b inhibitor slows down fibroblast apoptosis.However,interference with the circ004463/CADM3/MAP4K4 and mi R-23b mimics showed the opposite experimental results.In addition,overexpression of circ004463 increased the skin thickness and type I collagen content in mouse.Furthermore,the co-transfection of circ004463,mi R-23b,and CADM3/MAP4K4 significantly affected their expression levels,fibroblast activity,and type I collagen content.The circ004463-mi R-23b-CADM3/MAP4K4regulatory axis exerted its biological function by activating the downstream AKT/ERK signaling pathway.These results indicate that the circ004463-mi R-23b-CADM3/MAP4K4regulatory axis plays a critical regulatory role in the formation of the unique skin thickness trait in CH pigs.3.Single-cell transcriptome profiling reveals heterogeneity and gene expression patterns of fibroblasts in different skin anatomical locations of CH pigs.Skin tissue samples from six different anatomical locations(back,head,shoulder,leg,abdomen,and ear)of CH pigs and a control group of LW pigs back skin were sequenced using single-cell transcriptome sequencing technology.The results are as follows:A single-cell transcriptome map of skin tissue of CH pig from different anatomical locations and LW pigs back was constructed,with a total of 233,715 skin cells including seven types of cells such as fibroblasts,smooth muscle cells,and endothelial cells,and revealed heterogeneity of fibroblasts.Based on the analysis of skin tissues from different breeds and anatomical locations,genes associated with collagen synthesis were significantly upregulated in CH pigs back fibroblasts.And COL11A1,TNN,and INHBA were screened as key candidate genes affecting the formation of unique skin thickness trait in CH pigs.Moreover,Cell communication analysis revealed that the COLLAGEN and LAMININ signaling pathways mediated cell communication between fibroblasts and smooth muscle cells/epidermal stem cells.The above research results reveal the single-cell transcriptome expression information in CH pig skin tissue,screen key candidate genes that regulate the formation of unique skin thickness trait in CH pigs,namely COL11A1,TNN,and INHBA genes.4.Small extracellular vesicles derived from fibroblasts in the dermis of CH pigs efficiently promote fibroblast activity and type I collagen synthesis.Research results show that small extracellular vesicles derived from dermal fibroblasts of CH pigs and LW pigs(control group)were isolated and identified using differential ultracentrifugation,transmission electron microscopy,nanoparticle tracking analysis,flow cytometry and so on.Functional validation experiments were performed at the cellular in vitro(dermal fibroblasts of CH pigs,LW pigs and human)and mouse in vivo.RT-q PCR and ELISA results demonstrated that small extracellular vesicles derived from dermal fibroblasts of CH pigs significantly upregulated the expression of type I collagen in fibroblasts(P<0.05);CCK-8,Ed U,and flow cytometry analysis showed that these vesicles promoted fibroblast proliferation;wound healing experiments showed that they promoted fibroblast migration;collagen gel contraction experiments showed that they stimulated collagen gel contraction;subcutaneous injection experiments in mouse showed that they increased skin thickness and regulated the content of type I collagen.The above results indicate that small extracellular vesicles derived from dermal fibroblasts of CH pigs can more efficiently promote fibroblast activity.Then,proteomic analysis was performed on small extracellular vesicles derived from dermal fibroblasts of CH pigs and LW pigs,and 151 differentially expressed proteins were identified.The key candidate ITGBL1 protein molecule that regulates fibroblast activity was screened.In functional validation experiments at the cellular in vitro and mouse in vivo,RT-q PCR analysis showed that overexpression of the ITGBL1gene significantly upregulated the expression of type I collagen(P<0.05);Ed U and flow cytometry analysis showed that it promoted fibroblast proliferation;wound healing experiments showed that it promoted fibroblast migration;collagen gel contraction experiments showed that it stimulated collagen gel contraction;WB experiments showed that the ITGBL1 gene played its biological function by mediating the TGFβ-SMAD2/3signaling pathway.However,interference with the ITGBL1 gene showed the opposite experimental effect.Subcutaneous injection experiments in mice showed that overexpression of the ITGBL1 gene increased skin thickness and the content of type I collagen.These research results reveal that the unique thick skin trait of CH pigs endows small extracellular vesicles derived from autologous dermal fibroblasts were more efficiently promoting fibroblast activity and type I collagen synthesis.In summary,based on multiple omics analyses and in vivo and in vitro functional validation experiments,we have elucidated the molecular basis for the unique skin thickness trait in CH pigs.This includes the identification of the key regulatory axis circ004463-mi R-23b-CADM3/MAP4K4 involved in regulating fibroblast activity,type I collagen content,and skin thickness,the discovery of key candidate genes COL11A1,TNN,and INHBA associated with skin thickness formation based on single-cell transcriptomic profiling,and the identification of the active molecule ITGBL1 enriched in small extracellular vesicles derived from dermal fibroblasts.The findings of this study not only provide important practical value for the breeding of special pig breeds with thick skin traits,but also offer important theoretical references for human skin physiology and health research.