Analysis Of Molecular Regulatory Network Of Pig Ear Malformation Pathogenic Gene HOXA1 Based On RNA-Seq And CHIP-Seq

Our research group previously obtained a pig population with microtia.The affected individuals were mainly manifested as external auricular malformation and external auditory canal malformation,and the external ear completely disappeared in severe cases.All sick piglets were unable to eat and died within a week of birth.The research group found through various genetic techniques in the early stages that the transcription factor HOXA1 c.451 delins TC is a causal pathogenic site for the genetic disease.However,the molecular regulatory network of the pathogenic gene is still unclear.This study used ear tissue samples from individual piglets with congenital microtia and their normal littermates as research objects.It integrates histological analysis,RNA-seq,and CHIP-seq analysis to unravel the molecular regulatory network of the pathogenic gene HOXA1.First,ear tissue from an individual with the disease and ear tissue from a normal individual were used to make paraffin sections,which were stained with HE.By comparing and analyzing the panoramic sections of the ear tissue from the diseased and normal individuals,differences in the morphological characteristics of the ear tissue cells were identified.The results showed that the epidermis of the ear tissue from the diseased individual was significantly thinner（P < 0.05）,with a decrease of 1-2 layers of spinous cells;the area of the dermis was significantly different between the two groups（P < 0.05）;there were slightly more hair follicles and relatively less cartilage,but the changes were not significant.We hypothesize that the HOXA1 c.451 delins TC mutation causes abnormal ear development in affected individuals by affecting the proliferation or apoptosis of skin epidermal cells.Secondly,transcriptome（m RNA,lnc RNA,and mi RNA）analysis was performed using the outer ear tissues of diseased and normal piglets to detect genome-wide changes in transcription levels caused by HOXA1 pathogenic sites.Results A total of 419 differentially expressed m RNAs（dif-m RNAs,243 up-regulated and 176 downregulated in patients）were detected.Eight differentially expressed dif-mi RNAs（5 upregulated and 3 down-regulated in diseased individuals）,among which 3 were known mi RNAs and 5 were newly predicted mi RNAs.There were 3122 dif-lnc RNAs（1601 up-regulated and 1512 down-regulated）,of which 9 were known lnc RNAs and 3113 were newly predicted lnc RNAs.Twenty-three differentially expressed circ RNAs（16 up-regulated and 7 down-regulated in diseased individuals）.It was predicted that 138 target genes of differential lnc RNAs and overlapped genes of differential mi RNAs with dif-m RNAs（dif-lnc RNAs,112 up-regulated and 26 down-regulated in diseased individuals）and 8（dif-mi RNAs,26 down-regulated）,respectively.In diseased individuals,7 were up-regulated and 1 was down-regulated).Among the top10 genes before downregulation of m RNA expression levels in patients with the disease,the keratin gene KRT3（only highly expressed in the skin and esophagus and low expressed in the bone matrix）,the gene CRABP2（HOXA1 binding gene）and the gene ELOVL6 were most down-regulated.MYOC,MYT1 L,KRTAP7-1 and TAF7 L were among the top10 genes before up-regulation of m RNA expression in patients with the disease.difm RNAs,dif-lnc RNAs-dif-m RNAs and dif-lnc RNAs-dif-m RNAs have two difm RNAs,namely LTBP2 and PPP1R12 C,which are upregulated in the affected individuals.LTBP2,LTBP1 and LTBP4 belong to the family of potentially transforming growth factor β-binding proteins,which are known to be pathogenic genes of dermalysis.PPP1R12 C gene is a subunit encoding myosin phosphatase and can regulate the activity of myosin phosphatase.A ce RNA network,TCONS₀0238510-novel₃8-PRDX1,was discovered.The PRDX1 gene is an important member of the peroxiredoxin family and can regulate the repair function of the skin barrier.GO and KEGG analyses of target differentially expressed m RNAs and non-coding RNAs revealed significant differences in pathways such as DNA replication（PCNA,etc.）,longevity regulation（FOXO3,etc.）,and insulin resistance（CREB5,etc.）between affected and normal individuals.Thirdly,CHIP-seq of pathogenic gene HOXA1 was performed on the outer ear tissue of normal piglets to search for the possible binding genes of HOXA1,and 11036 genes were enriched in total.These genes were concentrated in GO entries related to cell proliferation and adhesion,protein anabolism,cell biosynthesis,and KEGG signaling pathways related to intracellular and intracellular membrane transport,cell proliferation and apoptosis,and calcium ion synthesis.RNA-seq and CHIP-seq joint analysis revealed that CHIP-seq enriched 246 difm RNAs,including 168 upregulated genes and 78 downregulated genes in affected individuals.These genes were also concentrated in the longevity regulation pathway,DNA replication,insulin resistance,and c GMP-PKG signaling pathway.Through PPI network interaction analysis using RNA-seq and RNA-seq/CHIP-seq joint analysis,it was discovered that CDC6,MCM4,MCM5,and PCNA genes played important roles in the protein network,with PCNA being a differentially expressed gene that was common between RNA-seq and CHIP-seq and a target gene of HOXA1.PCNA and FOXO3 are common genes in the PI3K-Akt signaling pathway and longevity regulation signaling pathway.GABARAPL1,a target gene of FOXO3,is a hallmark gene affecting autophagy in cells.Therefore,we speculate that the pathogenic gene HOXA1 downregulates the PCNA gene to affect the synthesis and repair of DNA.At the same time,the down-regulated PCNA activates the FOXO3 gene which further promotes the upregulation of autophagy gene GABARAPL1 expression.This ultimately leads to autophagy or apoptosis of the epidermis of the ear and esophageal epithelial cells in affected individuals,resulting in incomplete development of the ear and esophagus,causing microtia and an inability to eat normally.Finally,we used si RNA interference to target the HOXA1 gene in human epidermal cells,and the fluorescence quantitative PCR test showed that the proliferation markers（CDK8,PCNA,CCNB,and CCNE1）of epidermal cells were downregulated,as were the apoptosis markers（TP53,BAX,and PARP）.Therefore,it is speculated that the pathogenic gene affects the expression of proliferation or apoptosis markers,leading to incomplete development of the ear in affected individuals,ultimately resulting in microtia.In summary,this study analyzed the molecular regulatory network of the pathogenic gene HOXA1 at the transcriptional and protein levels,providing a theoretical basis for the study of the pathogenic mechanism of pig congenital ear malformation,and providing valuable reference for revealing the molecular mechanism of human congenital small ear malformation.