Analysis Of The Molecular Regulatory Network Of Differentially Expressed Genes Between Different Haplotypes Of The Bovine Prion Protein Gene(PRNP)

The etiology of bovine spongiform encephalopathy（BSE）,commonly referred to as mad cow disease,is attributed to the abnormal folding and accumulation of prion proteins within the brain,leading to the neurodegeneration of neurons and nerve fibers,ultimately causing fatal behavioral and coordination disorders in cattle.The highly infectious and lethal nature of this disease poses a significant threat to both the cattle industry and human health.While the PRNP gene encoding the prion protein is considered a pivotal effector gene in BSE,multiple studies have established a strong association between specific variants of PRNP,particularly insertion/deletion（Indel）of 23bp in the promoter region and 12 bp in the first intron,and the increased susceptibility to BSE.However,it has been demonstrated that BSE is associated with additional genes beyond PRNP,although the precise genetic factors contributing to BSE susceptibility remain unclear.In order to comprehensively and systematically identify the genes and molecular markers responsible for conferring resistance to BSE,as well as those that exert influence and mitigate BSE risk while simultaneously enhancing cattle quality,a meticulous investigation was conducted.The selection of indigenous Diqing cattle for this investigation was a meticulously considered decision,as these specimens were both abundant in number and readily accessible.As such,this investigation focused on Diqing cattle,and 921 samples of yellow cattle were obtained from Shangri-La City,Diqing,Yunnan.We analyzed their mtDNA and SRY gene sequences and subsequently selected 655 Diqing cattle to investigate the 23bp and 12bp Indel polymorphisms of the PRNP gene.Then,mRNA and miRNA sequencing was performed on 12 medulla oblongata samples of each of the 2 haplotypes,23I-12I and 23D-12D,of the PRNP gene of Diqing cattle.Construction and resolution of mRNA and miRNA molecular regulatory networks of these 2different haplotypes.Finally,using zebrafish as a model organism,we carried out functional validation of the identified key genes via CRISPR/Cas9-mediated gene knockdown.The key findings of this study are as follows:1.Identification of Diqing yellow cattle and its PRNP gene polymorphism analysisBy analyzing the high-variance region of the mtDNA D-loop in a cohort of 921 male bovine specimens,we constructed neighbor-joining（NJ）evolutionary trees to discern a subset of 655Diqing cattle that met our exacting criteria.Subsequently,we cloned the coding region of the SRY gene in 591 of these bulls and generated a mediated network map to effectively screen for Diqing cattle of the same paternal lineage.The 23 and 12 bp Indel polymorphisms of the Diqing cattle’s PRNP gene were then definitively established,with the 23 bp Indel genotype frequencies being0.162（I/I）,0.586（I/D）,and 0.252（D/D）,and their corresponding allele frequencies being 0.455（I）and 0.545（D）,respectively;the 12 bp Indel genotype frequencies were 0.657（I/I）,0.305（I/D）,and 0.038（D/D）,and their corresponding allele frequencies were 0.809（I）and 0.191（D）.Moreover,the haplotype frequencies of 23I-12I,23D-12D,23I-12D,and 23D-12I were 0.433,0.168,0.022,and 0.377,respectively,demonstrating that these four haplotypes,especially 23I-12I and 23D-12D,were relatively equilibrated.Particularly suitable for the next research to carry out.2.Construction and elucidation of mRNA and miRNA molecular regulatory networks in response to varied haplotypes of the PRNP geneA purposive sampling strategy was employed to select a dozen Diqing cattle with uniform sex,and age,all of which were homozygous for the 23I-12I and 23D-12D haplotypes of the PRNP gene,for deep sequencing of mRNA and miRNA in the medulla samples.Our mRNA sequencing analysis yielded a total of 23,031 expressed genes,of which 108 were found to be differentially expressed.Specifically,we observed up-regulation of cyclin dependent kinase inhibitor 2B（CDKN2B）,nucleoredoxin Like 2（NXNL2）,and CXC chemokine ligand 13（CXCL13）,while 58genes,including cyclic nucleotide gated channel subunit beta 1（CNGB1）,apolipoprotein B mRNA editing enzyme catalytic subunit 2（APOBEC2）,and phosphodiesterase 1C（PDE1C）,were down-regulated.Additionally,our miRNA sequencing analysis identified 42 differential miRNAs,including 24 that were up-regulated by bta-miR-1246,bta-miR-196b,and bta-miR-211,and 18that were down-regulated by bta-miR-122,bta-miR-219,and bta-miR-2316.In a combined analysis of mRNA and miRNA data,42 differentially expressed miRNAs were found to have predicted target genes in two databases,Target Scan and miRWalk,totaling 18,647 target genes.Furthermore,the intersect analysis of these target genes with the 108 differentially expressed genes identified from transcriptome sequencing led to the discovery of 52 differential genes,including APOBEC2,PDE1C,and protocadherin 19（PCDH19）,that had regulatory relationships with 12 differentially expressed miRNAs,such as bta-miR-122 and bta-miR-211.Subsequently,a protein-protein interaction network（PPI）was constructed using differential genes based on PRNP genes.Five algorithms,including maximum cluster centrality（MCC）,maximum domain fraction density（DMNC）,and maximum domain fraction（MNC）,were utilized to screen 10 candidate genes,namely myosin heavy chain 2（MYH2）,protocadherin 19（PCDH19）,myosin light chain 2（MYL2）,transient receptor potential cation channel subfamily V member 6（TRPV6）,leptin receptor（LEPR）,kinesin family member 12（KIF12）,cyclin-dependent kinase inhibitor 2B（CDKN2B）,C-type lectin domain family 5 member A（CLEC5A）,cyclic nucleotide gated channel subunit beta 1（CNGB1）,and dynein axonemal heavy chain 11（DNAH11）.Further,a machine learning algorithm was employed to target 7 key genes,namely PCDH19,MYL2,TRPV6,LEPR,CDKN2B,CNGB1,and DNAH11.Subsequently,a co-expression regulatory network was constructed between these 7 key genes and bovine transcription factors（TFs）.Gene set enrichment analysis revealed that the PCDH19 gene had the most transcription factor binding sites in the TF-mRNA regulatory network with 74,and was associated with 751 GO terms and 79KEGG pathways,including Parkinson’s disease-related pathways.Finally,it was found that PCDH19 positively regulated PRNP in the 23D-12D group,with a correlation coefficient of 0.43,while a significant difference（P<0.05）was also observed.These findings suggest that PCDH19may play a crucial role in the molecular regulatory network under different haplotypes of PRNP.3.Molecular cloning and functional validation of new anti-BSE-related genesIn this study,we identified eight genes（PRNP,APOBEC2,PCDH19,PDE1C,DCX,BNC2,BCO2,and OR9Q2）and five miRNAs（bta-miR-2316,bta-miR-211,bta-miR-449a,bta-miR-196b,and bta-miR-122）that were differentially expressed in the samples.Our analyses revealed a consistent pattern of expression across both mRNA and miRNA sequencing results.Notably,our functional assays demonstrated that bta-miR-196b and bta-miR-2368-3p had negative regulatory effects on PRNP,while bta-miR-196b had a similar effect on PCDH19,and bta-miR-122 and bta-miR-499a had negative effects on APOBEC2.Additionally,bta-miR-211 negatively regulated PDE1C.The overexpressed miRNAs effectively targeted and downregulated the expression of their respective target genes,as confirmed by dual luciferase reporter assays.To further substantiate the functional significance of PCDH19,we meticulously handpicked 52 samples for each of the 23I-12I and 23D-12D haplotypes of the PRNP gene,out of which 12 transcriptome sequencing samples for each haplotype were also included.After cloning the approximately 2,300bp sequence of the 5’regulatory region of the PCDH19 gene,we identified six SNPs loci,namely-1597A>G,-1495G>T,-1415C>G,-923A>C,-855C>T,and-176C>T,which constitute 12distinct haplotypes.The dominant haplotype in both groups was GTCACC,with frequencies of0.297 and 0.338 in the 23I-12I and 12D-23D groups,respectively.Notably,haplotype AGCACT exhibited a frequency of 0.150 and 0.047 in the 23I-12I and 12D-23D groups,respectively,whereas haplotype AGGATC demonstrated frequencies of 0.058 and 0.0001 in the 23I-12I and12D-23D groups,respectively,with statistically significant differences in frequency（P<0.05）.Employing CRISPR/Cas9 technology,we deleted a 1,400 bp segment of the 5’regulatory region of the zebrafish PCDH19 gene,followed by evaluating the brain development of zebrafish embryos at 20 hour post fertilization（hpf）,30 hpf,48 hpf,and 72 hpf time intervals while scrutinizing the expression of associated genes.Consequently,we acquired heterozygous mutants PCDH19^+/-with successful knockout and observed that mRNA expression of PCDH19 gene was down-regulated in all time intervals compared to the wild type group,displaying non-significant differences at 20 hpf and 72 hpf（P>0.05）,but significant differences at 30 hpf（P<0.01）and 48hpf（P<0.05）,with non-significant differences at 72 hpf（P>0.05）.Using q RT-PCR,we also confirmed the expression of seven key genes（PRNP（PRNPa and PRNPb）,MYL2a,TRPV6,LEPR,CDKN2B,CNGB1a,DNAH11）and discovered differential expression at different developmental time points,where the CDKN2B gene was notably upregulated and statistically significant in all four intervals following knockdown（P<0.01）.Additionally,zebrafish with knocked-down PCDH19 gene exhibited morphological abnormalities in the anterior neural plate region of the brain at 30hpf during early embryonic development,further confirming the significant role of the PCDH19 gene in the normal development and morphological maintenance of the anterior neural plate.Here,we present the first comprehensive mRNA and miRNA sequencing analysis of different haplotypes of the bovine PRNP gene as a collective,culminating in the construction of a molecular regulatory network centered around PRNP.Our study identifies seven genes,PCDH19,MYL2,TRPV6,LEPR,CDKN2B,CNGB1,and DNAH11,that exhibit putative associations with BSE,as well as eight miRNAs,including bta-miR-219,bta-miR-2316,and bta-miR-211.Further experimental validation,via dual-luciferase reporter gene technology and CRISPR/Cas9techniques,confirms that the PCDH19 gene likely plays a crucial role in the regulation of BSE in yellow cattle.Our findings not only provide a comprehensive overview of the genetic factors that underlie BSE,but also identify novel genes that may serve as potential targets for future intervention strategies aimed at mitigating the risk of BSE and improving cattle quality.