| Objectives:To screen differentially expressed miRNAs in sensitized acupoint tissues of rats with Knee Osteoarthritis(KOA)under different acupoint sensitization states and perform bioinformatics analysis.From the perspective of gene regulation,we study the occurrence of acupoint sensitization phenomenon and the miRNAs involved in the formation of different sensitization states and their biological functions,to provide some directions and references for explaining the molecular biological mechanism of acupoint sensitization phenomenon.Methods:1.Detection of acupoint sensitization and determination of sensitization status: 27 SD rats were randomly divided into normal saline(NS)group and KOA model group according to the ratio of 1:2.Rats in the KOA group were prepared by injecting 3mg/50μL MIA into the knee joint cavity,and rats in the NS group were injected with the same dose of normal saline.Electric Von Frey was used to detect the mechanical pain threshold of relevant acupoints of each rat before and after modeling,and the rate of change in mechanical pain threshold was calculated and used as the criterion for the degree of acupoint sensitization.The acupoints with the closest incidence of high acupoint sensitization to the incidence of low acupoint sensitization were screened,and the rate of change of mechanical pain threshold at this acupoint was used to distinguish rats with different acupoint sensitization states.2.High-throughput sequencing and biological information analysis:We extracted miRNAs from the subcutaneous connective tissue of sensitized acupoints from rats with three different sensitization states for high-throughput sequencing and screened for differentially expressed miRNAs,and then used q RT-PCR to validate the expression levels of miRNAs with significant differences in expression.Afterwards,the miRNAs with significant expression differences were analyzed bioinformatically.First,the miRanda database was used to predict target genes,while the Gene Ontology database was used for GO enrichment analysis of target genes,and KEGG pathway analysis was used for pathway analysis,so as to investigate the biological functions of differential miRNAs and the signaling pathways they regulate.Results:1.Comparison of mechanical pain threshold of acupoints before and after modeling between two groups(1)After modeling,compared with the NS group,the mechanical pain thresholds of ST34、SP10、EX-LE2、EX-LE5、ST35、ST36、SP6、SP9、GB34、GB39、LR3 of rats in the model group were significantly decreased,and the difference was statistically significant(p<0.05).(2)Compared with before modeling,the mechanical pain thresholds of ST34、SP10、EX-LE2、EX-LE5、ST35、ST36、SP6、BL23 of rats in the model group were significantly reduced after modeling,with statistically significant differences(p<0.05).2.Incidence of high and low acupoint sensitization of sensitized acupoints in ratsThe number of high and low acupoint sensitization occurred in each sensitized acupoint of rats in the model group was counted and the incidence was calculated,and we found that the incidence of high acupoint sensitization and low acupoint sensitization was the same in ST35 and ST36.However,because the incidence of sensitization at ST36 is more conducive to subsequent experiments,this experiment will take ST36 as the research object for high-throughput sequencing of rats with different sensitization states.3.Results of differential miRNAs Screening(1)A total of 32 differentially expressed miRNAs were screened in the low acupoint sensitization group compared with the non-sensitized group,with 22 miRNAs up-regulated and 10 miRNAs down-regulated.Of these,4 miRNAs were significantly up-regulated in the low acupoint sensitization group compared with the non-sensitized group: rno-miR-199a-3p,rno-miR-199a-5p,rno-miR-214-3p,and rno-miR-205.(2)A total of 20 differentially expressed miRNAs were screened from the high acupoint sensitization group compared with the non-sensitized group,with 7 miRNAs up-regulated and 13 miRNAs down-regulated.Among them,the expression of 4 miRNAs was significantly up-regulated in the high acupoint sensitization group compared with the non-sensitized group: rno-miR-199a-3p,rno-miR-199a-5p,rno-miR-214-3p,and rno-miR-205.(3)A total of 17 differentially expressed miRNAs were screened from the high acupoint sensitization and low acupoint sensitization group,with 10 miRNAs up-regulated and 7 miRNAs down-regulated.Among them,the expression of 1 miRNA was significantly down-regulated in the high acupoint sensitization group compared with the low acupoint sensitization group: rno-miR-543-3p.4.Results of Bioinformatics analysis(1)Sensitized group(high acupoint sensitization group and low acupoint sensitive group)compared with non-sensitized group,we subjected the four selected miRNAs with significant expression differences to target gene prediction,resulting in a total of 2107 target genes.We performed GO enrichment analysis of these target genes and found that the biological processes in which they are involved include negative regulation of receptor activity,negative regulation of alpha-beta T cell proliferation,etc.Cellular component includes AMP-activated protein kinase complex,ER-mitochondrion membrane contact site,etc.Molecular function includes receptor agonist activity,AMP-activated protein kinase activity,etc.We performed KEGG signaling pathway analysis of these target genes and found that the signaling pathways in which they are involved include p53 signaling pathway,Hedgehog signaling pathway,MAPK signaling pathway,etc.(2)high acupoint sensitization group compared with low acupoint sensitive group,we subjected the one selected miRNAs with significant expression differences to target gene prediction,resulting in a total of 25 target genes.We performed GO enrichment analysis of these target genes and found that the biological processes in which they are involved include positive regulation of inflammatory response,cellular response to hydrogen peroxide,etc.Cellular component includes spindle pole,etc.Molecular function includes single-stranded DNA binding,cytokine activity,etc.We performed KEGG signaling pathway analysis of these target genes and found that the signaling pathways in which they are involved include MAPK signaling pathway,FcεRI signaling pathway,Jak-STAT signaling pathway,etc.5.Results of QRT-PCR validationWe validated the 5 miRNAs with significant expression differences and the results showed: Compared with the non-sensitized group,the expression of rno-miR-199a-3p and rno-miR-205 was up-regulated in the low acupoint sensitive group,and the difference was statistically significant(p< 0.05),which was consistent with the results of high-throughput sequencing,the expression of rno-miR-199a-5p and rno-miR-214-3p was up-regulated,but the difference was not statistically significant;compared with the non-sensitized group,the expression of rno-miR-199a-3p,rno-miR-199a-5p,rno-miR-214-3p,and rno-miR-205 was up-regulated in the high acupoint sensitive group,but the difference was not statistically significant;compared with the low acupoint sensitive group,the expression of rno-miR-543-3p was down-regulated in the high acupoint sensitive group,but not statistically significant.Conclusion:1 Rno-miR-199a-3p,rno-miR-199a-5p,rno-miR-214-3p,and rno-miR-205 were significantly up-regulated in sensitized acupoint tissues of high and low acupoint sensitized rats compared with non-sensitized conditions,and these four differential miRNAs may be involved in the regulatory mechanism of acupoint sensitization;2.Rno-miR-543-3p was significantly down-regulated in the sensitized acupoint tissue of rats with high acupoint sensitization compared with low sensitization state,and this differential miRNA may be involved in the regulatory mechanism of the formation process of different sensitization states. |
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