Expression And Analysis Of PiR-36241、piR-36340 And Their Targeted MRNA In Pulpitis

BackgroundPulpitis is an inflammatory disease developed without timely and effective control and treatment of caries,trauma and other diseases.It is mainly caused by the host immune defense response activated by bacterial infection,with pain as the main symptom,and pulpitis is one of the most common oral diseases.At present,the preferred treatment for pulpitis is root canal therapy,which can reduce inflammation and relieve pain by removing the pulp and removing the infection.However,root canal therapy has its limitations:（1）treatment requires extraction of dental pulp,resulting in loss of blood supply,loss of sensation,and increased brittleness of teeth;（2）Inevitably,the tooth tissue is damaged during the treatment process,thus reducing the resistance to bending and increasing the risk of tooth splitting;（3）Tooth discoloration after treatment,which affects aesthetic appearance;（4）Due to the complexity of the root canal system,even though materials and equipment continue to improve,the success rate of root canal therapy is still only about 85%.Therefore,it is urgent to find a better method to prevent and cure pulpitis.Although revascularization has been used clinically for pulp regeneration,it is mainly suitable for young permanent teeth and has a narrow indication range.Therefore,it is of great clinical significance to further reveal the pathogenesis of pulpitis and find therapeutic targets,so as to achieve early inflammatory control,block the progression of inflammation,promote pulp regeneration and restore pulp vitality,and to transform the treatment strategy from"demedulation"to"pulp preservation”.piRNAs are a class of small non-coding RNAs with a length of about 26-33nt that regulate gene expression.Through specific binding with PIWI protein,it plays a biological role,and silences the transposon mechanism at the post-transcriptional level to maintain the stability and integrity of the genome,and plays an important role in the regulation of cell differentiation,embryo development,gene transcription and post-transcriptional modification.Although the research on inflammation and immunity is still in the initial stage,piRNAs play an important role in the occurrence and development of inflammation,revealing the changes and functions of piRNAs in the occurrence and development of pulpitis has clinical and scientific guiding significance for the prevention and treatment of pulpitis.ObjectiveIn this study,human pulpitis samples were collected for high-throughput sequencing and bioinformatics analysis to reveal the piRNAs expression profile in the occurrence and development of pulpitis,screen differential piRNAs and predict the target genes of these differentially expressed piRNAs,so as to further reveal the relationship between piRNA-mRNA and the molecular regulatory network system in pulpitis.To elucidate the etiology and pathogenesis of pulpitis to provide scientific basis.Material and MethodsThis research is divided into three parts:1.Expression profile analysis of piRNAs in human pulpitis（1）Clinical pulpitis tissue samples and normal pulp tissue samples were collected for high-throughput sequencing and histomathological analysis;（2）The differentially expressed piRNAs were obtained by high-throughput sequencing and target gene prediction,GO enrichment analysis and KEGG pathway analysis of target genes were performed by mi Randa and RNAHybrid..2.Establishment of experimental pulpitis model in mice（1）The pulpitis model of C57BL/6 mice was induced by pulp cavity exposure on the occlusal surface of the first maxilla of the mice by pulp opening method.The specimens were sacrificed at 0h,4h,8h and 16h after the exposure of the pulp cavity.（2）Maxillary bone was isolated for micro-CT observation,HE staining and quantitative analysis to analyze the occurrence,development and outcome of pulpitis.3.Expression and function prediction of piR-36241,piR-36340 and their targeted mRNA in human and mouse pulpitis（1）using samples of pulpitis|log₂FC|greater than 2 piRNAs and target genes of RT-q PCR validation and function prediction.（2）The differential piRNAs and their target genes obtained in step（1）of this part were verified and analyzed by RT-q PCR using the pulpitis tissues of mice..Results1.Expression profile analysis of piRNAs in human pulpitis（1）HE staining showed that during pulp inflammation,odontoblast cells were arranged disorderly,pulp vessels dilated,red blood cells exudated,and some pulp necrosis occurred.The number of inflammatory cells,including neutrophils and macrophages,was significantly more than that of normal pulp tissue.The expression of TNF-αand IL-10 was significantly higher in pulpitis samples,P<0.05。（2）High-throughput sequencing results showed that a total of 692 known piRNAs were identified in pulp tissues,including 453 piRNAs expressed in pulpitis group and 574 in normal pulp tissues.Among them,15 up-regulated piRNAs and 6down-regulated piRNAs were statistically significant.（3）Combined analysis of differentially expressed piRNAs and its target genes,KEGG pathway analysis showed that target genes were mainly concentrated in cytokine-cytokine receptor interactions,hematopoietic cell lineage and chemokine signaling pathways.GO enrichment results showed that signal transduction and multicellular development were the highest in the biological process of up-regulation and down-regulation of piRNAs target genes,respectively.The concentration of cell membrane was the highest in cell components.Receptor activity and calcium binding were the highest in the molecular function of up-regulated and down-regulated piRNAs target genes,respectively.2.Establishment of experimental pulpitis model in mice（1）In this study,the pulpitis model of mice was established by pulp opening method,and mice were killed successively according to different time periods.The HE staining results of 0 h,4 h,8 h and 16 h groups showed that with the extension of time after pulp opening,inflammation gradually developed from the crown to the root,until pulp cavitation and necrosis were completed.Pulpitis in mice was characterized by disordered arrangement of odontoblast cells,vascular dilatation,red blood cell exudation and partial pulp necrosis.The number of inflammatory cells in 4 h group,8h group and 16 h group was 2.4,3.3 and 1.3 times of that in 0 h group,respectively.The pathological process is basically consistent with human pulpitis.（2）Micro-CT scan of the maxillary bone taken at 0h,4h,8h and 16h groups showed that the pulp opening was located in the central fossa cavity of the maxillary first molar,and the size of the pulp opening was consistent,which met the experimental requirements.3.Expression and function prediction of piR-36241,piR-36340 and their targeted mRNA in human and mouse pulpitis（1）Clinical sequencing of|log2FC|greater than 2 piR-36241,piR-36340,piR-36252 and piR-31686.RT-q PCR showed that the expression of piR-36241 and piR-36340 in human pulpitis was significantly higher than that in normal pulp tissue.After the verification of target genes of piR-36241 and piR-36340,10 genes,including GFRA1,FAM19A3,LYPD6,MAPT,NOS1,ADGRG2,AJAP1,SPOCK1,SMPD3 and SNAP91,may be the potential target genes of piR-36241 for the regulation of pulpitis.CACNA2D2 may be a potential target gene of piR-36340 in the regulation of pulpitis.（2）The difference between piRNA and target mRNA was verified by RT-q PCR using mouse pulpitis samples,which showed that the expression of piR-36241 and piR-36340 in mouse pulpitis was significantly higher than that in normal pulp tissue,which was consistent with the results of clinical sample verification.After the target genes of piR-36241 and piR-36340 were verified by the mouse pulpitis tissue,9 target genes of piR-36241 were finally obtained in mouse pulpitis,including GFRA1,LYPD6,MAPT,NOS1,SMPD3,SNAP91,SPOCK1,ADGRG2,and AJAP1.ConclusionIn this study,piRNAs were detected in both inflammatory and normal dental pulp tissues and their target genes were predicted.It was found that piR-36241 and piR-36340 were significantly overexpressed in both human and mouse pulpitis.The 9target genes of piR-36241,GFRA1,LYPD6,MAPT,NOS1,SMPD3,SNAP91,SPOCK1,ADGRG2 and AJAP1,were all significantly down-regulated in human and mouse pulpitis.The expression levels of piR-36340 target genes in mouse pulpitis were not significantly different.This suggesting that piR-36241 may play a role in the occurrence and development of pulpitis.Open using the access method could effectively establish experimental pulpitis model in mice,the histopathological manifestations and clinical,indicated that the model was better to simulate the occurrence and development process of pulpitis,model good repeatability,stable effect,joint mice pulpitis histopathological and molecular pathological evaluation standard,the model was expected to be in the study of pulpitis of prevention and control has better application prospect.These new findings still need to be further studied to further clarify the role,function and molecular network mechanism of piR-36241 in the pulpitis process,so as to provide scientific guidance data for clinical pulpitis prevention and control and pulp regeneration.