Study On The Regulation Mechanism Of Acupuncture On Serum Exosomal MicroRNA In Patients With Knee Osteoarthritis

Objective:The serum exosomal microRNA differentially expressed in KOA patients was screened by small RNA sequencing technology,and the target gene and signal pathway are analyzed.To study the regulation mechanism of acupuncture treatment on serum exosomal microRNA in KOA patients.Methods:1.A total of 22 patients with KOA and 12 healthy subjects were included in the study.Patients with KOA were randomized into acupuncture treatment group(n=11)and waiting for treatment group(n=11).The acupuncture treatment group selected the acupoint of EX-LE2,EX-LE5,ST34,ST35,ST36,SP9,SP10 and GB34.3times/week for 4 consecutive weeks,a total of 12 times.The waiting for treatment group was not treated for the first 4 weeks,and the same acupuncture intervention was performed 4 weeks later.The effect of acupuncture was evaluated by WOMAC score at 0 weeks and 4 weeks after enrollment.2.Serum samples were collected from healthy subjects at 0 weeks after enrollment.Serum samples were collected from KOA patients before and after 4weeks,centrifuged in an ultracentrifuge,and precipitated under transmission electron microscopy to determine whether it was exosome based on morphology and size.The microRNAs in the isolated exosomes were sequenced by small RNA sequencing technology.The differentially expressed microRNAs were screened according to the principle of P?0.05,high difference fold and high expression abundance.The levels of screening and comparison were as follows:(1)comparing the content and type of serum exosomal microRNA in healthy subjects and KOA patients;(2)comparing the expression of serum exosomal microRNAs before and after treatment in acupuncture treatment group;(3)comparing the expression of serum exosomal microRNA before and after 4 weeks in waiting for treatment group.According to the above three comparison levels,wean map was used to screen out the serum exosomal microRNAsthat were mainly affected by acupuncture treatment in KOA patients.3.TargetScan and miRDB were used to predict target genes of differential serum exosomal microRNAs,and to further analyze the function and related pathways of target genes.To explore the regulation mechanism of acupuncture on serum exosomal microRNA in KOA patients,in order to provide new directions and references for basic research and clinical prevention of KOA.Results:1.Baseline data analysis results:Baseline data of KOA patients and healthy subjects were balanced and comparable(P>0.05).The baseline data of the acupuncture treatment group and the waiting treatment group were balanced and comparable(P>0.05).2.Clinical effect of acupuncture on patients with KOA:The pain score,joint function score and WOMAC total score of the acupuncture treatment group decreased after acupuncture(P<0.05).There was no significant difference in pain,stiffness,joint function score,and WOMAC total score in the waiting for treatment group before and after 4 weeks.In the control group between the two groups,the pain,joint function score and WOMAC total score of the acupuncture treatment group were lower than those of the waiting for treatment group(P<0.05).3.Screening results of serum exosome microRNA expression:(1)Compared with healthy subjects,there were 236 differentially expressed serum exosomal microRNAs in KOA patients,of which 114 were up-regulated and122 were down-regulated;(2)Compared with 0 weeks after enrollment,there were 60 serum exosomal microRNA expression differences after 4 weeks of acupuncture treatment,of which32 were up-regulated and 28 were down-regulated;(3)Compared with 0 weeks after enrollment,there were 110 exogenous microRNAs differentially expressed after 4 weeks of treatment in the waiting treatment group,of which 59 were up-regulated and 51 were down-regulated;(4)After the above three levels of comparison and screening analysis,it wasfinally determined that the three serum exosomal microRNAs with the most significant differential expression after acupuncture intervention were screened:hsa-miR-1228-5p,hsa-miR-1296-5p,hsa-miR-3168.4.Bioinformatics analysis results:(1)Target gene prediction results: A total of 73 target genes for 3 exosomal microRNAs were predicted.(2)Analysis of target gene function enrichment analysis: Functional analysis of target genes from three exosomal microRNAs revealed that it may be involved in multiple biological processes: regulation of RNA metabolism,regulation of nucleobase-containing compound metabolism,regulation of RNA polymerase II transcription,regulation of nucleic acid template transcription,regulation RNA biosynthesis,etc.,and play a variety of molecular functions.(3)Pathway analysis results of target genes: The target genes of the three exosomal microRNAs are enriched in 37 pathways,including biochemical metabolic pathways such as apoptosis,autophagy,hepatitis B,non-small cell lung cancer,type II diabetes,hypertrophic cardiomyopathy,regulation of lipolysis in adipocytes,etc,and including signal transduction pathways such as MAPK signaling pathway,p53 signaling pathway,ErbB signaling pathway,oxytocin signaling pathway,etc.Conclusions:1.Acupuncture treatment can effectively relieve knee pain and joint function in KOA patients;2.The differential expression of serum exosome microRNA between KOA patients and healthy people was significant,of which 114 were up-regulated and 122 were down-regulated;3.Acupuncture can down-regulate the expression of serum exosomes hsa-miR-1228-5p,hsa-miR-1296-5p and hsa-miR-3168 in KOA patients.4.By bioinformatic analysis of three differentially expressed serum exosomal microRNAs,acupuncture may play a therapeutic role by regulating MAPK signaling pathway,apoptosis,autophagy and metabolism in KOA patients.