Screening,Identification And Bioinformatics Analysis Of MicroRNAs On Abnormal Phlegmatic Syndrome And It's Medicine Intervention Rats's Penile Tissue

Objective:To screen the microRNA profile on abnormal phlegmatic syndrome rats?s penile tissue,establish the candicate miRNA markers,identificate the expression of miR-140-3p and miR-486,to predict the function of miR-140-3p and miR-486.Methods:80 healthy mature male SD rats were enrolled in this study,10 of them were taken randomly as the control group?N?,the remainder 70 were in the model group.To established animal models of abnormal phlegmatic syndrome by treated with spinach and coriander diet in the cold humid environment.Observing biological characterization of rats weekly until the model was established.APO erectile experiment and mating experiment was performed to select impotence rats.The impotence rats were randomly divided into importence model group?BM?and importence model's medicine intervention group?BY?.The non-impotence rats were randomly divided into abnormal phlegmatic syndrome model group?ZM?and abnormal phlegmatic syndrome model's medicine intervention group?ZY?.Medicine intervention was performed for 3 weeks,while observing biological characterization and evaluating erectile function of the rats,then penis tissue of the rats was taken.The total RNA was extracted and then subjected to Illumina Hiseq technology to screen differentially expressed microRNA in penis tissue of the rats,cluster analysis was performed,the candidate microRNA was selected and viladated by qPCR with amplified subjects 20 rats.The target genes regulated by differentially expressed microRNA were predicted by the software of Targetscan,miRDBand miRanda.Bioinformatics analysis was performed to analyze the enriched gene ontology?GO?and signaling pathway?KEGG pathway?.Results:?1?The results of biological characteristics:compare with control group,with the lapse of time,the rats is sluggish with dull and random hair,clear urine,soft feces,white greasy tongue coating on the tongue,dark-purple tongue.The weight gain is slower than normal,with increased food intake,decreased water intake,urine and feces output increased.After treatment withYimusake,the symptoms were impoved.?2?the results of APO experiment and mating experiment:compare with control group,the erectile latency was increased and the number of erections was reduced in the ZM group;the incubation periods of straddle,insertion and ejaculation time were prolonged,and the frequency of straddle increased;the frequency of insertion and ejaculation decreased,after treatment with Yimusake,compare to abnormal phlegmatic syndrome model group,the incubation periods of straddle,insertion and ejaculation decreased;and the frequency of straddle,insertion and ejaculation increased in the ZY group.?3?The results of Illumina Hiseq showed that:There were 147 miRNAs were diffentially expressed in ZM group compared to N group.Among them,15 miRNAs were identified by absolute value of log₂ Ratio>0.8,among which 13 miRNAs were up-regulated and 2 miRNA downregulation;There were 131miRNAs with significant difference in ZY group compared with ZM group.3 miRNAs were selected by absolute value of log₂ Ratio>0.8,all miRNAs were downregulation.2miRNA were inversely regulated by Yinusake tablets treatment.?4?The results of real-time qPCR showed that:The trend of the results of miR-140-3p and miR-486 were consistent with the that of high-throughput sequencing technology.Compared to N group,the expression level of miR-140-3p was significantly increased in ZM group?P?29?0.05?,the expression level of miR-486 was significantly increased?P<0.05?.Compared to ZM group,the expression level of miR-140-3p and miR-486 was significantly decreased in ZY group?P<0.05?.Conclusions:?1?Rats model of abnormal phlegmatic syndrome was successfully established.The biologic characteristics of abnormal phlegmatic syndrome was consistent with the clinical manifestation of Uygur's medicine,the latency of erection was significantly prolonged and the function of sexual behavior was significantly decreased.?2?A total of 15 miRNAs such as miR-140-3p?miR-143-3p?miR-486 were identified as molecular marker of miRNAs of abnormal phlegmatic syndrome,miR-434-3p,miR-486 and other three miRNAs can be used as candidate molecular markers for the intervention of yimusake tablet.?3?expressed miRNA were selected which is related to targets of medicine by comprehensive analysis.miR-486 may be a marker in the development of abnormal Phlegmatic syndrome,miR-140-3p,miR-486 may be treated as target of Yimusake intervention.miR-140-3p may be involved in the formation of fibrosis through its target gene TGF-?3,and miR-486 may be involved in the regulation of glycolipid metabolism through its target gene PTEN,but it need to validation.