Expression And Significance Of LncRNA ASO3491 And E2F2 In Non-obstructive Azoospermia

Objective 1.To study the expression profiles and expression differences of long noncoding RNA(lncRNA)in non-obstructive azoospermia(NOA)testis tissue,and the potential function of lncRNA in NOA.2.To study the mRNA expression profile and expression characteristics of testicular tissue in NOA patients,and explore the role and significance of mRNA in the important pathogenesis of NOA.3.To detect the expression of ASO3491 in serum and spermatozoon.4.To explore the expression of E2F2 in NOA,further providing a new index for the clinical diagnosis of NOA,and providing a theoretical basis for the follow-up study of ASO3491 and E2F2 in the pathogenesis of NOA,providing a new treatment for the clinical treatment of NOA.Methods Experimental studies.1.Collection of crystallographic lncRNA+mRNA expression from testicular tissues of patients with NOA(n=3),OA patients(n=3)and testicular abscess with normal fertility(n=3)from December 2014 to December 2016 Spectral chip detection.2.Screening candidate genes by gene Go enrichment,KEGG pathway analysis,disease enrichment,and gene targeting prediction.3.The accuracy of microarray sequencing was further verified by qRT-PCR detection of gene expression.4.Re-collecting NOA patients(n=35),OA patients(n=35),normal patients(n=35)serum and seminal plasma from March 2017 to December 2017 in Ningxia Medical University General Hospital Reproductive Medicine Center,using qRT-PCR and ELISA to detect serum the expression ofASO3491 and E2F2 in the seminal plasma was compared and analyzed.5.Analysis of the correlation between ASO3491,E2F2 levels and sex hormones,testicular total volume.Results 1.Compared with the normal control group,there were 4356 lncRNA high expression and 715 lncRNA low expression in testicular tissue of NOA patients;928 lncRNA high expression and 20 lncRNA low expression in testicular tissue of OA patients;In contrast,there were 8437 lncRNA high expression and 3808 lncRNA low expression in testicular tissues of NOA patients.Differentially expressed lncRNA mostly belonged to intergenic lncRNA.2.Compared with the normal control group,there were 2971 mRNA high expression and 1509 mRNA low expression in testicular tissue of NOA patients;342 mRNA expression and 108 expression in testicular tissue of OA patients Low expression of mRNA;compared with OA group,there were 6040 mRNA high expression and 5303 mRNA low expression in testicular tissue of NOA patients.3.Three candidate lncRNA include ASO3491,ENSG00000232527.3 and ENSG00000227372.5,six candidate mRNA include PRKX,AKT2,MLLT4,E2F2,SCD5 and ACSS1 were screened out.The results of gene-targeted bioinformatics analysis indicated the target gene of ASO3491 is E2F2,the target gene of ENSG00000232527.3 is AKT2,and the target gene of ENSG00000227372.5 is SCD5.4.PCR verified that the expression levels of 9 candidate genes in testis tissue were consistent with the results of gene chip detection.5.ASO3491 was expressed in male serum and seminal plasma.Serum ASO3491 was significantly expressed in NOA compared to the normal group and the OA group.There was no significant difference in ASO3491 between the normal group,the OA group and the NOA group in the seminal plasma.6.E2F2 was expressed in male serum and seminal plasma.Compared with the normal group and the OA group,E2F2 in the seminal plasma wassignificantly lower in NOA.There was no significant difference in serum E2F2 between normal group,OA group and NOA group.7.There was a significant negative correlation between serum ASO3491 and seminal plasma E2F2 expression(r=-0.699,p=0.000).Serum ASO3491 was negatively correlated with serum T level(r=-0.363,P=0.000);serum ASO3491 and testicular total volume were negative.Correlation(r=-0.446,P=0.000).There was a significant positive correlation between seminal plasma E2F2 and serum T level(r=0.344,P=0.000);seminal plasma E2F2 was positively correlated with total testicular volume(r=0.360,P=0.000).Conclusion 1.The expression profile of lncRNA is significantly changed in the NOA group,indicating that lncRNA plays a very important role in the occurrence of NOA and may provide new biomarkers for the clinical diagnosis of NOA.2.The mRNA expression profile was significantly altered in the NOA group,and the differentially expressed genes were mainly enriched in the reproductive disease pathway,indicating that differentially expressed genes are important in the growth and development of germ cells.3.ASO3491 of serum and E2F2 of seminal plasma are differentially expressed in NOA,which may become a new biomarker for clinical diagnosis of NOA.4.Serum ASO3491 is negatively correlated with seminal plasma E2F2.5.Nine candidate genes ASO3491,ENSG00000232527.3 and ENSG00000227372.5,PRKX,AKT2,MLLT4,E2F2,SCD5 and ACSS1 may all become new biomarkers for NOA clinical diagnosis.