Screening Differential Expression Profiles Of Serum Lncrna In Patients With Non-obstructive Azoospermia Based On A New Generation Of Sequencing

Objective: In this study,we used a new generation of sequencing to find the differential expression profiles of LncRNAs in patients with non-obstructive azoospermia(NOA),find out the significant differences in LncRNAs,study the influence of differentially expressed LncRNAs on the occurrence and development of NOA,and find out the target genes of LncRNAs related to the pathogenesis of NOA,so as to provide theoretical and experimental support for the early diagnosis and treatment of NOA.Methods:(1)Collect NOA serum samples and semen samples from normal male donors,improve their data,and initially establish a complete blood sample bank and semen sample bank;(2)Extract RNA from them,detect the purity of total RNA,and use a new generation of sequencing technology to analyze and obtain the differential expression profiles of LncRNA in NOA serum,and preliminarily screen the differentially expressed serum LncRNA;(3)Use real-time fluorescence quantitative PCR(R)T-PCR was used to verify the stability and repeatability of the results.(4)Bioinformatics software was used toanalyze the differentially expressed LncRNA target genes and obtain signal pathways related to the development of NOA.(5)The relative expression levels of Lnc-CDC27-2 and Lnc-BCL6-9 in serum samples were detected by RT-PCR.Results:1.Total transcriptome sequencing of LncRNA results:A total of 593 LncRNAs were expressed in NOA and healthy subjects(P <0.05,and the absolute value of log2(fold change)was more than 1),including321 up-regulated LncRNAs and 272 down-regulated LncRNAs.2.Bioinformatics Software AnalysisThere are 628 genes regulated by 593 lncRNAs(some genes can be regulated by multiple lncRNAs).These genes were imported into DAVID database and analyzed.554 of them belonged to human beings.GO analysis showed that there were 10 biological processes involved BP;cell localization analysis(CC)found that most genes(150/554)were located in the nucleus and cytoplasm(146/554);and molecular function analysis(MF)found that the most involved genes were protein binding(247/554).Four key signaling pathways were identified by BIOCARTA software:abnormal regulation of CDK5 in Alzheimer's disease;activation of Ca+/calmodulin-dependent protein kinase;signal pathway induced by bioactive peptide;and effect of calcineurin on differentiation of keratinocytes.KEGG software analysis obtained 16 key signaling pathways: Alzheimer's disease;Axon-guided renin secretion;Hedgehog signaling pathway;Parkinson'sdisease;adhesion;circadian rhythm cycle;cAMP signaling pathway;oxytocin signaling pathway;alcohol abuse;prion disease;viral carcinogenesis;nodular receptor signaling pathway;platelet activation;oxidative phosphorylation.3.Real-time fluorescence quantitative PCR to verify the differential expression profiles:The most obvious up-regulation of Lnc-BCL6-9 and the most obvious down-regulation of Lnc-CDC27-2 were selected and their expressions in NOA and healthy blood samples were detected.The results showed that the up-regulation and down-regulation trends of Lnc-CDC27-2 and Lnc-BCL6-9 were consistent with the sequencing results.The expression of Lnc-CDC27-2 and Lnc-BCL6-9 in NOA and healthy people.Lnc-CDC27-2 was used to detect the specificity and sensitivity of NOA.Lnc-BCL6-9 was used to detect the specificity and sensitivity of NOA.Conclusion:1.LncRNAs differentially expressed in NOA and healthy individuals were obtained.2.Lnc-CDC27-2 and Lnc-BCL6-9 can be used as potential markers of NOA.