Identification Of LncRNA In Human Mature Spermatozoa And The Correlation Between Its Abnormal Expression And Asthenospermia

Background:All the time sperm has been thought to only transport DNA into the occyte,while other components such as RNA molecules are only residues and do not have physiological functions.In recent years,studies have shown that sperm RNA molecules can be passed to offspring by fertilization,and regulated the development of fertilized eggs and embryo.Sperm RNA can be divided into mRNA and ncRNA,and a large number of mRNAs and small non-coding RNAs?nucleotides less than 200?have been identified by microarray and RT-PCR in human and other mammal animals.However,human mature sperm whether exist long non-coding RNA?lncRNA?that is longer than 200 nucleotides,and its functions are far from elucidated.Research into lncRNAs has made significant progress in recent years.It has many functions in a lot of life processes,and one of the currently best characterized role of lncRNAs is the regulation of epigenetic dynamics.It could form functional complex with chromatin regulatory protein,RNA binding protein and small RNA,which can regulate the biological process.For example,studies have shown that testis-specific lncRNA is essential for male reproduction and the key lncRNA loss will lead to male sterility,suggesting that the abnormal expression of lncRNA may be closely related to male infertility.Male sterility has become serious disease which affected the health of people.Asthenospermia accounted for a large proportion of infertility problems.But the etiology of asthenospermia has been unknown.Therefore,combined with research hotspots and clinical needs in these areas,this study is intended to be work out.Whether there is lncRNA in mature sperm,how is its expression spectrum? Whether lncRNA is associated with asthenospermia? In this study,the expression profile of lncRNA in human mature spermatozoa would be analyzed,and several lncRNAs which associated with asthenospermia were screened out and identified.This study could provide scientific basis for revealing the etiology of asthenospermia and offer new clues to the mechanism of male infertility.Method:A total of 84 samples of spermatozoa were collected.36 semen samples of asthenospermia were randomly selected 12 samples which be divided into three parts;48 semen samples of normal sperm motility were randomly selected 12 samples which were mixed into four parts.All samples were purified and extracted total RNA,then detected its RNA's purity and quality.Finally,seven mixed samples were selected,and numbered as: NS-1,NS-2,NS-3,NS-4,ATNZ-1,ATNZ-2,NS-1,NS-1,NS-1,NS-1,NS-1,ATNZ-3.The expression profiles of lncRNA in human mature spermatozoa was obtained by transcriptome sequencing.Then we screened out significantly differential expressed lncRNAs?Fold-change > 2.0,P < 0.01?.And we used GO analysis?Gene Ontology,gene ontology?and KEGG pathway analysis for further study.And the presence of intact lncRNA transcripts in human mature sperm was confirmed by RT-PCR and fluorescence in situ hybridization?FISH?.We used a lot of samples to validate lncRNAs which were significantly differential expressed and specific in sperm and testis.We confirmed lncRNAs existed in human mature sperm by real-time fluorescence quantitative PCR?QPCR?.Analysis of the correlation between the differential expression of lnc RNAs and the Progressive motility?PR%?of asthenospermia.At the same time,a co-expression analysis network of lncRNA and mRNA was constructed to analyze the function and possible mechanism of these differentially expressed lnc RNAs.Results:1.There were 38307 known lncRNAs and 27472 novel lncRNAs in human mature spermatozoa,and the two representative lncRNAs were confirmed by RT-PCR and fluorescence in situ hybridization?FISH?.2.In the NS group?normal samples?and the ATNZ group?asthenospermia samples?,there were 2768 differential expression of lncRNAs?Fold-change > 2.0,P < 0.01?.Among them,2445 lncRNAs were up-regulated and 323 lnc RNAs were down-regulated;615 m RNAs were differentially expressed,of which 393 mRNAs were up-regulated and 222 mRNAs were down-regulated.3.GO analysis shows that m RNA is divided into three categories: biological processes,cellular components,and molecular functions.And m RNAs were significantly enriched in the biological process,including protein kinase C-activation,G protein-coupled receptor signaling pathway,actin cytoskeletal tissue,RNA metabolic process regulation and cell junction.KEGG pathway analysis showed that differentially expressed mRNA was involved in cGMP-PKG signaling pathway,NOD-like receptor signaling pathway,apoptosis and so on.4.Compared the NS group with the ATNZ group,we predicted the cis-regulated target genes of differentially expressed lncRNAs?the upstream 10 kbp sequence of the coding gene and the 100 kbp sequence downstream of the lncRNA?and the trans-regulated genes?free energy less than-30 kj/mol?.The results showed that there were 13131 target genes for lncRNA.The GO enrichment analysis showed that the biological processes involved in these target genes include: organelles,RNA transport,RNA metabolic processes,cell composition involved in morphogenesis,myofibrillar assembly,actin structural tissue,actin filaments Reorganization and other processes.KEGG pathway analysis showed that differentially expressed lncRNA target genes involved in protein digestion and absorption,RNA transport,actin cytoskeleton regulation,PI3K-Akt signaling pathway and other pathways.5.There were 48 lncRNAs are specificity in sperm and testes.Identified by quantitative PCR,we find only five sperm and testis specific lncRNAs : NONHSAG032058,NONHSAG009522,NONHSAG072018,LXLOC₀98487,LXLOC₀77183.Then we collected lots of clinical samples?51 normal samples and 51 asthenospermia samples?to verified by Qrt-PCR.And the expression of four lncRNAs are increased in the asthenospermia samples,and they are same as the sequencing results.They are LXLOC₀98487,NONHSAG009522,NONHSAG072018 and NONHSAG032058.Indicating that they may be involved in the regulation of mature sperm function.6.Combined with the above results,there were significant differences in the expression levels of LXLOC₀98487 between the normal samples and asthenospermia samples?Mann-Whitney,P < 0.001;Kolmogorov-Smirnov,P=0.033?;there were significant differences in the expression levels of NONHSAG009522 between the normal samples and asthenospermia samples?Mann-Whitney,P < 0.001;Kolmogorov-Smirnov,P < 0.001?;there were significant differences in the expression levels of NONHSAG072018 between the normal samples and asthenospermia samples?Mann-Whitney,P < 0.001;Kolmogorov-Smirnov,P < 0.001?;there were significant differences in the expression levels of NONHSAG032058 between the normal samples and asthenospermia samples?Mann-Whitney,P=0.030;Kolmogorov-Smirnov,P=0.037?;The relationship between the expression of four lncRNAs and the basic parameters of semen was analyzed,and the expression level of four lncRNAs and the rate of progressive motility?PR?were not correlated with the results.7.Tissue-specific and differentially expressed LncRNA: NONHSAG072018,we found it's target gene is associated with the channel protein and may be involved in the regulation of the target gene of the channel protein in multiple levels and plays an important role in it.Tissue-specific and differentially expressed LncRNA: NONHSAG009522 and its target gene is involved in amino acid synthesis,pantothenic acid metabolism,mitochondrial metabolism and so on.Conclusion:1.This study confirmed the presence of lncRNAs in human mature spermatozoa and the expression profile of human mature sperm lncRNAs.The results could enrich RNA types in mature spermatozoa,for in-depth studies of sperm RNA function,and promoted the study of male infetility,human assisted reproductive technology,and nuclear transfer techniques.2.The above results showed that we screened out 5 lncRNAs which are specificity in the sperm and testis.The results showed that the expression of lncRNAs was different from the normal group.And the expression levels of lncRNAs in the asthenospermia were higher than those in the normal group,and four lnc RNAs were in accordance with the sequencing results.At present,five candidate lncRNAs have been shown to have no correlation with the PR value of asthenospermia,but there is a significant difference between the asthenospermia and the normal population,indicating that lncRNA may be a contributing factor to the asthenospermia.Due to the relatively less sample,the resulting results require reliable,abundant large clinical samples and more accurate experimental and statistical analyzes to be further validated.The corresponding target genes were predicted by RNA-PLEX software for the five candidate lncRNAs.Only two of them identified the target genes and analyzed by GO and KEGG showed that they could participate in the target gene of channel proteins and participate in amino acid Synthesis,pantothenic acid metabolism,mitochondrial metabolism,and in which play an important role.The results provide an experimental basis for the molecular mechanism of the depressive symptoms.It is of great theoretical significance for the study of spermatogenesis and sperm function regulation.Therefore,they are likely to become a new member of " asthenospermia-related molecular markers" and play its clinical value.