| Infertility is defined as a failure to achieve a pregnancy after 12 months or more of regular unprotected sexual intercourse.Nowadays,infertility is a global public health concern and affects about 15% of couples worldwide.Nearly half of infertility is attributed to the male.The potential causes of male infertility are multifaceted,including environmental pollution,congenital malformations,chromosomal abnormalities,etc.In the last decades,some male infertility patients have been well treated with the development of functional genomics technologies.However,the situation of male infertility has not been fundamentally changed due to its underlying mechanism remains elusive.Therefore,exploring the molecular events of male infertility will lead to new strategies for the diagnosis and the treatment of male infertility.Long non-coding RNAs(lncRNA)are a class of RNA molecules arbitrarily defined as being longer than 200 nucleotides and not translated into a protein.lncRNAs regulate gene expression mainly on three levels,including epigenetic modification,transcription,and post transcription,through DNA methylation,histone modification,and chromatin remodeling.Therefore,lncRNAs are involved in many physiological processes including cell survival,proliferation,differentiation,and motility,as well as cancer progression.A large number of studies have shown that the expression of lncRNA is tissue-specific and,furthermore,abnormal expression of lncRNAs might be associated with numerous diseases,including reproductive system diseases.lncRNA Gas5(growth arrest-specific transcript 5)is located on human chromosome 1q25.1 and produces a 650 bp transcript.Researchers have proved that Gas5 can as a potential tumor suppressor gene,can act as a tumor suppressor gene,and its expression is decreased in tumor tissues relative to normal tissues.Moreover,recent studies reported that Gas5 can promote the self-renewal of embryonic stem cells,affects the proliferation of smooth muscle cells and female germ line stem cells,and regulates insulin secretion to maintain the function of β cells.ObjectiveIn our previous studies,we have found that the expression of Gas5 changes significantly when spermatogenesis was impaired,which indicated that Gas5 is closely related to male reproductive function.Thus,in this study,we aimed to investigate the expression of Gas5 in testis and explored the possible biological roles of Gas5 in leydig cells.MethodsSmiFISH was performed to detect the expression of LncRNA Gas5 in testicular tissue and leydig cells.Leydig cells were transduced with either Gas5-overexpression or Gas5-shRNA lentivirus for the overexpression and knockdown of Gas5,respectively.Subsequently,EdU and MTT assays were used to determine the effect of Gas5 on the proliferation of leydig cells.Next,flow cytometry was used to assess the potential effect of Gas5 on the cell cycle and cell apoptosis.Differentially expressed proteins were characterized with mass spectrometry(MS)between Gas5 knockdown group and control group.The GO term enrichment and STRING analysis were subsequently performed to get insight into the interactions between the proteins of interest.Immunofluorescence staining was performed to detect nucleolar damage in Leydig cells.The expression levels of ribosomal RNA were measured in leydig cells by RT-qPCR.Finally,RIP analysis followed by qRT-PCR analysis was performed to confirm that Gas5 directly interacts with NCL.Results 1.SmiFISH studies showed that Gas5 was localized in germ cells,Leydig cells and Sertoli cells.In Leydig cells,we observed that Gas5 was expressed in both the nucleus and the cytoplasm.2.Leydig cells were sucessfully transduced with lentivirus for the overexpression and knockdown of Gas5,respectively.MTT and EdU assays revealed that Gas5 overexpression promoted cell proliferation,whereas Gas5 knockdown inhibited this property in leydig cells.3.Cell cycle analysis showed that Gas5 overexpression regulated Leydig cell proliferation by increasing the percentage of cells in S-phase.In the Gas5 knockdown group,cell cycle arrest at G2 phase.Apoptosis analysis showed that Gas5 had no obvious effects on apoptosis of Leydig cells.4.Through GO term enrichment and STRING analysis of differentially expressed proteins,we found that Gas5 knockdown mainly affects ribosome biogenesis.5.Immunofluorescence analysis of Fibrillarin revealed that nuclear area markedly increased in Gas5 overexpressing leydig cells and decreased in Gas5 knocked down cells.6.RT-qPCR results showed that the expression levels of ribosomal RNA were down-regulated after Gas5 being down-regulated,whereas that the expression levels of ribosomal RNA were up-regulated in Gas5 overexpressing Leydig cells.7.RIP experiments showed that Gas5 may interact with NCL.Conclusion 1.In Leydig cells,Gas5 was expressed in the nucleus and cytoplasm.2.Gas5 can promote the proliferation of testicular leydig cells.3.The effect of Gas5 on leydig cell proliferation may be achieved by binding to NCL to regulate ribosome biogenesis. |
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