Study On The Roles Of LncRNA Gas5 In Regulating Sertoli Cell Proliferation And Its Molecular Mechanisms

Male reproductive function in the general population have attracted increasing attention due to numerous reports indicating declining sperm counts and diminishing sperm quality.Spermatogenesis occurs in testicular seminiferous epithelium containing germ cells and somatic cells,such as Sertoli cells,and there appears to be extensive cross-talk between the developing germ cells and Sertoli cells.Thus,Sertoli cells play an indispensable role in spermatogenesis.In fetal and neonatal period,FSH and other factors stimulate the proliferation of Sertoli cells.Growing evidence suggests that failure of Sertoli cells to proliferate normally may result in male inferlity.Therefore,it is of great significance to further investigate the molecular mechanism of proliferation and functional maturation of Sertoli cell.ObjectiveOur previous research has confirmed that Gas5 is expressed in testicular Sertoli cells and Leydig cells,and its expression level might be related to reproductive function.This prompted us to explore whether Gas5 was involved in the growth and proliferation of Sertoli cells.Methods1.The localisation of LncRNA Gas5 in testicular tissue and Sertoli cells were analyzed by smiISH method.2.Stable Gas5 expressing TM4 cell lines as well as Gas5 knock-down cells lines were established by transfection of either Gas5-overexpression or Gas5-sh RNA lentivirus,followed by puromycin antibiotic selection.3.MTT,EdU and flow cytometry were employed to detect cell proliferation,cell cycle,and apoptosis.4.To gain a global insight on Gas5 related genes,we used RNA-seq to analyze the transcriptomic changes in Sertoli cells.5.Immunofluorescence staining was performed to demonstrate nucleolar changes in Sertoli cells;Furthermore,expression levels of ribosomal RNA were measured using quantitative RT-PCR.6.To identify Gas5-proteins complexes,we present an RNA pull-dwo n assay using streptavidin(SA)-binding RNA aptamer termed S1 m.Results1.smiISH expriment showed that Gas5 was localized in both the nucleus and the cytoplasm of testicular Sertoli cells.2.Stable Gas5 over-expression and knock-down cell lines were established to characterize the direct implication of Gas5 on cell proliferation.MTT assays revealed that cell growth was inhibited in Sertoli cells transfected with Gas5-sh RNA lentivirus compared with controls;In contrast,over-expression of Gas5 promoted cell proliferation.This promoting effect of Gas5 was further proven by the results of EdU analysis.Flow cytometric assay showed that Gas5 over-expression deregulated Sertoli cell cycle,as it increased the proportion of S phase,while Gas5 knockdown decreased the proportion of Sertoli cells in S phase compared to untreated cells.Furthermore,up-or down-regulation of Gas5 did not affect TM4 cell apoptosis.3.A total of 99 genes were altered significantly,in which 67 genes were down-regulated and 32 genes were up-regulated,in Sertoli cells upon Gas5 knock-down.KEGG analysis revealed that the most significant pathway were enriched in cytokine-cytokine receptor interaction,NOD-like receptor signaling pathway,Hippo signaling pathway-multiple species,etc.,indicating that down-regulation of Gas5 affect the immune reponse of Sertoli cells.In Gas5 over-expressed Sertoli cells,597 genes were identified as differentially expressed compared to the control group.These differentially expressed genes were significantly enriched in sterol biosynthetic process,cholesterol biosynthetic process,defense response to virus,and steroid metabolic process,which are mainly classified into metabolism-related pathways.Further GSEA analysis showed marked enrichment for ribosome biogenesis both in Gas5 up-and down-regulated Sertoli cells,indicating that Gas5 may affect cell proliferation through regulating ribosome biogenesis.4.Immunofluorescence analysis of FBL indicated that hibiting Gas5 caused a reduction of nucleolar area,while increasing Gas5 extenuate nucleolar area.Moreover,Real-time PCR results showed that silencing of Gas5 leaded to down-regulation of rRNA levels,while forced expression of Gas5 increased rRNA production.5.By quantitative mass spectrometry,we detected multiple proteins may be associated with Gas5,and these identified proteins were significantly enriched in spliceosome and ribosome.Conclusion1.At the subcellular level,Gas5 is expressed in the cytoplasm and the nucleus of Sertoli cells.2.Gas5 can promote the proliferation of testicular Sertoli cells.3.Gas5 participates in the immune response of Sertoli cells,and its proliferation effect on Sertoli cells may be related to the regulation of ribosomal biogenesis.