Functional Mechanism Of Non-coding RNA In Cerebellar Neuron And Testicular Cancer

Noncoding RNAs(ncRNAs)are usually defined as transcripts that do not encode proteins(some may encode small peptides).A large number of ncRNAs have been found to have regulatory roles in eukaryotes,which are involved in many processes such as gene transcription,post-transcriptional modification,protein translation and degradation.Although there are an increasing number of reports on ncRNA,the biological functions of most ncRNA,especially their tissue-specific functions,still need to be further explored.LncRNAs(long noncoding RNAs,lncRNAs)are a class of RNA molecules with transcripts more than 200 nucleotides in length.LncRNAs are abundantly enriched in the mammalian central nervous system.However,whether and how these lncRNAs function in modulating neuronal synapses remain unclear.In this study,we found a highly expressed lncRNA Gm2694 which was decreased with age in the cerebellum from the whole brain tissue ribo-minus RNA-sequencing(rmRNA-seq)database of young and old mice,and three of its isoforms were named lncRNA Synage.Through sequence alignment analysis,we found that the lncRNA Synage is not conserved in sequence,but its genomic location(all of which are adjacent genes of the Cblnl gene)in mice,rhesus monkeys and humans and its specific distribution of high expression in cerebellar tissue are very conserved.To study the biological function of lncRNA Synage in vivo,we successfully constructed lncRNA Synage knockout(KO)mice by the CRISPR-Cas9 system and found that lncRNA Synage deletion causes a full-spectrum cerebellar ablation phenotype that proceeds from cerebellar atrophy through neuron loss,cell senescence,synapse density reduction,synaptic vesicle loss,and finally a reduction in synaptic activity in mice by immunofluorescence,β-Gal staining and transmission electron microscopy.In addition,the results of the animal behavior analysis experiments,such as new object recognition and balance beam,showed that motor coordination and cognitive function of lncRNA Synage knockout mice are severely impaired.These deficits can be rescued by AAV-mediated Synage overexpression from birth.In addition,we performed molecular experiments,such as RNA pull-down,RIP,co-IP,IF-FISH and EMSA,in vivo and in vitro and found that lncRNA Synage can regulate cerebellum synaptic stability via at least two mechanisms.One is through the function of lncRNA Synage as a ceRNA by competing with mmu-miR-325-3p to regulate the expression of the known cerebellar synapse organizer Cbln1.The other function is to serve as a scaffold for organizing the assembly of the LRP1-HSP90AA1-PSD-95 complex in Purkinje cell synapses.Thus,our results show roles for the lncRNA Synage in regulating synaptic stability and function during cerebellar development.In addition to lncRNAs,piRNAs(PIWI interacting RNAs,piRNAs)are also a class of ncRNAs,which are single-stranded noncoding RNAs with a length of 23-36 nt.piRNAs were first discovered in the reproductive system of mammals,and subsequent studies found that piRNAs were also expressed in other organs.Studies have found that the abnormal expression of piRNAs is significantly related to the occurrence and development of various cancers.However,little is known about the role of piRNAs in male testicular cancer.In this study,we found significantly downregulated piR-36249 in human testicular cancer tissues compared with tumor-adjacent tissues.Overexpression of piR-36249 in NT2 cells significantly inhibited cell proliferation,migration and promoted cell apoptosis.Moreover,we found that piR-36249 may be involved in regulating OAS2 mRNA and up-regulating OAS2 protein expression by RNA-seq analysis and Western blot assay.Knockdown of OAS2 in NT2 cells significantly promoted cell proliferation and migration,which was consistent with the cell phenotype after inhibition of piR-36249.Previous studies have revealed that OAS2 is a tumor suppressor modulating the occurrence and development of some tumors.Therefore,piR-36249 can inhibit testicular cancer progression by promoting the expression of OAS2,These results together suggest that piR-36249 is a tumor suppressor and may serve as a potential predictor for the prognosis of testicular cancer.In conclusion,the results of this dissertation reveal the molecular mechanism by which ncRNA(lncRNA Synage)regulates synaptic stability and neuronal function in the mouse cerebellum and find that ncRNA(piR-36249)can be used as a tumor suppressor factor to regulate the development of testicular cancer.These studies will provide an important scientific basis for further understanding the functional mechanisms of ncRNAs in the nervous system and reproductive system.