Analyses And Functional Studies Of Differentially Expressed MiRNAs Between Mouse Spermatogonia And Spermatocytes

MicroRNAs (miRNAs) are a class of widely distributed and highly conserved non-coding small RNAs about 22 nucleotides (nt) long. They are thought to play negative regulatory roles posttranscriptionally, causing mRNA cleavage or translation repression, by targeting the imperfect complementary sequences, usually located in the 3"-untranslated regions (UTRs) of mRNAs. It has been reported that miRNAs are involved in various biological processes, including the cell cycle, differentiation and tumorigenesis. Additionally, the temporal and spatial specificity that is frequently associated with development and differentiation is another characteristic of miRNA expression.As a multilevel differentiation process, spermatogenesis functions to perform cyclic gamete production in which diploid spermatogonia eventually differentiate into haploid spermatozoa. Spermatogenesis can be theoretically separated into 3 phases:the mitotic, meiotic and spermiogenesis phases. For mice, the first wave of spermatogenesis takes about 35 days, during which the first generation of spermatogonia emerge at the 6th day post-partum (dpp), and the type B spermatogonia first appear at the 8th dpp, whereas the pre-leptotene spermatocytes emerge around the 10th dpp and the round spermatids at the 20th dpp.Nowadays, it is clear that the developmental process involves in diverse transcriptional and posttranscriptional regulations. As a newly defined regulatory layer, miRNAs have drawn more and more attentions since discovered. Studies on miRNA regulation in spermatogenesis have rapidly become one of the focal points in studies on male reproduction. For instance, miR-122a was thought to be involved in the direct regulation of Tnp2, a testis-specific, crucial protein involved in chromatin remodelling in postmeiotic germ cells. miR-34c was recently suggested to enhance the late stages of spermatogenesis by reinforcing the germ cell phenotype.In this work, a miRNA microarray analysis was performed using two lines of mouse germ cells, GC-1 spg and GC-2 spd, which were type B spermatogonia and premeiotic spermalocytes, respectively, to determine the profiles of differentially expressed miRNAs (DEMs). During spermatogenesis, these cells are representative of two successive differentiation stages prior to the onset of meiosis, or rather as two transitional stages in the shift from mitosis to meiosis. The profiling of these stages may allow for some insight into the nascent period of meiosis.Analysis based on the outputs of the miRNA microarrays indicated that 28 miRNAs stood out according to the inclusion criteria, i.e., GC-1/GC-2 fold change>2 or<0.5, among which 20 miRNAs were higher in GC-1 cells, while the other 8 were higher in GC-2 cells. Via the method of real time PCR, we subsequently identified 4 miRNAs, i.e.. miR-15a, miR-125a-5p,miR-184 and miR-468, which were actually expressed at highei levels in GC-1 cells.Target prediction was carried out with the widely used algorithms:miRanda, PicTar and TargetScan. Dual Luciferase Reporter (DLR) assays were performed using the constructs containing the predicted sites to evaluate the authenticity of the putative targets, among which only the 3"-UTR of Ccnt2, one of the putative targets of miR-15a, caused a marked reduction in luciferase activity.As a member of the miR-15a/miR-16-1 cluster, miR-15a has long been considered an important tumour suppressor associated with the inhibition of cell proliferation, the promotion of apoptosis and the suppression of tumorigenesis by specifically targeting numerous carcinogenic genes since it was identified. miR-15a downregulation has been associated with various cancers. Ccnt2, composed of two splice variants (T2a and T2b), is a member of the T-type cyclins, which have been identified only in the past decade and are widely expressed among tissues. Coupled with CDK9, Ccnt2 forms a complex known as'positive transcription elongation factor b'(P-TEFb), which is thought to facilitate the transition from abortive to productive elongation catalytically by phosphorylating the earboxyl-terminal domain (CTD) of RNA polymeraseâ…¡. The CDK9/Cyclin T complex is generally considered to function in a cell cycle-independent manner and is involved in muscle differentiation.Eight mouse cell lines were collected for the detection of Ccnt2 and miR-15a as a result to observe an inverse relationship existed between them. Alignments of the potential target site in Ccnt2 3'-UTR indicated that the sequence was highly conserved across species. To confirm the regulatory relationship between miR-15a and Ccnt2, DLR assays were performed with mutants containing different mutagenic target sites. Of note, our results indicated that the base pairing at the 3'end of miR-15a was more essential for Ccnt2 recognition. Furthermore, transfection of a miR-15a mimic or inhibitor into GC-1 cells resulted in a marked reduction or increase of Ccnt2 protein levels, respectively, which supported the conclusion that Ccnt2 acted as a novel target gene of miR-15a.Based on the involvement of the CDK9/Ccnt2 complex in muscle differentiation, we hypothesised that miR-15a may have an effect on this process. We observed this process in a differentiation-inducible myoblast, C2C12. A miR-15a mimic or siRNA from Ccnt2 were transfected into C2C12 cells in advance. After a 48-hour incubation, miR-15a was exclusively overexpressed in corresponding cells, while Ccnt2 mRNA was downregulated in both siRNA and mimic treated cells compared with controls, suggesting that a cleavage of Ccnt2 mRNA was induced by miR-15a. Myodifferentiation was induced after transfection, with early and late differentiation markers, myogenin and myosin heavy chain (MHC), respectively, being detected at the appropriate times. Following the progress of differentiation, the levels of myogenin and MHC increased continuously, whereas that of Ccnt2 decreased gradually, suggesting that Ccnt2 was important tor the initiation of cellular differentiation. Compared to the negative control, miR-15a led to a truncated build-up of myogenin and MHC. Morphologically, the formation of myotubes was delayed by miR-15a. Briefly, miR-15a negatively affected muscle differentiation by targeting Ccnt2.Do Ccnt2 and miR-15a make a difference during spermatogenesis? Ccnt2 was detected in developing mice testes at different levels. Ccnt2 mRNA rose continually in the postnatal 3-week period and suddenly dropped off from the 4th week, suggesting its importance in early spermatogenesis. As for the Cent2 protein, the larger form (T2a in mice) increased markedly from the 3rd week, while the smaller form (T2b in mice) decreased simultaneously, implying that they affect different stages of spermatogenesis Parallel detections of miR-15a showed that it dropped continuously after birth, especially within the postnatal 3-week period, the inverse of what was observed with Ccnt2. Based on these results, Ccnt2 and miR-15a might be crucial in early spermatogenesis.In summary, we identified 4 DEMs between two premeiotic male germ cells, made predictions about their putative targets, and confirmed Ccnt2 as a direct target of miR-15a. We also report that miR-15a inhibited muscle differentiation by targeting Ccnt2. which represents a novel interaction. Subsequently, miR-15a and Ccnt2 were profiled in developing mice testes as a result to observe their inverse correlations in the postnatal 3-week period, which suggested their importance in early spermatogenesis. In view of our results and its tumour suppressor characteristics, miR-15a is quite promising and may be applied in male contraception and in the treatment of testicular cancer or male sterilily in the future.