Positional Cloning And Functional Study Of Puberty Related Gene MiR-505-3p In Mice

In recent years, with the improvement of our living standards, the incidence of children precocious puberty was increased. So the studies of puberty onset receive extensive concerns. Puberty is a process of individual maturity and acquirement reproductive ability. As a complex trait, puberty is affected by many factors such as heredity and environment. In mammals, puberty is regulated by the hypothalamus-pituitary-gonadal axis(HPG axis), the secretion of various hormones directly controls puberty onset and maintains reproductive function. Before puberty onset, HPG axis was suppressed, and the hormone secretion is insufficient, so it cannot maintain the development of gonad and the synthesis of sex hormone. Gn RH neurons are key roles of puberty, and they can secrete gonadotropin-releasing hormone(Gn RH) by pulse. When puberty is onset, the frequency and pulse amplitude of Gn RH secretion were significantly increased; therefore this phenomenon is the landmark event of puberty onset.Based on the deep research into the puberty, a lot of key genes which can regulate puberty onset are defined, such as Gn RH, Kiss1 and GPR54, etc. As puberty involves a variety of cell types, numerous intracellular and intercellular signaling pathways, thus the onset of puberty is regulated by a highly coordinated gene network. There are at least three gene networks that may contribute to control the timing of puberty. The first was tumor related genes network, the second and the third gene network was respectively Lin28 b and zinc-finger protein gene network. The dynamic regulation of these gene networks can also be regulated by epigenetics. It is worth noting that genes in networks are always transcriptional and post transcriptional regulation factors.Micro RNAs(miRNAs) is a class of important transcription factor that regulates gene expression at multiple hierarchical levels. It has been proved that the miRNAs can participate in regulatory of puberty onset. In this study, we refined a QTL on X chromosome that can regulate puberty onset. By positional cloning we find mir-505-3p can inhibit the puberty onset in female mice and perform its function in cell model and mouse model.Since 2008, our laboratory performed a genome-wide scanning for linkage in reciprocal crosses between two strains, C3H/He J(C3) and C57BL6/J(B6), which differed significantly in the pubertal timing. A genome-wide search was performed in phenotypically extreme F2 mice. Then we find a 25 c M QTL in X chromosome located in DXMit103~DXMit119 and the result had been published in PLo S ONE. In order to find out the functional gene in this QTL, we use a specific segment of chromosome substitution method to fine mapping the QTL. Through continuous backcross with C3 mice, we narrowed the QTL into 1c M located in rs13483770~rs2905584 on the seventh generation(N7).In this study we selected the N7 mice that carry the QTL to continuously backcross with C3 mice until N10. In N10 we chose the female mice which genotype were XBXC, and the male mice which genotype were XBY and XCY. Then we crossed them in XBXC×XBY and XBXC×XCY ways. The offspring of them denoted N11. The three genotypes of N11 female offspring were XBXB, XBXC and XCXC. By comparing the VO time of the female mice, we confirmed that the QTL could inhibit the puberty onset in female mice. In order to find out the functional gene in this QTL, we studied the DNA level and the m RNA level of seven protein-coding genes and one miRNA gene in this QTL. We found that miR-505-3p in the hypothalamus of B6 mice were significantly higher than that of C3 mice before puberty onset, while there was no significant difference of other genes expression between these two stains. Therefore we think miR-505-3p maybe the gene that related to inhibit puberty onset in this QTL on X chromosome.In order to confirm the miR-505-3p inhibition of puberty onset, we verified the function of miR-505-3p in cell model and mouse model. At the cellular level, we chose immortalized Gn RH neurons GT1-7 cell for cell model, and transfected with lentivirus to stable express miR-505-3p. In this model, we found that when the expression of miR-505-3p was increased, the expression of key genes such as Gn RH, GPR54, Kiss1, which regulate puberty onset in GT1-7 cells was significantly decreased. This indeed shows that miR-505-3p could inhibit puberty-related genes in cell model. In order to know the mechanism which miR-505-3p inhibited puberty-related genes, we profile m RNA expression on cell model that overexpressed miR-505-3p. We found that there was 1490 genes changing more than 2 times in GT1-7 cell which overexpressed miR-505-3p. GO and KEGG analysis of these differentially expressed genes found that overexpression of miR-505-3p could inhibit Gn RH signaling pathway. To confirm the target gene of miR-505-3p, we use Target Scan database to predict and we also did experiments to confirm. Among the target genes, we found Srsf1 can active m TOR pathway, which can regulate pubety onset. Then we overexpressed Srsf1 in GT1-7 cell, we notice the expression of Gn RH, Kiss genes is increased. So we come to a conclusion that miR-505-3p can inhibit the puberty related genes through Srsf1.Then we study the function of miR-505-3p on animal level. We use Crispr/Cas9 system successfully knockout miR-505-3p in B6 mice, and we check puberty phenotypes of them. Compare to the wild type, VO of miR-505-3p knockout mice was advanced significantly. The body weight and gonad weight of female mice are also significantly heavier than the wild type. In addition, the reproductive ability has also been affected; the knockout mice show a higher corpus luteum number, a higher rate of dystocia and postnatal death and a higher kisspepetin concentration. Then we tested the expression of puberty related genes in hypothalamus of knockout mice, and we found that mouse hypothalamus Srsf1, Kiss1, GPR54 gene expression increased significantly after knockout miR-505-3p. So we believe miR-505-3p can inhibit puberty onset through regulate the expression level of Srsf1 in mouse model.Beside the miR-505-3p, we also found a set of miRNAs, which may participate in the puberty process through miRNA array method, and in this study we proposed an optimized way to improve the detection methods of miRNAs and the typing method of Cripsr/Cas9 mice.In conclusion, we start our research from positional cloning and end it with a new regulator of puberty onset: miR-505-3p. It can inhibit puberty onset in cell model as well as mouse model. These studies further reveal the mechanism of puberty onset and the function of miRNAs.