Study Of Post-transcriptional Regulation Of Sexual Development-related Genes Regulated By SRSF1 In GT1-7 Cell Model

Puberty is a process of individual maturity and acquirement reproductive ability.As a complex trait,puberty is affected by many factors such as heredity,nutrition and environment.The regulation of puberty is a complex network that contains multiple pathways.In the previous study,we found a miR-505 gene that delayed the onset of mouse puberty on the X chromosome of inbred mice and confirmed that SRSF1 is the target gene of miR-505.Moreover,SRSF1 can alleviate the inhibitory effect of miR-505 on GnRH and Kiss1.In this study,stable cell clone with low expression of SRSF1 was constructed by lentivirus-mediated shRNA in GT1-7 cells to determine the relationship between SRSF1 and sexual development-related genes,like GnRH and Kiss1.Stable cell clone and pGT1-7 cells were used to study the possible signaling pathways of puberal genes influenced by SRSF1 through RNA-seq and study of proteomics.Furthermore,explore of miR-505 and SRSF1 affect the molecular mechanisms of sexual development related genes.The stable cell clone was established by infected immortalized neuronal cell line use lentivirus containing the SRSF1-shRNA vector to obtain a stable and low expression SRSF1 cell lines.The expression levels of GnRH and Kiss1 in the stable cell clone were also tested.At the mRNA level,expression profiling of stable cell clone was performed using RNA-seq,which is high-throughput sequencing technology.The results of sequencing were analyzed by bioinformatics.Tuxedo was used to compare the reads to the genome.The aligned reads were transformed into Cufflinks to generate transcripts.The differentially expressed genes were obtained by Cuffdiff.Then,differentially expressed genes were deal with GO analysis and KEGG pathway analysis.At the protein level,screening of differentially expressed proteins was carried out on the stable cell clone using iTRAQ,a high-throughput screening technique.The Ingenuity Pathway Analysis(IPA)software was used to analyze the molecular functions,networks and pathways of the differentially expressed proteins.Real-time PCR and Western blot were performed to detect the changes of the mRNA and protein levels of Srsf1.The knock-down efficiency of SRSF1 was 45%(P < 0.001)and 42%(P < 0.05),respectively.The stable cell clone were established successfully,be known as shRNA-SRSF1-GT1-7.GnRH and Kiss1,which are the genes of neutral development in stable transgenic plants,were reduced by 35%(P < 0.01)and 46%(P < 0.001)at mRNA level,respectively.It shows that SRFS1 directly affects the expression of sexual development related genes.A total of 3161 genes were detected in shRNA-GT1-7-SRSF1 by RNA-seq,of which 1407 were up-regulated and 1754 down-regulated.A total of 3034 genes with differential expression were detected in pGT1-7 cells with overexpressing mir-505,including 1357 up-regulated genes and 1677 down-regulatedgenes.Most of the differential display genes in both cells are located in the cytoplasm,which have the function of binding and activating.In the KEGG analysis,the metabolic pathway,the cancer pathway,the gamma-aminobutyric acid synapse,and the PI3K-Akt signaling pathway are all enriched in both cells,and the differential genes of pGT1-7 cells are also involved in insulin secretion and GnRH signaling.iTRAQ detected 4945 proteins in both cells,102 differential proteins in shRNA-GT1-7-SRSF1(fold change 1.3-fold and p< 0.05),and 173 differential proteins in pGT1-7(fold change above 1.3 times,p< 0.05).More than half of these differential proteins are located in the cytoplasm,just like RNA-seq.The expression difference proteins in shRNA-GT1-7-SRSF1 are mainly involved in the signaling pathway of ?-aminobutyric acid degradation,the insulin receptor signaling pathway and the AMPK signaling pathway.Gamma-aminobutyrate degradation and PI3K-Akt signaling also appear in pGT1-7 cells.Real-time PCR showed that the experimental results were consistent with the sequencing results and iTRAQ results,indicating that the results of RNA-seq and iTRAQ are reliable.In summary,we speculate that SRSF1 may function through the ?-aminobutyric acid signaling pathway,the AMPK signaling pathway,and the PI3K-Akt signaling pathway.This will provide a theoretical basis for further research on the molecular mechanism of puberty effected by SRSF1.