Research On The Influence Of MiRNA On The Puberty Onset Using Genetically Engineered Mice And GT1-7 Cell Line

Background:MicroRNAs are ～22nt, non-coding small RNAs, which regulate a series of biological processes including cell proliferation and apoptosis, neuron differentiation, tumorigenesis and individual development by targeting the 3’UTRs of messenger RNAs. Our previous research isolated a puberty-related gene:miR-505 by comparing the difference of vaginal opening between two kinds of inbred female mice:C3H/HeJ and C57BL/6. Furthermore, some studies showed that miR-29a, one member of miR-29 gene family, is highly expressed in the mammalian brain and its expression significantly fluctuates with the growth and development of young individuals, which indicates miR-29a probably functions in mammalian neural system to regulate the individual development. Gene knockout is one of the most popular reverse genetics technologies which investigate the miRNA biological functions. CRISPR-Cas9 derived from immune system of bacteria and archaea is a novel gene-editing technology which can efficiently induce mutation and loss-of-function on targeted gene.Objective:In this research, miR-505 knockout mice were used to study the influence of miR-505 deficiency on the pubertal timing and fertility of female mice. CRISPR-Cas9 technology was utilized to abolish the miR-29a gene in GT1-7 cells. And the phenotype change of miR-29a knockout GT1-7 cells was detected to investigate the relationship between miRNAs and puberty.Methods:In animal experiments, miR-505 gene modified mice were obtained by embryo injection of sgRNA/Cas9. And the steadily hereditable gene modified mice were obtained by the three-generation backcross between gene modified founders and wild-type C57BL/6 mice. Offsprings which had three genotypes were obtained by inbreeding. Finally, the expression level of miR-505, the pubertal timing and fertility of female mice with different genotypes were compared. In cellular experiments, Cas9 steadily expressed GT1-7 cells were constructed. And sgRNA29a-EGFP plasmids were constructed and transfected into above-mentioned GT1-7 cells, flow cytometry was employed to enrich positive cells and test positive cell clones. Finally, the detection of miR-29a expression, phenotype analysis and target gene prediction and validation were applied in positive cell clones.Results:In animal experiments,16 miR-505 genetically engineered mice were obtained by embryo injection. Among these mice,2 genetically engineered mice had 17bp and 23bp deletions on miR-505 loci, respectively. The miR-505 expression of these mice which had large fragment deletions decreased significantly. Although the significant difference of miR-505 expression among different genotype female mice failed to cause statistically significant change of the pubertal timing and fertility, the population means showed that the deficiency of miR-505 in female mice had a tendency to advance the vaginal opening and enhance partial reproductive indexes. In cellular experiments, Cas9 steadily expressed GT1-7 cells model was successfully constructed. And multiplex gene editing was fulfilled using this cell model. Furthermore, the mutation rate and miR-29a expression of enriched EGFP-expressing positive GT1-7 cells changed significantly. A miR-29a gene-modified GT1-7 cell clone was obtained by flow cytometry, which had 2 bp deletions on miR-29a loci. Moreover, compared with enrich cells, the miR-29a expression of this cell clone declined by higher degrees, which slowed down the growth rate of GT1-7 cells and also caused its phenotype change. The results of target gene validation showed that DCX gene was affected by miR-29a.Conclusion:Although CRISRP-Cas9 system in this research mainly induced deletions in the processing regions of miRNAs, it could still lead to the decreased expression of miRNAs efficiently by gene editing. Cas9 steadily expressed GT1-7 cell model significantly improved the efficiency of gene editing and facilitated to enrich cells by flow cytometry in the following work. The knockout of miR-505 failed to significantly affect puberty, which was observed through miR-505 knockout mouse models. However, the population means showed the vaginal opening and partial reproductive indexes of miR-505 knockout female mice had consistent variation trends. In comparison, miR-29a had obvious biological functions in GT1-1 cells, which probably contributed to the regulatory role of miR-29a on DCX gene.