Characterization Of MiRNA Maturity Related Proteins And The Role Of MiR-133b In Tilapia Oogenesis

Epigenetics is the study of heritable changes in gene expression caused by mechanisms other than changes in the DNA sequence,while noncoding RNAs(nc RNAs)have emerged as important players of epigenetic regulation.micro RNAs(miRNAs)are noncoding small RNAs with length of about 22 nt,accounting for 1-3% of all vertebrate genes.miRNAs regulate gene expression at the posttranscriptional level by base pairing with complementary target messenger RNA(m RNA)molecules,causing either m RNA cleavage or translational inhibition.It modulates gene expression in many homeostasis processes and pathological states in cells,and participates in a variety of life activities.dicer,drosha and xpo5 are responsible for synthesizing proteins which play key roles in miRNA biogenesis to produce mature miRNAs from their precursor molecules.Although drosha,dicer and xpo5 have been widespread in vertebrates,the expression pattern of these three genes are not clear,particularly in gonads.We identified drosha,dicer and xpo5 in vertebrates,and analyzed their expression by in situ hybridization.The miRNA transcriptome indicated miR-133 b was highly expressed in early development of tilapia ovary.miR-133 b belongs to the miR-133 family.Current research on miR-133 b indicated its important role in the muscle differentiation and tumors occurrence in model animals,the only study indicated that miR-133 b regulated estrogen level by targeting foxl2 in mouse,while its function in fish gonad development is unknown.In this study,complete sequence of miR-133 b which was isolated from Nile tilapia and other animals,which revealed miR-133 b is conserved and shared by invertebrates and vertebrates.The expression pattern of miR-133 b in different tissues,embryos and ovary of tilapia was detected by Real-time PCR and in situ hybridization.Target genes of miR-133 b were predicted by bioinformatics method and verified by luciferase reporter gene assay.The morphology of gonad,gene expression and serum estrogen level of fish treated by the antagomir and agomir of miR-133 b were analyzed in vivo.The main results were as follows:1)Firstly,the coding sequences of drosha,dicer and xpo5,the key enzymes involved in miRNA splicing and transport,were identified in the Nile tilapia genome.The transcriptome data from different tilapia tissues indicated that these three genes were abundantly expressed in the gonads,followed by the brain.The gonadal transcriptome data at different developmental stages indicated that these three genes were expressed higher in the ovary and testis at 90 and 180 dah(days after hatching)than 5 and 30 dah,among which the drosha was highly expressed in the ovary,while dicer and xpo5 were highly expressed in the testis.And drosha,dicer,xpo5 were all expressed in germ cells and somatic cells of the tilapia gonads by in situ hybridization.2)Sequence analysis of mir-133 b precursors in vertebrates and invertebrates revealed that miR-133b-3p was more conserved than miR-133b-5p.The result of synteny analysis showed that the adjacent genes of miR-133 b had been changed in tetrapods and fishes.The results of Real-time PCR showed that the expression of miR-133 b could be detected in both embryos and unfertilized eggs.miR-133 b was highly expressed in early stages of tilapia ovary.miR-133 b was found to be expressed mainly in phase I,II oocytes and somatic cells of the ovary by in situ hybridization.The results indicated the miR-133 b was conserved,possibly a maternal miRNA,which might play an important role in the oogenesis of Nile tilapia.3)Bioinformatics prediction and dual luciferase report system have proved that transgelin 2(tagln2)was the target gene of miR-133 b.Conservative analysis of binding sites revealed that tagln2 was a target gene for mir-133 b in both mammals and teleost.Based on in situ hybridization,miR-133 b and tagln2 m RNA were expressed in the same cell types in ovary.The XX fish at 3 dah were microinjected with miR-133b-3p antagomir every 8 days for six times.Histological analysis indicated no phase I and II oocytes were observed in the ovaries treated with antagomir-133 b compared with control group.Besides,the expression level of target gene tagln2 in the treatment group were significantly increased.And the m RNA level of foxl2,cyp19a1 a and the level of estradiol(E2)in serum were significantly higher in the treatment group.Consistently,the 2-month-old XX fishes were injected with the agomir of miR-133 b for 6 consecutive days.We found that the level of estradiol(E2)in serum of agomir-133 b treated group was significantly lower than control group.The expression level of tagln2,foxl2 and cyp19a1 a in each group were consistent with the level of E2 in serum.The results of this study indicated that miR-133 b plays an important role in regulating estrogen synthesis and ovarian development of Nile tilapia.In summary,the spatiotemporal expression patterns of drosha,dicer and xpo5 were analyzed in tilapia gonad,which laid a foundation for further studies on the role of miRNA in fish reproduction.Our results also demonstrated miR-133 b might regulate oogenesis by regulating the release of estrogen via regulating its target gene tagln2 in tilapia,which provides a new perspective for the function of miR-133 b in ovarian development.