The Function Of Let-7/lin-28 Pathway In Metamorphosis Of Japanese Flounder

The Japanese flounder(Paralichthys olivaceus), one of the important marine commercial fishes in our country, undergs many postembryonic metamorphosis. It has been very hot to study the mechanisms of those metamorphosis of Japanese flounder. The heterochronic genes, let-7, lin-28 a, lin-28 b, lin-41 and lin-29, are highly conversed and exhibit similar spatial and temporal expression profile in many vertebrates. Negative or positive regulatory relationships among these genes enabled the extremely precision development of organisms. Studying of the regulatory networks composed of heterochronic genes provides an important guidance to revealing the mechanisms of metamorphosis in Japanese flounder.let-7 mi RNAs can exert their functions by binding with 3’-untranslated regions(UTRs) of target genes. LIN-28 a and LIN-28 b, RNA binding proteins, together with cytoplasmic Protein LIN-41 and LIN-29, are all essential factors of temporality and spatiality of bological development. We have identified and analyzed the roles of ten let-7 mi RNAs in Japanese flounder. In this study we cloned full c DNA sequences of lin-28 a, lin-28 b and lin-29 and coding sequence(CDS) of lin-41 in flounder, and detected the expressions of these genes in flounder. We also tested the effects of the TH and the TU on these heterochronic genes and analyzed the regulations of them during early development in Japanese flounder.In this study we used PCR and other technologies to clone c DNA sequences of lin-28 a, lin-28 b, lin-41 and lin-29 and analyzed the characterizations, homology alignment, protein structures, evolutionary tree, and functions of their amino acid sequences by bioinformatics methods, as well as their relative expressions in different tissues of adult fishes and each stages of early development in Japanese flounder. We also studied the influences of TH and the TU treatments on expressions of these heterochronic genes in Iapanese flounder. We predicted the target sites of let-7 in lin-28 a and lin-28b’s 3’-UTRs and authenticated the regulation between them using target genes validation kit. We constructed eukaryotic expression vectors of lin-28 a and lin-28 b and then transfected them as well as let-7 Micro RNAs mimicis, into flounder embryonic cells(FECs) and subsequent tests. All these were done to determin the functions of these heterochronic genes and their regulatory relationships in development of Japanese flounder.From the results, we knew that c DNA sequence of lin-28 a had a full length of 1617 bp, which included 118 bp of 5’-UTR, 890 bp of 3’-UTR and 609 bp of open reading frame(ORF) that could encode 202 amino acids; lin-28 b included 218 bp of 5’-UTR, 2075 bp of 3’-UTR and 705 bp of open reading frame(ORF) that could encode 234 amino acids; lin-29 included 112 bp of 5’-UTR, 328 bp of 3’-UTR and 1329 bp of open reading frame(ORF) that could encode 234 amino acids; the length of lin-41 ORF was 2502 bp that encoded 833 amino acids. The homologies of lin-28 a, lin-28 b, lin-41 and lin-29 among various species were 62%~94%, 63%~93%, 70%~93% and 68%~95%, and they all were belong to the group of fish in their own evolutionary tree, which indicated that their sequences and functions were conserved in many vertebrates.The expression levels of lin-28 a, lin-28 b, lin-41 and lin-29 of flounder were very high in brains and gonads in comparison to muscles(P < 0.05), which implied the importance of these genes in developments of brain and gonad. The reason why lin-41 and lin-29 were widely expressed in many tissues maybe reasulted from the diversity of their domains and functions(P < 0.05). In addition, lin-28 a, lin-28 b and lin-41 had extremely high expression levels in embryonic period while specially low levels during metamorphosis period(P < 0.05), which contrary to the expression trend of let-7 in flounder, this result indicated that there were some negative regulations or targeted inhibitions between the three genes and let-7. In the experiment, TH had some effects on the expressions of these genes but in the absence of clear rules while TU could promote their expressions(P < 0.05), which manifested that TH could regulate these heterochronic genes indirectly.In the validation test of target genes, only the second predicted site in lin-28 a 3’-UTR was targeted by let-7(P < 0.05), and there would be the most strong targetting when all the three sites came to together(P < 0.01). We could conclude that lin-28 a was one of the target gene of let-7 in flounder and there were more than one target binding site of let-7 and lin-28 a. Moreover, the degrees of targetting were different among different members of let-7 family in Japanese flounder.After the recombinant eukaryotic expression vectors of lin-28 a and lin-28 b and let-7 mi RNAs mimicis were transfected into FECs and overexpressed, we could see the green fluorescence in cells if the recombinant plasmids had been transfected and expressed in cells successfully. Overexpressions of lin-28 a and lin-28 b up-regulated the levels of pre-let-7 and lin-29(P < 0.05), while down-regulated the level of mature let-7(P < 0.05), which indicated that lin-28 a and lin-28 b participated in the post-transcriptional regulation of the let-7 family members in FECs. And overexpression of lin-28 a also made more FECs divide. Above all, let-7 and lin-28 a formed a negative feedback pathways in Japanese flounder.In conclusion, we analyzed the expression profiles of heterochronic genes lin-28 a, lin-28 b, lin-41 and lin-29 in adult fish tissues and early development of Japanese flounder and the effect of exogenous TH on expressions of these heterochronic genes. We also preliminary investigated that lin-28 a was a target gene of let-7. After that we constructed eukaryotic expression vectors of lin-28 a and lin-28 b and revealed their funtions on cell cycles and relationships with let-7. Finaly we preliminarily established the negative feedback regulation between let-7 and lin-28 a in development of Japanese flounder.