Characterization And Function Analysis Of MiRNA In The Transplantation Immunity In Pinctada Fucata Martensii

Transplantation operation was one of the crucial process during pearl production.After the transplantation operation,the immune rejection response would be induced by the transplanted mantle tissue and nucleus,it is crucial for the cultivation of high-quality pearls to regulate the immune response effectively after transplantation.Our previous research showed the genes expression of microRNA(miRNA)processing related enzymes(Dicer and Drosha)of Pinctada fucata martensii significantly changed after transplantation,indicating that miRNA is involved in the regulation of the transplantation immunity during pearl production.The aim of this study was to systematically study the dynamic changes of miRNAs after transplantation,combine transcriptome analysis to obtain key miRNAs,and verify their functions.The main findings are as follows:1.MiRNA transcriptome analysis:In this study,a total of 14 miRNA transcriptomes were constructed,including the control group,pre-operative treatment group,allograft and xenograft at 6 time points(6 h,12 h,24 h,3 d,6 d and 12 d),in which the xenografted mantle tissue was from Pinctada maxima.A total of 186 miRNAs were obtained,including 99 known miRNAs and 87 unknown miRNAs.Among them,the expression of 135 miRNAs changed in both the allograft and xenograft group.There were 20 miRNAs specifically changed in allogeneic group,and 17 miRNAs specifically changed in xenograft group.Functional enrichment analysis indicates that the Notch signaling pathway regulated by miRNA has an important regulatory role in transplantation immunity2.Function analysis of pma-miR-1985:After over-expression of pma-miR-1985 in Pinctada fucata martensii,the activity of immune and antioxidant enzymes(SOD,CAT,ACP,etc.)in the serum were significantly improved.The expression level of pma-miR-1985 increased after the stimulation of pathogenic model molecules(LPS,PGN,Poly I:C).Expression profile analysis showed that pma-miR-1985 may participate in the regulation of transplantation immunity through ECM-receptor interaction and Relaxin signaling pathway.Expression profile and targeting prediction showed the target genes of pma-miR-1985 were pma-GATA-3 and pma-IFI44L.After overexpression of pma-miR-1985,the expression of its target genes was down-regulated,and the dual luciferase system verified their target relationship.We speculate that pma-miR-1985 may inhibit excessive inflammation after transplantation through pma-GATA-3 and pma-IFI44L.3.Function analysis of pma-miR-184-3p:After overexpression of pma-miR-184-3p in Pinctada fucata martensii,the activity of immune and antioxidant enzymes in the serum were significantly improved.The expression level of pma-miR-184-3p increased after the stimulation of pathogenic model molecules.Expression profile analysis showed that pmamiR-184-3p can activate the cell cycle and participate in the regulation of transplantation immunity by regulating cell proliferation.Expression profile and targeting prediction showed the target genes of pma-miR-184-3 p were pma-TLR3 and pma-FN.After overexpression of pma-miR-184-3p,the expression of its target genes was down-regulated,and the dual luciferase system verified their target relationship.We speculate that pma-miR184-3p may reduce rejection caused by transplantation through pma-TLR3 and pma-FN.4.Function analysis of pma-novel-63:After overexpression of pma-novel-63 in Pinctada fucata martensii,the activity of immune and antioxidant enzymes in the serum were significantly improved.The expression level of pma-novel-63 increased after the stimulation of pathogenic model molecules.Expression profile analysis showed that pmanovel-63 can participate in the regulation of transplant immunity through Notch signaling pathways and mRNA surveillance signaling pathways.Expression profile and targeting prediction showed the target genes of pma-novel-63 were pma-GDP-FucTP,pma-CysLTR2 and pma-RLR like.After overexpression of pma-novel-63,the expression of its target genes was down-regulated,and the dual luciferase system verified their target relationship.Pmanovel-63 may increase the production of interferons and inflammatory cytokines after the transplantation of pearl oyster through regulate the pma-GDP-FucTP,pma-CysLTR2 and pma-RLR like genes,and enhance the immune response of the shellfish.