Sequence-reads were in turn trimmed according to BWAs C q algorithm [55] and the sequence reads were quantified using the transcriptome assembly (Kallisto version 0.43.0) [56, 57]. participate in ion uptake/excretion, which is essential for the re-establishment of fluid and electrolyte homeostasis. The osmoregulatory plasticity of gills provides an excellent model to study the role of microRNAs (miRs) in adaptive osmotic responses. The present study is to characterize an ex-vivo gill filament culture and using omics approach, to decipher the CA-4948 interaction between tonicity-responsive miRs and gene targets, in orchestrating the osmotic stress-induced responses. Results Ex-vivo gill filament culture was exposed to Leibovitzs L-15 medium (300 mOsmol l??1) or the medium with an adjusted osmolality of 600 mOsmol l??1 for 4, 8 and 24?h. Hypertonic responsive genes, including osmotic stress transcriptional CA-4948 factor, Na+/Cl?-taurine transporter, Na+/H+ exchange regulatory cofactor, cystic fibrosis transmembrane regulator, inward rectifying K+ channel, Na+/K+-ATPase, and calcium-transporting ATPase were significantly upregulated, while the hypo-osmotic gene, V-type CA-4948 proton ATPase was downregulated. The data illustrated that the ex-vivo gill filament culture exhibited distinctive responses to hyperosmotic challenge. In the hyperosmotic treatment, four key factors (i.e. drosha RNase III endonuclease, exportin-5, dicer ribonuclease III and argonaute-2) involved in miR biogenesis were dysregulated (adapted at different salinities [i.e. freshwater (FW), brackish-water or seawater (SW)] were described [15]. Japanese eels are euryhaline fish. The osmoregulatory tissue – gills provides an excellent model to study role of miRs in the regulation on plasticity of adaptive osmotic responses in vertebrates. The present study aimed to identify and characterize the involvement of miRs and messenger RNAs (mRNA) under osmotic perturbations. In this report, we used both ex-vivo gill filament and primary gill cell culture models, accompanied with miR-, transcriptome-sequencing and miR inhibition, to identify tonicity-sensitive miRs and to characterize their expressions in gill filaments. Results Hyperosmotic treatment induced differential expression of transcriptional Factor-1/regulators, ion channels/transporters and miR biogenesis factors in ex-vivo gill filament culture Gill filaments of eels were ex-vivo cultured in isotonic (300 mOsmol l??1) or hypertonic (600 mOsmol l??1) L-15 medium (penicillin-streptomycin (PS), gentamycin, FBS) at 23?C for 4, 8 and 24?h. Transcripts levels of expression was used as internal control for normalization of target gene expression. Results (mean??s.e.m.) were from five independent experiments. * transcripts were significantly reduced under hyperosmotic stress. The expression levels of was used as internal control. Results (mean??s.e.m.) were from five independent experiments. * and slc(Fig. ?(Fig.6d).6d). The binding site of is at 3UTR between 447 to 468, while the binding site of is at the 3UTR between 53 to 73. Open in a separate window Fig. 6 Inhibition miR-29b-3p/miR-200b-3p in primary gill cell culture. To determine the biological effects of miR-29b-3p and miR-200b-3p through loss-of-function, two specific miRNA inhibitors (miR-29b-3p or miR-200b-3p) were transfected in gill cell culture. a qRT-PCR was performed to determine the efficiency of inhibition on miR-29b-3p and miR-200b-3p. Venn diagram shows the overlay of up-regulated genes in 4/8-h hypertonic (Hyper) treatment and b miR-29b-3p inhibition, and c miR200b-3p. The figures in parentheses represent the numbers of co-expressed genes. The potential target genes of miR-29b-3p and miR-200-3p were listed in the table. d Using miRanda algorithm, the full length of the miR was aligned against Rabbit polyclonal to AARSD1 the target gene sequences. Thermodynamic stability of the RNA duplexes is based on these alignments and strict alignment in the seed region at position 2C7?bp of the miR to the mRNA sequence. * [20, 21], [22], [23] are regulatory proteins known to modulate gene transcription, apical trafficking of transmembrane G-protein coupled CA-4948 receptors/ion-transporters and epithelial chloride (Cl?) transport respectively. The hyperosmotic induction was demonstrated in intact fish and primary gill epithelial cell CA-4948 culture [24]. Its expression could also be stimulated by the seawater-adapting hormone,.
