MicroRNAs (miRNAs) are short (~22 nucleotides) non-coding RNAs that act as post-transcriptional regulators of mRNA. Therapeutic delivery of miRNAs has been studied in several human diseases as it associates with diverse biological processes including immune responses. However, miRNA based therapy in teleost is yet to be discovered. Therefore, we encapsulated the selected miRNA mimics into chitosan nanoparticles (CNPs) to investigate the expression of endogenous miRNAs and their role on immune modulation in zebrafish model.
Total of five miRNA mimics (miR-24, miR-146, miR-155, miR-462 and miR-734) were encapsulated with CNPs (miRNAs-CNPs). All five different miRNAs-CNPs were intraperitoneally injected (20 µg/fish) to adult zebrafish separately. CNPs and water (nuclease free) were injected as controls. Tissue samples (gills) were collected at 12, 24, and 48 hours post injections (hpi) and expression of endogenous miRNAs and specific immune regulatory genes (il-1β, ifn γ) were analyzed by qRT-PCR.
Results showed significantly upregulated (p<0.05) miR-24 (3.3-fold) and miR-462 (2.8-fold) expression levels at12 hpi in gills of miR-24-CNPs and miR-462-CNPs injected fish compared to the negative control. Moreover, miR-734 was significantly (p<0.05) upregulated at 12 (8.8-fold) and 24 (7.2-folds) hpi upon injection of miR-734-CNPs to zebrafish. At 24 hpi, miR-24, miR-146, miR-155, miR-462 and miR-734 expression had >1.5 fold of expression upon respective miRNAs-CNPs administration. Among the miRNA regulatory genes examined, il-1β was downregulated (p<0.05) in miR-146-CNPs injected fish at 12 hpi. Moreover, ifn γ was downregulated in miR-24, 146, 155, 462-CNPs injected fish at 24 hpi compared to control (CNPs). Overall outcome of this revealed that the delivery of encapsulated miRNAs (with CNPs), could be able to modulate the expression of endogenous miRNAs and thereby regulate the immune responses by inhibiting the expression of proinflammatory cytokines in zebrafish.
Keywords: Cytokines; chitosan nanoparticles; encapsulation; miRNA mimics; zebrafish