Temporal regulation of miRNA 92a and miRNA 279d on PaEno1 expression and dynamic responses of metal-stress-related genes to cadmium exposure in Propsilocerus akamusi (Diptera: Chironomidae) larvae.

Previous studies have indicated that Eno1 may play an important role in Cadmium (Cd) resistance. However, the potential regulatory mechanisms of Eno1 in response to Cd2+ stress are still limited. In this study, we hypothesised that Cd-induced miRNA alteration could effectively regulate PaEno1 (Eno1 in Propsilocerus akamusi (Diptera: Chironomidae)) under Cd2+ stress. By integrating Illumina miRNA sequencing, real-time quantitative Polymerase chain reaction (PCR) (RT-qPCR) validation, PaEno1 target prediction and in vivo functional assays, we verified that PaEno1 is a putative target of miR-92a and miR-279d, which potentially mediate the post-transcriptional regulation of PaEno1 under Cd2⁺ stress. RT-qPCR revealed significant downregulation of miR-92a and miR-279d by 2.825-4.225-fold upon long-term Cd exposure. Agomir/antagomir-mediated overexpression and knockdown of the two miRNAs exerted no significant impact on larval survival (Log-rank test, p > 0.05). The hazard ratios were 0.715 and 0.609 for miR-279d (agomir and antagomir), and 0.810 and 0.869 for miR-92a, respectively. The 95% confidence intervals spanned 0.266-1.921 and 0.217-1.712 for miR-279d agomir and antagomir, and 0.399-1.642 and 0.434-1.740 for miR-92a agomir and antagomir, with all intervals crossing the null value of 1. Agomir-induced upregulation of miR-92a and miR-279d dynamically altered the expression of PaEno1, HbV, HbVII, alpha-esterase, and carboxylesterase. Overall, miR-92a and miR-279d act as temporal post-transcriptional regulators of PaEno1 rather than key modulators of Cd tolerance. This work underscores the complexity of miRNA-regulated networks in P. akamusi responding to Cd stress.