Diesel Exhaust‐Induced miR ‐133b‐3p Downregulation and CTGF ‐Linked Hippo Pathway Inactivation Pr

ABSTRACT

Exposure to diesel exhaust (DE) significantly contributes to air pollution. DE dysregulates miRNA expression, miRNA‐target interactions promoting tissue remodeling and lung inflammation. In the present study, C57BL/6 mice were divided into two groups: one was given DE for 30 min/day, 5 days/week, for 8 weeks whereas the control group was given filtered air. BALF and lungs were collected for cytokine analysis. MiRNA expression was analyzed in lungs using microarrays. qRT‐PCR and western blotting validated miRNAs and target genes. Functional impacts of miRNA‐target gene interaction were studied using in vitro and in vivo models. DE exposure downregulated miR‐133b‐3p (> 2‐fold, p  < 0.05) prompting investigation into downstream effects. In silico analysis identified 371 potential targets of miR‐133b‐3p, with Hippo signaling as one of the most enriched pathways wherein CTGF, FGF1 , and TCF7 genes shared a common predicted binding site for miR‐133b‐3p. Functional validation using A549 cells showed transfection with miR‐133b‐3p mimic reduces CTGF expression at both mRNA ( p  = 0.0054) and protein ( p  = 0.0001) levels, while inhibition of miR‐133b‐3p restored CTGF expression, confirming it as a direct target. In vivo, DE exposure increased CTGF mRNA & protein expression whereas decreased P‐ YAP/YAP ratio was consistent with Hippo pathway dysregulation and co‐occurred with miR‐133b‐3p downregulation. DE exposure also increased pro‐inflammatory cytokines (IL‐18, IL‐1β, GM‐CSF) in BALF, indicating increased inflammatory response. Furthermore, DE exposure decreased LOXL1 and LOXL4 mRNA and increased MMP‐3 , MMP‐7 , and MMP‐12 protein levels therefore altering ECM turnover potential corroborated with STRING analysis. Cumulatively, DE‐induced miR‐133b‐3p downregulation and CTGF alteration are associated with Hippo pathway dysregulation, increased inflammation, and MMP/LOX ‐driven ECM remodeling in mouse lungs.