Exposure to airborne PM2.5 suppresses microRNA expression and deregulates target oncogenes that cause neoplastic transformation in NIH3T3 cells

Abstract

Long-term exposure to airborne PM2.5 is associated with increased lung cancer risk but the underlying mechanism remains unclear. We characterized global microRNA and mRNA expression in human bronchial epithelial cells exposed to PM2.5 organic extract and integrally analyzed microRNA-mRNA interactions. Foci formation and xenograft tumorigenesis in mice with NIH3T3 cells expressing genes targeted by microRNAs were performed to explore the oncogenic potential of these genes. We also detected plasma levels of candidate microRNAs in subjects exposed to different levels of air PM2.5 and examined the aberrant expression of genes targeted by these microRNAs in human lung cancer. Under our experimental conditions, treatment of cells with PM2.5 extract resulted in downregulation of 138 microRNAs and aberrant expression of 13 mRNAs (11 upregulation and 2 downregulation). In silico and biochemical analyses suggested SLC30A1, SERPINB2 and AKR1C1, among the upregulated genes, as target for miR-182 and miR-185, respectively. Ectopic expression of each of these genes significantly enhanced foci formation in NIH3T3 cells. Following subcutaneous injection of these cells into nude mice, fibrosarcoma were formed from SLC30A1- or SERPINB2-expressing cells. Reduced plasma levels of miR-182 were detected in subjects exposed to high level of PM2.5 than in those exposed to low level of PM2.5 (P = 0.043). Similar results were seen for miR-185 although the difference was not statistically significant (P = 0.328). Increased expressions of SLC30A1, SERPINB2 and AKR1C1 were detected in human lung cancer. These results suggest that modulation of miR-182 and miR-185 and their target genes may contribute to lung carcinogenesis attributable to PM2.5 exposure.

Keywords: PM2.5; bronchial epithelial cell; gene expression; microRNA expression; neoplastic transformation.

Figure 1. Altered global microRNA a. and mRNA b. expression in human bronchial epithelial cells exposed to DMSO extracts of airborne PM_2.5, and potential interactions between microRNAs and mRNAs suggested by integrate and in silico analysis c. The experimental conditions are described in Materials and Methods

Figure 2. Relative activity of reporter gene constructed with wild type of 3′UTR of SLC30A1 a. SERPINB2 b. or AKR1C1 c. gene or their mutant types d. cotransfected with miR-182 or mir-185 or their inhibitors in A549 and H446 cells. Results are mean ± SEM obtained from three experiments and each had six replicates. *, P < 0.05 and **, P < 0.01 compared with without microRNA control or wild type. Mutations in the core microRNA binding sites are shown e

Figure 3. Suppression of endogenous mRNA (up panel) and protein (lower panel) of SLC30A1 a. SERPINB2 b. and AKR1C1 c. in A549 and H446 cells transfected with miR-182 mimic, miR-185 mimic or their inhibitor. Results of mRNA levels are mean ± SEM obtained from three experiments. *, P < 0.01 and **, P < 0.001 compared with control or inhibitor

Figure 4. Foci formation ability of SLC30A1, SERPINB2 or AKR1C1 ectopically and stably expressed in NIH3T3 cells. a. Establishment of NIH3T3 cells stably expressing SLC30A1, SERPINB2 or AKR1C1. b. Colony number (mean ± SE) of cells with ectopic expression of each of the three genes. *, P < 0.05 compared with vector control

Figure 5. Xenograft tumor formation in nude mice of NIH3T3 cells ectopically and stably expressing SLC30A1, SERPINB2, AKR1C1, or vector control a. Histological analysis showed that all tumor cells had similar morphology and were diagnosed as fibrosarcoma b. Immunohistochemical staining c. demonstrated a high expression of SLC30A1 (up panel) or SERPINB2 (lower panel), respectively, in tumors induced by each of these two genes

Figure 6. Aberrant expression of SLC30A1 a. SERPINB2 b. and AKR1C1 c. in human lung cancer and paired normal tissues (up panel, squamous cell carcinoma and lower panel, adenocarcinoma)

Data were retrieved from the TCGA database