d-Limonene inhibits the occurrence and progression of LUAD through suppressing lipid droplet accumulation induced by PM(2.5) exposure in vivo and in vitro

BACKGROUND: PM(2.5) exposure is associated with lung adenocarcinoma (LUAD), but the mechanism is unclear. The lack of understanding impedes our effort on prevention. This study examined a possible mechanism of lung cancer caused by PM(2.5) exposure, and aimed to find a potential intervention for people living in PM(2.5) polluted regions. METHODS: Electron microscopy and oil-red staining were conducted to examine the lipid droplet accumulation. Masson’s trichrome staining, colony forming, scratch assay and transwell experiment were conducted to evaluate the effect of PM(2.5) exposure and d-limonene intervention on the occurrence and progression of LUAD. Potential intervention targets were found by RNA-Seq and verified by luciferase reporter assay. MiR-195 KO mice constructed with CRISPR/Cas9 technology were used to investigate the pivotal role of d-limonene-miR-195-SREBP1/FASN axis. Cohort analysis of lung cancer patients, human LUAD tissues staining and human intervention trial were also conducted to validate the results of cell and animal experiments. RESULTS: Our results showed that PM(2.5) exposure induced accumulation of lipid droplets in LUAD cells which accompanied by increased malignant cellular behaviors. PM(2.5) exposure led to cleaved N-SREBP1 translocation into nucleus, which activated the de novo lipogenesis pathway. Same changes were also observed in normal lung epithelial cells and normal lung tissue, and mice developed pulmonary fibrosis after long-term exposure to PM(2.5). Furthermore, in a cohort of 11,712 lung cancer patients, significant lipid metabolism disorders were observed in higher PM(2.5) polluted areas. In view of that, d-limonene was found to inhibit the changes in lipid metabolism through upregulating the expression of miR-195, which inhibited the expression of lipogenic genes (SREBF1/FASN/ACACA) specifically. And a small human intervention trial showed that serum miR-195 was upregulated after oral intake of d-limonene. CONCLUSION: Our findings reveal a new mechanism of pulmonary fibrosis and LUAD that is related to PM(2.5) exposure-induced lipid droplet accumulation. We also demonstrate that d-limonene-miR-195-SREBP1/FASN axis is a potential preventive intervention for mediating the progression and development of LUAD induced by PM(2.5) exposure. Trial registration Chinese Clinical Trial Registry, ChiCTR2000030200. Registered 25 February 2020, http://www.chictr.org.cn/showproj.aspx?proj=48013 GRAPHICAL ABSTRACT: [Image: see text]