MiR-361-5p/abca1 and MiR-196-5p/arhgef12 Axis Involved in γ-Sitosterol Inducing Dual Anti-Proliferative Effects on Bronchial Epithelial Cells of Chronic Obstructive Pulmonary Disease

PURPOSE: Chronic obstructive pulmonary disease (COPD), a progressive and irreversible respiratory disease, becomes the third leading cause of death and results in enormous economic burden on healthcare costs and productivity loss worldwide by 2020. Thus, it is urgent to develop effective anti-COPD drugs. MATERIALS AND METHODS: In the present study, two published GEO profiles were used to re-analyze and ascertain the relationships between circulating miRNAs and bronchial epithelial cells (BECs) mRNAs in COPD. The microRNA levels of miR-361-5p and miR-196-5p in plasma of COPD patients and healthy volunteers were detected by qRT-PCR. Next, the effects of γ-sitosterol (GS) on the expression of miR-361-5p and miR-196-5p and cell proliferation were investigated in BEC and H292 cell lines. Finally, whether specific miRNA-mRNA pathways involved in the effect of GS on BECs was assayed using Western Blot, real-time PCR and immunofluorescence. RESULTS: miR-196-5p and miR-361-5p were, respectively, up- and down-regulated in COPD patients compared with healthy controls. Luciferase assays demonstrated that miR-361-5p and miR-196-5p were, respectively, targeting abca1 and arhgef12 3ʹUTR in BEAS-2B cells. GS significantly suppressed miR-196-5p and promoted miR-361-5p levels in BEAS-2B cells and inhibited BECs proliferation in vitro. GS promoted miR-361-5p expression, which inhibited BCAT1 mRNA and protein levels and weaken mTOR-pS6K pathway, resulted in anti-proliferation in BEAS-2B cells. In addition, RhoA was activated by ARHGEF12 due to the inhibitory effect of miR-196-5p on arhgef12-3ʹUTR which was partially abolished by GS suppressing miR-196-5p expression. Activated RhoA further activated ROCK1-PTEN pathway and finally inhibited mTOR pathway, resulting in induced BECs proliferation. The anti-proliferation effect of GS was not observed in H292 cells. CONCLUSION: These findings indicate that miR-361-5p/abca1 and miR-196-5p/arhgef12 axis mediated GS inducing dual anti-proliferation effects on BECs.