Fig. 4.

miR-181c negatively regulates the expression of CCN1 by directly targeting the CCN1–3′-UTR. a miR-181c binding sites in the CCN1 3′-UTR. Mutations in the complementary site for the seed region of miR-181c in 3′-UTR of CCN1 gene are indicated. b Wild-type or mutant reporter plasmids were cotransfected with agomiR-181c or antagomiR-181c into 293 T cells. After 48 h transfection, luciferase activity was determined. Western blot (c) and qRT-PCR (d) analyses of the expression of CCN1 in HBECs exposed to 2.5% CSE or control medium and transfected with agomiR-181c or scramble. Western blot (e) and qRT-PCR (f) analyses of CCN1 expression in lung tissues of mice treated with PBS/Air, CS, agomiR-181c/CS, and antagomiR-181c/CS. g The relative expression levels of CCN1 were examined by qRT-PCR in lung tissues of 8 never smokers and 18 COPD patients. h Pearson correlation analyses between miR-181c levels and mRNA expression levels of CCN1 in human COPD tissues. Data represent mean ± SD from three independent experiments; *p < 0.05, **p < 0.01