Figure 6

Inhibition of let-7d in OS-RC-2 cells. (A, B) Real-time RT-PCR quantification of relative expression of let-7d (A), COL3A1 and CCL7 (B) in let-7d inhibitor or negative control transfected OS-RC-2 cells. (C) Western blot and (D) ELISA of COL3A1 and CCL7 expression. Corresponding densitometry of each band is presented in a bar graph. Data presented are the mean ± SD of three independent experiments. (E) Proliferation assay was performed by CCK-8. (F, G) Representative images and quantitative data of migrated negative control- (a) or let-7d inhibitor- (b) transfected OS-RC-2 cells evaluated by Boyden chamber assay. Original magnification: ×100. (H) Representative images of wound gaps in let-7d inhibitor- and control-transfected OS-RC-2 cells at different time points. Original magnification: ×40. (I) The quantification results of percent wound healing are represented as the mean ± SD of three independent experiments. (J) PBMC chemotaxis was evaluated by chemotaxis assay with the presence of 1 μg/mL CCL7 neutralizing antibody or control IgG in the conditioned medium. Results are expressed as mean ± SD of three independent experiments. *P < 0.05. (K, L) The linear regression and correlation between let-7d and COL3A1 mRNA levels in all 80 RCC tissues (K) and between let-7d and CCL7 mRNA levels in all 30 T3 stage RCC tissues from the same set (L). Expression status is shown as the tumor/non-tumor ratio in a log scale.