Figure 1.

miR-506 induces G1 phase arrest in lung cancer cells by targeting CDK6 expression

(A) The result of the CDK6 3′UTR reporter assay. A diagram of the wild-type or the mutant luciferase reporter vector encompassing the binding site of miR-506 in the CDK6 3′UTR is shown (right). The bold characters in miR-506 indicate the seed region. The bold italic characters show the binding site of the miR-506 seed region binding site in the CDK6 3′UTR. The luciferase reporter assay was conducted 48 h after transfection of luciferase reporter constructs containing the wild-type or mutated miR-506 binding sites from the CDK6 3′UTR. Data are presented as the mean ± SD (n = 4). ∗p < 0.05, Mann-Whitney U test.

(B) CDK6 mRNA levels were measured by RT-qPCR in 95D cells. Cells were transfected with no target control (NC) and miR-506. Forty-eight hours after transfection, cells were collected for RT-qPCR analysis. mRNA expression was normalized to β-Actin. Data are presented as the mean ± SD (n = 3). ∗p < 0.05, two-tailed Student’s t test.

(C) Western blot analysis of CDK6 protein levels in 95D cells. Cells were transfected with NC, miR-506, and Antagomir (A-506). Forty-eight hours after transfection, cells were harvested for western blot analysis. β-Actin was used as a reference gene for western blot. Band densities were quantified using Image J, and normalized band densities are shown on the bottom. Data are presented as the mean ± SD (n = 3). ∗p < 0.05, two-tailed Student’s t test.

(D) FACS (fluorescence-activated cell sorting) analysis of cell-cycle phases in 95D and H1299 lung cancer cells. The cell was harvested for cell-cycle testing 48 h after transfection of NC, miR-506, miR-506 Antagomir (A-506), and siRNA-CDK6 (si-CDK6).