miR-205 regulates A549 cells proliferation by targeting PTEN

Abstract

miR-205 is an epithelial-specific miRNA and has been shown to orchestrate some cellular processes such as epithelial mesenchymal transition (EMT) and differentiation fate of stem cells in mammary gland. miR-205 play a part of a tumor suppressor in human cancers. However, the role of miR-205 in lung cancer is unclear. In this study, we detected the expression level of miR-205 in 46 cases clinical lung cancer specimens and adjacent normal tissues by stem-loop RT-PCR. We found that the expression of miR-205 was significantly increased in lung cancer specimens compared to adjacent normal tissues (P < 0.01). Furthermore, we observed the expressions of PTEN protein and mRNA in lung cancer tissues and adjacent normal tissues by methods of western blot and Real time PCR respectively. We found that the expressions of PTEN protein and mRNA was significantly decreased in lung cancer specimens compared to adjacent normal tissues. And then, we found there is a negative relationship between the expression of miR-205 and PTEN mRNA in lung cancer by analyzed. To validate whether PTEN was direct targets of miR-205, a dual-luciferase reporter assay was employed, the result showed that PTEN is a target gene of MiR-205. In subsequent experiments, we examined the expressions of PTEN protein and mRNA after transfection of miR-205 mimics or inhibitor into A549 cells, and A549 cell proliferation was measured by CCK-8 tests. We found that the expression of PTEN protein and mRNA in A549 cells were significantly down-regulated or up-regulated after miR-205 mimics and miR-205 inhibitors transfected into, and miR-205 could inhibits A549 cells proliferation. These results indicate that miR-205 might inhibitor the proliferation of A549 cells by regulating the expression of PTEN.

Keywords: Lung cancer; PTEN; miR-205; proliferation.

Figure 1

The expression of MiR-205 was up-regulated in lung cancer tissue. U6 RNA was used as an endogenous normalizer and the relative combined expression levels of miR-205 are shown. 2^-ΔΔCt values ± SEM, n = 46, *P < 0.01.

Figure 2

The expression of PTEN is downregulated in lung cancer tissue. U6 RNA was used as an endogenous normalizer and the relative combined expression levels of miR-205 are shown. 2^-ΔΔCt values ± SEM, n = 46, *P < 0.01.

Figure 3

The expression of PTEN protein is downregulated in lung cancer tissue. (A) PTEN protein expression was determined by Western blot in lung cancer (C) and normal tissues (N). (B) Optical density values for combined PTEN protein relative to GAPDH in lung cancer tissues. N = 46. *P < 0.01.

Figure 4

The scatter diagram of the expression of miR-205 and PTEN.

Figure 5

PTEN as a target gene of miR205. A. Schematic diagram of the predicted miR-205 binding site in the PTEN 3’-UTR. B. Luciferase reporter assays in untreated HEK-293T cells, or cotransfected with wild type (wt) or mutant (mut) PTEN 3’-UTR, and miR-133b or miR-133b negative control (NC). *P < 0.01.

Figure 6

Determination of miR-205 mimics or miR-205 inhibitors transfection efficiency. A. The expression of miR-205 was significantly increased after miR-205 mimics’ transfection into A549 cells. B. The expression of miR-205 was significantly decreased after miR-205 inhibitors transfection into A549 cells. *P < 0.01.

Figure 7

Effect of miR-205 mimics and inhibitor on PTEN mRNA and protein levels in A549 cells. A549 cells were untreated (A, D), treated with miR-205 mimics (B), treated with miR-205 NC (C), treated with miR-205 inhibitors (E), treated with miR-205 inhibitors NC(F). (A, C) The expression of PTEN protein was determined by Western blot. (B, D) Optical density values for combined PTEN protein relative to GAPDH. (E) The expression of PTEN mRNA was determined by Realtime PCR, (F) Optical density values for combined PTEN mRNA relative to U6, n = 46. *P < 0.01.

Figure 8

Effect of miR-205 on proliferation of A549 cells. A. A549 cells were untreated or transfected with miR-205-NC or miR-205-mimics for up to 72 h. B. A549 cells were untreated or transfected with miR-205-inhibitor-NC or miR-205- inhibitor for up to 72 h. Cell proliferation was measured by CCK-8. *P < 0.05.