MicroRNA-205 targets SMAD4 in non-small cell lung cancer and promotes lung cancer cell growth in vitro and in vivo

Abstract

Despite advances in diagnosis and treatment, the survival of non-small cell lung cancer (NSCLC) patients remains poor; therefore, improved understanding of the disease mechanism and novel treatment strategies are needed. Downregulation of SMAD4 and dysregulated expression of miR-205 have been reported. However, the relationship between them remains unclear. We investigated the effect of microRNA (miR)-205 on the expression of SMAD4 in NSCLC. Knockdown and overexpression of SMAD4 promoted or suppressed cellular viability and proliferation, and accelerated or inhibited the cell cycle in NSCLC cells, respectively. The 3'-untranslated region (3'-UTR) of SMAD4 was predicted as a target of miR-205. Luciferase assays validated that miR-205 binds directly to the SMAD4 3'-UTR. Protein and mRNA expression analyses confirmed that miR-205 overexpression in NSCLC cells inhibited the expression of SMAD4 mRNA and protein. In human NSCLC tissues, increased miR-205 expression was observed frequently and was inversely correlated with decreased SMAD4 expression. Ectopic expression of miR-205 in NSCLC cells suppressed cellular viability and proliferation, accelerated the cell cycle, and promoted tumor growth of lung carcinoma xenografts in nude mice. Our study showed that miR-205 decreased SMAD4 expression, thus promoting NSCLC cell growth. Our findings highlighted the therapeutic potential of targeting miR-205 in NSCLC treatment.

Keywords: NSCLC; SMAD4; cell cycle; miR-205; proliferation.

Figure 1. Expression of SMAD4 is reduced in NSCLC cells and human NSCLC tissues

(A) SMAD4 mRNA levels in 52 NSCLC tissues and paired noncancerous lung tissues. (B) Box plots showing relative SMAD4 mRNA expression levels of NSCLC tumors and adjacent normal lung tissues in a public data set (GSE19188). (C) Quantitative real-time reverse transcription PCR analysis of SMAD4 mRNA levels in HBE cells and NSCLC cells (A549, H1299, A1650, SPC-A1, H460, 95d, 95C, H226, H520 and SK-MES-1). SMAD4 mRNA levels are expressed as a relative index normalized against the expression of ACTB (β-actin). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2. Silencing of SMAD4 promotes NSCLC cell viability and proliferation and overexpression SMAD4 inhibits NSCLC cell viability and proliferation

(A) SMAD4 mRNA and protein levels in A549 cell lines either silenced for SMAD4 expression or overexpressing SMAD4, respectively. (B) CCK-8 assay of cell viability in A549 cells; the results were detected at 24, 48 and 72 h. (C) Representative images of clonogenic analysis for cell proliferation in A549 cells. Bar charts showing clonogenic growth of A549 cells. Values are the means ± SE from three measurements. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. Knockdown of SMAD4 or overexpression of SMAD4 had no effect on cell apoptosis, whereas it promotes or inhibits the cell cycle in NSCLC cells

(A) and (D) Flow cytometry cell cycle analysis of A549 cells (silenced for SMAD4 or overexpressing SMAD4 and compared with NC or Vector controls). Cells were harvested at 72 h post-transfection and stained with propidium iodide. The percentages of cell cycle phases are shown in the insets of each panel, in which values represent mean ± SD of three measurements. (B) and (E) Flow cytometry apoptosis assay of A549 cells (silenced for SMAD4 or overexpressing SMAD4 and compared with NC or Vector controls). Cells were harvested at 72 h post-transfection and stained with Annexin V/FITC and propidium iodide (PI). (C) and (F) Expression of p21 (an inhibitor of cell proliferation) was analyzed by western blotting. Densitometry values for each protein were normalized to β-actin. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4. MiR-205 reduces SMAD4 expression by directly targeting its 3

′-UTR. (A) Schematic diagram showing the subcloning of the predicted miR-205 binding site at position 262–269 of the SMAD4 3′-UTR into a psiCHECK-2 luciferase construct. Predicted duplex formation between miR-205 and the wild-type or mutant of miR-205 binding site is indicated. (B) Luciferase activities of the construct containing the wild-type or mutant Smad4 3′-UTR reporter gene in A549 cells cotransfected with negative control (NC) or miR-205. Scrambled sequences were used as the NC. Relative Renilla luciferase activity was determined followed by normalization against the firefly luciferase activity.

Figure 5. Expression of miR-205 is upregulated in NSCLC tissues and inversely correlated with SMAD4 expression

(A) Relative miR-205 levels in 52 NSCLC tissues (T) and paired noncancerous lung tissues (N). (B) Scatter diagram showing relative SMAD4 mRNA expression levels of NSCLC tumors and adjacent normal lung tissues in a public data set (GSE36681). (C) Correlation between the miR-205 level and SMAD4 mRNA expression in 52 paired NSCLC tissues. MiR-205 and SMAD4 mRNA levels are expressed as relative indexes normalized against U6 and β-actin, respectively. The x and y-axes represent the log10 transformed fold change of T/N mRNA expression ratios of miR-205 and SMAD4, respectively. (D) Relative expression of miR-205 levels and SMAD4 mRNA in 52 paired NSCLC tissues. The y-axis represents the log10 transformed fold change of T/N expression ratios of miR-205 levels and SMAD4 mRNA. The number of each specimen is indicated below the x-axis. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6. Overexpression of an miR-205 mimic inhibits NSCLC cell viability and proliferation

(A) QRT-PCR analysis of miR-205 levels in A549 cells transfected with miR-205 mimics or NC for 72 h. U6 was used as the internal control. SMAD4 mRNA expression in A549 cells transfected with miR-205 mimics or NC. β-actin was used as the internal control. (C) The SMAD4 protein levels in A549 cells transfected with miR-205 mimics or NC. (B) CCK-8 assay of cell viability in A549 cells transfected with miR-205 mimics or NC for 96 h. B Representative images of the clonogenic analysis for cell proliferation in A549 cells transfected with miR-205 mimics or NC. Bar charts showing clonogenic growth of A549 cells transfected with miR-205 mimics or NC. Values are represented as means ± SE from three measurements. **P < 0.01; ***P < 0.001.

Figure 7. Overexpression of an miR-205 mimic had no effect on cell apoptosis, but promoted the cell cycle in NSCLC cells

(A) Flow cytometry cell cycle analysis of A549 cells with miR-205 mimics or NC. Cells were harvested at 72 h post-transfection and stained with propidium iodide. Shown in the inset of each panel are the percentages of cell cycle phases, in which values represent the mean ± SD of three measurements. (B) Flow cytometry apoptosis assay of A549 cells with miR-205 mimics or NC. Cells were harvested at 72 h post-transfection and stained with Annexin V/FITC and propidium iodide (PI). (C) Expression of p21 (an inhibitor of cell proliferation) was analyzed by western blotting. Densitometry values for each protein were normalized to that of β-actin. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 8. Overexpressing SMAD4 in NSCLC cells promotes tumor growth in vivo

(A) At the experimental endpoint, tumors were dissected and photographed as indicated. (B) Tumor growth curves in mice (n = 3/group) inoculated with the indicated cells at the indicated days; each tumor formed by the indicated cells was weighed. (C) Hematoxylin and eosin (H and E) staining confirmed tumor cells in slices of the indicated tumor sections. Immunohistochemical staining for SMAD4 was quantified using its staining intensity.

Figure 9. Overexpressing miR-205 in lung carcinoma xenografts promotes tumor growth in vivo

(A) QRT-PCR analysis of miR-205 levels and SMAD4 mRNA expression in excised tumors transfected with miR-205 agomir and NC agomir; U6 and β-actin were used as internal controls, respectively. (B) At the experimental endpoint, tumors were dissected and photographed as indicated. (C) Tumor growth of miR-205 agomir and NC agomir-treated A549 xenografts in nude mice (n = 3). The graph show the tumor growth curves at sacrifice with respect to the first measurements, using the administration of 1 nmol miR-205 agomir or NC agomir per mouse every 4 days for seven times; the arrows indicate the weight of the excised tumors (mean ± SD, n = 3). (D) Hematoxylin and eosin (H and E) staining confirmed tumor cells in slices of the indicated tumor sections. Immunohistochemical staining for SMAD4 and PTEN was quantified using the staining intensity.