MiR-130 exerts tumor suppressive function on the tumorigenesis of human non-small cell lung cancer by targeting PTEN

Abstract

MicroRNAs (miRNAs) have been involved in some human malignancies and correlated with tumor progression. The dysregulation of miR-130 is found in various cancers and correlated with tumor proliferation and apoptosis. However, its expression and function in non-small cell lung cancer (NSCLC) have not been investigated yet. In this study, we demonstrated that miR-130 is significantly down-regulated in NSCLC tissue samples and cell lines. Low miR-130 expression was closely associated with lymph node metastasis, late stages of disease progression and diminished survival in NSCLC patients. The up-regulation of miR-130 could significantly inhibit NSCLC cell growth and enhance cell apoptosis both in vitro and in vivo. Whereas inhibition of miR-130 exerted opposite effects. Furthermore, dual-luciferase reporter assay confirmed that PTEN was regulated by miR-130 directly, and the knockdown of PTEN markedly abrogated the anti-growth effect of miR-130. Additionally, miR-130 was found positively correlated with PTEN in NSCLC specimens. In conclusion, our results suggested that the expression of miR-130 is significantly associated with the growth and apoptosis of NSCLS cells by targeting PTEN, whilst miR-130 may be a potential therapeutic target for NSCLC treatment.

Keywords: Non-small cell lung cancer; PTEN; apoptosis; miR-130; proliferation.

Figure 1

MiR-130 is down-regulated and associated to NSCLC progression. A: The relative level of miR-130 was decreased in 75% (67/89) cases of NSCLC specimens. B: Relative level of miR-130 was down-regulated in NSCLC tissues compared with the normal tissues. C: The level of miR-130 was significantly decreased in five human NSCLC cell lines than in 16HBE using qRT-PCR. D: Kaplan-Meier analyses of cumulative survival for patients with NSCLC according to the expression of miR-130. ***P < 0.001.

Figure 2

Effect of miR-130 on the cell growth and apoptosis of NSCLC cells in vitro. A: Relative expression of miR-130 in NSCLC cells transfected with the miR-130, anti-miR-130 or their matched controls. B: CCK-8 assay was used to evaluate the effect of miR-130 on the cell growth of NSCLC cells. C: NSCLC cells were processed for annexin V/PI staining and analyzed with flow cytometry after transfected with miR-130 or anti-miR-130. D: The levels of PCNA and cleaved PARP fragment in NSCLC cells were examined by western blot analysis. Data were derived from 3 experiments with 6 replicates. ***P < 0.001, **P < 0.01.

Figure 3

MiR-130 directly targets PTEN. A: The putative miR-130 binding sequences in the 3’-UTR of PTEN. B: Analysis of the relative luciferase activities of PTEN-wt and PTEN-mut in A549 cells. C: The level of PTEN in A549 and H1299 cells was analyzed by western blot. D: Knockdown of endogenous PTEN markedly attenuated the anti-proliferative effect of miR-130 on A549 cells. E: Silencing of PTEN inhibted the apoptosis of A549 cells induced by miR-130, the arrows shown the typical apoptotic cells. Results are reported as the mean ± SD of three independent experiments, **P < 0.01.

Figure 4

Overexpression of miR-130 significantly suppressed the growth of NSCLC subcutaneous tumor. A: Necropsy photographs of tumor-bearing mice showing therapeutic benefit of miR-130 overexpression. B: The relative level of miR-130 was analyzed in tumor tissues by qRT-RCR. C: Survival of mice from miR-130 group was significantly prolonged in comparison with those of the miR-NC group. D: Up-regulation of miR-130 remarkably reduced PCNA positive expression in xenograft tumors. E: Apoptosis of NSCLC cells in vivo was detected by TUNEL assay. F: Western blot showing that the expression of PTEN was decreased in the subcutaneous tumors of miR-130 group. ***P < 0.001, **P < 0.01.

Figure 5

The level of PTEN is positively correlated with miR-130 expression in NSCLC specimens. A: Examination of PTEN expression in the NSCLC tissues and non-cancer tissues. B: An inversely correlation between miR-130 and PTEN expression in NSCLC tissues.