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. 2015 Feb 15;5(3):1180-9.
eCollection 2015.

MiRNA-1469 promotes lung cancer cells apoptosis through _targeting STAT5a

Chengshan Xu  1 , Ling Zhang  1 , Hengheng Li  1 , Zhihua Liu  2 , Lianning Duan  1 , Chengrong Lu  1
Affiliations

MiRNA-1469 promotes lung cancer cells apoptosis through _targeting STAT5a

Chengshan Xu et al. Am J Cancer Res. .

Abstract

MicroRNAs play key roles in cell growth, differentiation, and apoptosis. In this study, we described the regulation and function of miR-1469 in apoptosis of lung cancer cells (A549 and NCI-H1650). Expression analysis verified that miR-1469 expression significantly increased in apoptotic cells. Overexpression of miR-1469 in lung cancer cells increased cell apoptosis induced by etoposide. Additionally, we identified that Stat5a is a downstream _target of miR-1469, which can bind directly to the 3'-untranslated region of the Stat5a, subsequently reducing both the mRNA and protein levels of Stat5a. Finally, co-expression of miR-1469 and Stat5a in A549 and NCI-H1650 cells partially abrogated the effect of miR-1469 on cell apoptosis. Our results show that miR-1469 functions as an apoptosis enhancer to regulate lung cancer apoptosis through _targeting Stat5a and may become a critical therapeutic _target in lung cancer.

Keywords: MicroRNA; Stat5a; apoptosis; lung cancer.

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Figures

Figure 1
Figure 1
MiR-1469 is upregulated during apoptosis of lung cancer cells. A. The apoptosis of A549 cells was induced by treating with VP16 (100 μM) for 48 h. The histogram shows the apoptotic cell percentage detected by FCM, and error bars denote mean ± SD (right panel). B. Flow cytometry showed apoptosis of H1650 cells after VP16 (100 μM) treatment. The histogram shows the apoptotic cell percentage detected by FCM, and error bars denote mean ± SD (right panel). C. Western blot analysis was used to detect the expression of BcL-2 and γ-H2AX in A549 and H1650 cells both treated by VP16 (100 μM) for 48 h. b-actin was detected as a loading control. D. The histogram shows the expression of miRNA-1469 in A549 (left panel) and H1650 cells (right panel) 48 h after VP16 treatment (100 μM). *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 2
Figure 2
Overexpression of miRNA-1469 promotes VP16-induced cell apoptosis. A. The apoptosis of A549 cells was induced by treatment with VP16 (100 μM) for 48 h after miRNA-1469 mimics (20 nM) transfection for 24 h. The histogram shows the apoptotic cell percentage detected by FCM, and error bars denote mean ± SD (right panel). B. Flow cytometry was used to detect apoptosis of H1650 cells induced by treating with VP16 (100 μM) after miRNA-1469 mimics (20 nM) transfection for 24 h. The histogram shows the apoptotic cell percentage and error bars denote mean ± SD (right panels). **, p < 0.01; ***, p < 0.001. C. Flow cytometry was used to detect the cell cycle of A549 and H1650 cells 48 h after miRNA-1469 mimics (20 nM) transfection.
Figure 3
Figure 3
Stat5a is a direct _target of miRNA-1469. A. MiR-1469 _targeting site resides at nucleotides 226-232 of Stat5a-3’UTR and is highly conserved in different species. Upper panel: sequence alignment of miR-1469 with binding sites on the Stat5a-3’UTR. Bottom panel: sequence of the miR-1469 binding site within the Stat5a-3’UTR of three species (human, chimpanzee and rhesus). B. Diagram of the luciferase reporter plasmids including plasmid with the full-length Stat5a-3’UTR insert (pIS0-Stat5a-3’UTR) and plasmid with a mutant Stat5a-3’UTR (pIS0-Stat5a-3’UTR-mut) which carried a substitution of three nucleotides within the miR-1469 binding site. C. Luciferase activity assay demonstrates a direct _targeting of the Stat5a-3’UTR by miR-1469. A549 cells (left panel) and H1650 cells (right panel) were transfected with miR-1469 mimics (20 nM) and pIS0-Stat5a-3’UTR /pIS0-Stat5a-3’UTR-mut. pRL-SV40 Renilla was used for the normalization of transfection efficiency. After 48 h, the luciferase activities were measured. D. Western blotting was used to detect the expression of the Stat5a protein after miRNA-1469 mimics (20 nM) or miRNA-1469; Inhibitor (40 nM) transfection of A549 (left panel) or H1650 (right panel) cells. E. Stat5a mRNA in the A549 (left panel) or H1650 (right panel) cell lines treated as above was measured with real-time RT-PCR. β-actin was used as internal control. F. Overexpression of miR-1469 mimics (20 nM) or miRNA-1469.Inhibitor (40 nM) in A549 or H1650 cell lines transfected miRNA-1469 mimics or miRNA-1469. Inhibitor was measured by real-time RT-PCR. U6 was used as internal control. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 4
Figure 4
MiR-1469 regulates apoptosis through Stat5a. A. Apoptosis of A549 cell were detected by FCM 48 h after miRNA-1469 mimics and combined with Stat5a transfection (left panel). Error bars denote mean ± SD (right panel). B. The apoptosis of H1650 cells 48 h after transfection of miRNA-1469 mimics and combined with Stat5a were detected by FCM (left panel). Error bars denote mean ± SD (right panel). **, p < 0.01; ***, p < 0.001.
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