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. 2016 May 3;17(5):515-25.
doi: 10.1080/15384047.2015.1095405. Epub 2015 Oct 22.

Decreased MicroRNA-26a expression causes cisplatin resistance in human non-small cell lung cancer

Yong Yang  1 , Peng Zhang  1 , Yanfeng Zhao  1 , Jie Yang  1 , Gening Jiang  1 , Jie Fan  1   2
Affiliations

Decreased MicroRNA-26a expression causes cisplatin resistance in human non-small cell lung cancer

Yong Yang et al. Cancer Biol Ther. .

Abstract

Background: Lung cancer is the most common cancer that is caused by perturbation of regulatory pathways rather than dysfunction of a single gene. Cisplatin (CDDP; cis-diamminedichloroplatinum II) is the first member of a class of platinum-containing anti-cancer medication, which binds to DNA and triggers apoptosis. CDDP-based chemotherapy is used to treat various types of cancers. However, the efficacy of CDDP in the treatment of non-small-cell lung cancer (NSCLC) is limited by acquired drug resistance. MicroRNAs have recently emerged as key regulators of cancers, and miR-26a is one of down-regulated miRNAs in A549/CDDPres cell line. This study aimed to investigate the role of miR-26a in CDDP resistance in NSCLC as well as the underlying mechanisms.

Methods: In this study, we analyzed expressional profiles of CDDP resistance-related mRNA, miRNA, and transcription factors (TF) that regulate miRNA expression in NSCLC. A549 cells were treated with CDDP, miR-26a mimic, or miR-26a inhibitor, and followed by biological analysis including drug sensitivity assay, colony formation assay, terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) assay, and cell cycle analysis. Luciferase assay was used to determine the target of miR-26a. The regulation of miR-26a in Akt pathway was measured by western blot.

Results: High mobility group A (HMGA) 2 was identified as the target of miR-26a. Overexpression of miR-26a in A549 cells inhibited G1-S transition, increased cell death in response to CDDP treatment, and decreased the colony formation of A549 cells. MiR-26a significantly decreased the expression of E2F1, diminished Akt phosphorylation, and downregulated Bcl2 expression. Cell growth was suppressed by inhibiting HMGA2-mediated E2F1-Akt pathway.

Conclusion: MiR-26a is responsible for A549 cell sensitivity in the treatment of CDDP through regulating HMGA2-mediated E2F1-Akt pathway.

Keywords: Cisplatin resistance; E2F1; HMGA2; miR-26a; non-small-cell lung cancer.

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Figures

Figure 1.
Figure 1.
The union of active TF-miRNA regulatory pathway in CDDP resistance. Yellow nodes represent the known CDDP associated genes and red nodes represent the direct curated genes.
Figure 2.
Figure 2.
Role of HMGA2 and miR-26a in lung cancer. (A) Kaplan–Meier survival analysis of lung adenocarcinoma patients with high or low HMGA2 expression (N=35). The results shows that the patients with low HMGA2 exhibited significantly longer overall survival (OS) (log-rank test, P=0.03). (B) Validation of miR-26a expression in A549 versus A549/CDDPres by realtime RT-PCR. The relative amount of miR-26a was normalized to U6 snRNA. (C) Validation of HMGA2 expression in A549 vs. A549/CDDPres by Western blot. The relative amount of HMGA2 was normalized with GAPDH. Data in histograms are means ± SD, **P<0.01 compared with A549 (t test).
Figure 3.
Figure 3.
miR-26a directly targets HMGA2 in NSCLC cells. (A) Predicted miR-26a target sequence in the 3′ UTR of HMGA2. (B) Realtime PCR and western blotting analysis of HMGA2 expression in A549 cells transfected with miR-26a mimic or the miR-26a inhibitor, GAPDH served as the loading control. (C) 3′ UTR reporter vector model. The LightSwitch 3´UTR Reporter vector contains a 3124 bp human HMGA2 3´UTR sequence cloned downstream of the RenSP luciferase gene in the pLightSwitch_3UTR reporter vector. A constitutive promoter drives expression of the hybrid RenSP- 3´UTR transcript. (D) Luciferase reporter assay of the A549 cells transfected with the HMGA2–3′ UTR reporter and increasing amounts (30, 60, 90 nM) of miR-329 mimics. Bars represent the mean ± SD of 3 independent experiments. * P<0.05.
Figure 4.
Figure 4.
miR-26a overexpression reduces CDDP resistance in NSCLC. (A) Drug sensitivity assays revealed that upregulation of miR-26a decrease IC50 of A549/CDDPres to CDDP. (B) Representative micrographs and quantification of crystal violet stained cell colonies. (C) Flow cytometric analysis for TUNEL assay of A549 cells transfected with NC or miR-26a mimics. Each bar represents the mean of 3 independent experiments. (D) Cell cycle analysis of A549 cells transfected with NC or miR-26a mimics. Each bar represents the mean of 3 independent experiments. * P< 0.05.
Figure 5.
Figure 5.
miR-26a inhibition increase CDDP resistance in NSCLC. (A) Drug sensitivity assays revealed that downregulation of miR-26a increase IC50 of A549 to CDDP. (B) Representative micrographs and quantification of crystal violet stained cell colonies. (C) Flow cytometric analysis for TUNEL assay of A549 cells transfected with NC or miR-26a inhibitor. Each bar represents the mean of 3 independent experiments. (D) Cell cycle analysis of A549 cells transfected with NC or miR-26a inhibitor. Each bar represents the mean of 3 independent experiments. * P< 0.05.
Figure 6.
Figure 6.
miR-26a inhibits E2F1 and Akt pathway. (A) Real time PCR analysis of E2F1 expression in A549 cells transfected with miR-26a mimic or the miR-26a inhibitor, GAPDH served as the loading control. (B) Western blotting analysis of E2F1, phosphor-Akt and Bcl2 expression in A549 cells transfected with miR-26a mimic or the miR-26a inhibitor, GAPDH served as the loading control. (C) Kaplan-Meier survival analysis of lung adenocarcinoma patients with high or low E2F1 expression (N=113). Adenocarcinoma patients with high E2F1 exhibited significatly shorter OS (log-rank test, P = 0.0257). (D) Western blotting analysis of E2F1, phosphor-Akt and Bcl2 expression in A549 cells transfected with siRNA HMGA2, GAPDH served as the loading control.
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