miR‑139‑5p enhances cisplatin sensitivity in non‑small cell lung cancer cells by inhibiting cell proliferation and promoting apoptosis via the targeting of Homeobox protein Hox‑B2

Abstract

The development of chemotherapeutic dug resistance hinders the clinical treatment of cancer. MicroRNAs (miRNAs/miRs) have been revealed to serve essential roles in the drug resistance of numerous types of cancer. miR‑139‑5p was previously reported to be associated with cisplatin (DDP) sensitivity in human nasopharyngeal carcinoma cells and colorectal cancer cells. However, the effect and underlying mechanism of miR‑139‑5p in DDP sensitivity in non‑small cell lung cancer (NSCLC) cells has not yet been fully elucidated. In the present study, the expression of miR‑139‑5p and Homeobox protein Hox‑B2 (HOXB2) in NSCLC tissues was examined by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blotting. Subsequently, the effect of miR‑139‑5p on the DDP sensitivity of NSCLC cells in vitro was investigated. Cell proliferation was examined using a Cell Counting Kit‑8 assay. Western blotting was used to evaluate the protein expression of HOXB2, phosphorylated (p)‑PI3K, p‑AKT, caspase‑3 and cleaved‑caspase‑3, and RT‑qPCR was used to evaluate the expression of miR‑139‑5p, and the mRNA expression levels of HOXB2, PI3K, AKT and caspase‑3. The apoptotic rate of the cells was detected using flow cytometry. miR‑139‑5p expression in NSCLC tissues was shown to be significantly lower compared with that in adjacent tissues. Additionally, miR‑139‑5p increased cell apoptosis and inhibited NSCLC cell proliferation induced by DDP in vitro via modulating the PI3K/AKT/caspase‑3 signaling pathway. Furthermore, HOXB2 was identified to be a target of miR‑139‑5p, and miR‑139‑5p was revealed to sensitize NSCLC cells to DDP via the targeting of HOXB2. Taken together, the results of the present study demonstrated that regulating the expression of miR‑139‑5p could provide a novel approach to reverse DDP resistance and increase chemosensitivity in the treatment of NSCLC.

Keywords: microRNA‑139‑5p; non‑small cell lung cancer; cisplatin sensitivity; PI3K/AKT; cleaved‑caspase‑3.

Figure 1.

Expression of miR-139-5p and HOXB2 in lung cancer tissues. The relative expression levels of miR-139-5p and HOXB2 in tissues were determined by reverse transcription-quantitative PCR and western blotting. Compared with the control tissues, miR-139-5p was significantly downregulated and HOXB2 was significantly upregulated in lung cancer tissues. Relative mRNA expression of (A) miR-139-5p and (B) HOXB2 in tumor and paracarcinoma tissues. (C) Relative protein expression of HOXB2 in tumor and paracarcinoma tissues. (D) Semi-quantitative analysis of western blotting. **P<0.01, tumor vs. control group. miR, microRNA; HOXB2, Homeobox protein Hox-B2.

Figure 2.

Expression of miR-139-5p after transfection with miR-139-5p mimics. The relative expression of miR-139-5p in tissues was determined by reverse transcription-quantitative PCR after transfection with mimics followed by incubation at 37°C with 5% CO₂. Compared with the miR-NC mimics group, transfection with miR-139-5p mimics induced a significant increase in the expression of miR-139-5p. The three groups used were the Control group (non-treated group), the NC group (cells transfected with control plasmids) and the mimics group (cells transfected with miR-139-5p mimics). **P<0.01, miR-139-5p mimics group vs. miR-NC mimics group. miR, microRNA; NC, negative control.

Figure 3.

Target verification via a luciferase reporter assay and HOXB2 protein expression in A549 cells. HOXB2 was found to be a direct target of miR-139-5p and the expression of HOXB2 was downregulated significantly in DDP-treated A549 cells after transfection with miR-139-5p mimics. (A) TargetScan was used for target prediction. (B) Relative luciferase activity after transfection. (C) Relative protein expression of HOXB2 in A549 cells and (D) semi-quantitative analysis of western blotting. **P<0.01. miR, microRNA; HOXB2, Homeobox protein Hox-B2; NC, negative control; WT, wild-type; UTR, untranslated region; DDP, cisplatin.

Figure 4.

Effects of miR-139-5p on cell proliferation and apoptosis in DDP-treated cells. Non-small cell lung cancer cell proliferation was inhibited and apoptosis was increased significantly in DDP-treated A549 cells after transfection with miR-139-5p mimics. (A) Cell proliferation was measured by a Cell Counting Kit-8 assay. (B) Cell apoptosis was measured by flow cytometry. (C) The rate of apoptosis. **P<0.01. miR, microRNA; NC, negative control; DDP, cisplatin.

Figure 5.

Effects of miR-139-5p on the expression of AKT, PI3K and caspase-3 mRNA in DDP-treated cells. The relative expression levels of PI3K, AKT and caspase-3 in cells were determined by reverse transcription-quantitative PCR. mRNA expression of (A) PI3K and (B) AKT were not significantly altered, whereas (C) caspase-3 was upregulated significantly in DDP-treated A549 cells after transfection with miR-139-5p mimics. **P<0.01. miR, microRNA; NC, negative control; DDP, cisplatin.

Figure 6.

Effects of miR-139-5p on the expression of p-AKT, p-PI3K and cleaved-caspase-3 protein in DDP-treated cells. Protein expression of p-AKT, p-PI3K were downregulated and cleaved-caspase-3 expression was upregulated significantly in DDP-treated A549 cells after transfection with miR-139-5p mimics. (A) The relative expression levels of p-AKT and p-PI3K in cells were determined by western blotting. (B) The relative expression levels of caspase-3 and cleaved-caspase-3 in cells were determined by western blotting. (C) Semi-quantification of p-AKT and p-PI3K expression. (D) Semi-quantification of caspase-3 and cleaved-caspase-3 expression. *P<0.05, **P<0.01, DDP + miR-139-5p mimics group vs. DDP + miR-NC mimics group, DDP group vs. control group. miR, microRNA; NC, negative control; p-, phosphorylated; DDP, cisplatin.