miR-1260b Activates Wnt Signaling by Targeting Secreted Frizzled-Related Protein 1 to Regulate Taxane Resistance in Lung Adenocarcinoma

Abstract

Objectives: MicroRNAs (miRNAs) have been demonstrated to contribute to carcinogenesis; however, their association with tumor chemoresistance is not fully understood. In this study we aimed to investigate the molecular mechanisms involved in resistance to taxane-based chemotherapy in lung adenocarcinoma (LAD). Methods: We established paclitaxel-resistant A549 cells (A549/PTX) and docetaxel-resistant H1299 cells (H1299/DTX). In order to hit the mark, we employed multiple methods including qRT-PCR, western blotting analysis, loss/gain-of-function analysis, luciferase assays, drug sensitivity assays, animal experiment, wound-healing assay, and invasion assay. Results: Bioinformatics analysis and a luciferase reporter assay revealed that secreted frizzled-related protein 1 (SFRP1) is a direct target of miR-1260b. By qRT-PCR analysis, we found that miR-1260b was significantly upregulated in taxane-resistant cells as compared to parental cells. Suppression of miR-1260b reversed the chemoresistance of human LAD cells to taxanes both in vitro and in vivo, whereas ectopic miR-1260b expression decreased the sensitivity of parental LAD cell lines to taxanes. Downregulation of miR-1260b expression inactivated the Wnt signaling pathway and reversed the epithelial-mesenchymal transition (EMT) phenotype of taxane-resistant LAD cells. In clinical tumor tissue samples, high miR-1260b expression was detected in tumors of non-responding patients treated with taxane-based chemotherapy and was associated with low SFRP1 expression and poor prognosis. Conclusions: Our findings reveal that targeting of the miR-1260b/SFRP1/Wnt signaling axis might provide a novel strategy for overcoming chemotherapy resistance in LAD.

Keywords: SFRP1; Wnt signaling; chemoresistance; lung adenocarcinoma; miR-1260; taxane.

Figure 1

Different sensitivity to paclitaxel and docetaxel between A549/PTX cells and parental A549 cells and SFRP1 was a direct target of miR-1260b in LAD cells. (A) IC₅₀ values for paclitaxel (left) and docetaxel (right) in A549 and A549/PTX cells as determined by MTT assays. (B) Proliferation ability of A549 and A549/PTX cells as determined by colony formation assays. (C) Cell cycle analysis of A549 and A549/PTX cells by flow cytometry. (D) Consensus sequences for miR-1260b in the SFRP1 3′-UTR were predicted by bioinformatics analysis. (E) A reporter vector containing the SFRP1 3′-UTR (wild-type or mutant constructs) was cotransfected with miR-1260b mimic in A549 cells, which were then analyzed using a dual-luciferase reporter assay. (F) SFRP1 protein levels in parental and taxane-resistant cells after transfection with miR-1260b mimic or inhibitor as determined by western blotting. (G) Expression of miR-1260b in A549/PTX and H1299/DTX cells compared with A549 and H1299 cells as indicated by qRT-PCR. miRNA abundance was normalized to the U6 RNA level. The experiment was repeated at least three times. Student t-test was employed to compare the differences between two groups. The data are represented as mean ± SD. **p < 0.01 compared with control group.

Figure 2

Inhibition of miR-1260b reversed the in vitro chemoresistance of A549/PTX and H1299/DTX cells to paclitaxel or docetaxel. (A) Expression levels of miR-1260b in A549/PTX and H1299/DTX cells transfected with mimic NC or miR-1260b inhibitor as determined by qRT-PCR. IC₅₀ values for paclitaxel or docetaxel in A549/PTX (B) and H1299/DTX (C) cells transfected with mimic NC or miR-1260b inhibitor as determined by MTT assays. (D) Colony formation assays revealed proliferation ability of A549/PTX (upper) and H1299/DTX (lower) cells transfected with miR-1260b inhibitor, following treatment of paclitaxel (200 μg/L) or docetaxel (50 μg/L), respectively. Flow cytometric analysis indicated cell cycle (E) and apoptosis (F) in A549/PTX and H1299/DTX cells transfected with mimic NC or miR-1260b inhibitor, following treatment of paclitaxel (200 μg/L) or docetaxel (50 μg/L), respectively. Data between two groups were tested using independent sample t-test. The data are based on 3 to 5 independent experiments and are shown as mean ± SD. **p < 0.01 compared with NC group.

Figure 3

Ectopic miR-1260b expression decreased the chemosensitivity of A549 and H1299 cells to paclitaxel or docetaxel. (A) Expression levels of miR-1260b in A549 and H1299 cells transfected with mimic NC or miR-1260b mimic were determined by qRT-PCR assay. IC₅₀ values for paclitaxel or docetaxel in A549 (B) and H1299 (C) cells transfected with mimic NC or miR-1260b mimic as determined by MTT assays. (D) Colony formation assays revealed proliferation ability of A549 (upper) and H1299 (lower) cells transfected with mimic NC or miR-1260b mimic following treatment of paclitaxel (20 μg/L) or docetaxel (5 μg/L), respectively. (E) Flow cytometry indicated cell cycle of A549 (upper) and H1299 (lower) cells transfected with mimic NC or miR-1260b mimic following treatment of paclitaxel (20 μg/L) or docetaxel (5 μg/L), respectively. Paired t-test was applied to compare the differences between two groups. Similar results were obtained in 3 independent experiments and are shown as mean ± SD. *p < 0.05; **p < 0.01 compared with NC group.

Figure 4

Inhibition of miR-1260b increased the chemosensitivity of A549/PTX and H1299/DTX cells to taxanes in vivo. Nude mice were subcutaneously injected with A549/PTX or H1299/DTX cells that had been infected with lentivirus carrying miR-1260b inhibitor and treated with paclitaxel (15 mg/kg) or docetaxel (1 mg/kg) when the mean tumor volume reached ~100 mm³. (A) Representative picture of tumors from A549/PTX/Lv-NC or A549/PTX/Lv-anti-miR1260b group with paclitaxel treatment. (B) Representative picture of tumors from H1299/DTX/Lv-NC or H1299/DTX/Lv-anti-miR1260b group with docetaxel treatment. (C) Growth curve of tumors derived from A549/PTX/Lv-NC or A549/PTX/Lv-anti-miR1260b cells. (D) Growth curve of tumors derived from H1299/DTX/Lv-NC or H1299/DTX/Lv-anti-miR1260b cells. Paired t-test was performed for comparisons between two groups. Each data point represents the mean ± SD of 7 mice. The three independent experiments gave similar results. *p < 0.05; **p < 0.01 compared with Lv-NC group. (E) H&E- (upper) and PCNA-stained (lower) sections of the transplanted tumors from A549/PTX/Lv-NC or A549/PTX/Lv-anti-miR1260b cells upon paclitaxel treatment. (F) H&E- (upper) and PCNA-stained (lower) sections of the transplanted tumors from H1299/DTX/Lv-NC or H1299/DTX/Lv-anti-miR1260b cells upon docetaxel treatment. Photomicrographs were taken at 400x magnification.

Figure 5

miR-1260b affected Wnt signaling activity by directly targeting SFRP1 in LAD Cells. (A) mRNA expression levels of β-catenin, cyclin D1, and c-myc in A549/PTX and H1299/DTX cells transfected with miR-NC or miR-1260b inhibitor as determined by qRT-PCR. (B) Protein expression levels of p-GSK3β, GSK3β, β-catenin, cyclin D1, and c-myc in A549/PTX and H1299/DTX cells transfected with miR-NC or miR-1260b inhibitor as determined by western blotting. (C) TCF/LEF reporter assay to assess the effect of miR-1260b on Wnt signaling activity in A549 (left) and H1299 (right) cells. Data are represented as mean ± SD of three independent experiments (Student t-test). *p < 0.05; **p < 0.01.

Figure 6

Inhibition of miR-1260b reversed the EMT phenotype and suppressed the invasive ability of A549/PTX cell line. (A) The phenotype of A549, A549/PTX, A549/PTX/AmiR-NC, A549/PTX/AmiR-1260b cells. Photomicrographs were taken at 200x magnification. (B) mRNA and protein expression levels of E-cadherin, N-cadherin, and vimentin in A549/PTX/AmiR-NC and A549/PTX/AmiR-1260b cells as determined by qRT-PCR (left) and western blotting (right). Cell migration and invasion capacities of A549/PTX/AmiR-NC and A549/PTX/AmiR-1260b cells as determined by wound healing (C) and invasion (D) assays, respectively. Photomicrographs were taken at 200x magnification. (E) Eight weeks after tail vein injection of A549/PTX/Lv-NC or A549/PTX/Lv-anti-miR1260b cells, metastatic potential was determined by H&E staining. (F) Human-specific GAPDH (hGAPDH) expression relative to mouse-specific GAPDH (mGAPDH) in circulating tumor cells as determined by qRT-PCR. (G) Human-miR-1260b (hmiR-1260b) expression relative to mouse-miR-1260b (mmiR-1260b) in circulating tumor cells as determined by qRT-PCR. The data are represented as mean ± SD of triplicate samples performed in two independent samples (Student t-test). *p < 0.05; **p < 0.01 compared with control group.

Figure 7

miR-1260b was associated with SFRP1 expression and poor prognosis in LAD tumor tissue samples. (A) mRNA expression levels of miR-1260b (left) and SFRP1 (right) in sensitive (n = 18) and insensitive (n = 20) LAD tissues as detected by qRT-PCR. U6 RNA and GAPDH were used as internal controls. The data express as the mean ± SD (Student t-test). **p < 0.01. (B) Linear regression analysis revealed an inverse correlation between miR-1260b and SFRP1. (C) Kaplan–Meier survival analysis of LAD patients according to the level of miR-1260b expression in tumor tissues. The p-value was determined with the log-rank test.

Figure 8

Schematic diagram for miR-1260b modulation of chemoresistance in NSCLC. miR-1260b regulates chemoresistance by targeting SFRP1 and thereby activating Wnt signaling.