Overexpression of the lung cancer-prognostic miR-146b microRNAs has a minimal and negative effect on the malignant phenotype of A549 lung cancer cells

Abstract

Introduction: Expression levels of miR-146b-5p and -3p microRNAs in human non-small cell lung cancer (NSCLC) are associated with recurrence of the disease after surgery. To understand this, the effect of miR-146b overexpression was studied in A549 human lung cancer cells.

Methods: A549 cells, engineered with lentiviruses to overexpress the human pre-miR-146b precursor microRNA, were examined for proliferation, colony formation on plastic surface and in soft agar, migration and invasiveness in cell culture and in vivo in mice, chemosensitivity to cisplatin and doxorubicin, and global gene expression. miR-146b expressions were assessed in microdissected stroma and epithelia of human NSCLC tumors. Association of miR-146b-5p and -3p expression in early stage NSCLC with recurrence was analyzed.

Principal findings: A549 pre-miR-146b-overexpressors had 3-8-fold higher levels of both miR-146b microRNAs than control cells. Overexpression did not alter cellular proliferation, chemosensitivity, migration, or invasiveness; affected only 0.3% of the mRNA transcriptome; and, reduced the ability to form colonies in vitro by 25%. In human NSCLC tumors, expression of both miR-146b microRNAs was 7-10-fold higher in stroma than in cancerous epithelia, and higher miR-146b-5p but lower -3p levels were predictive of recurrence.

Conclusions: Only a minimal effect of pre-miR-146b overexpression on the malignant phenotype was seen in A549 cells. This could be because of opposing effects of miR-146b-5p and -3p overexpression as suggested by the conflicting recurrence-predictive values of the two microRNAs, or because miR-146b expression changes in non-cancerous stroma and not cancerous epithelia of tumors are responsible for the prognostic value of miR-146b.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Generation of A549-derived cell-lines.

A. Most stable mfold-predicted secondary structures of human pre-miR-146b and a variant pre-microRNA examined in this study. The 5′ and 3′ ends, nucleotide positions, and segments corresponding to mature miR-146b-5p and -3p sequences (5p and 3p) are indicated. B. Histograms for red fluorescence quantified by flow cytometry of A549 and A549-derived cell-lines stably transduced with lentiviruses bearing constructs engineered for expression of human pre-miR-146b (A549/146b), or its variant shown in A (A549/v146b), or neither (A549/vec). C. Comparison of miR-146b-5p and -3p levels (5p and 3p), normalized to that of the RNU6B small nucleolar RNA, in the A549-derived cell-lines as assessed using reverse transcription followed by PCR (RT-PCR). Means and their standard errors for measurements from three different experiments are shown. D. Standard curves showing the relationship between quantification cycle (Cq) value and concentration of RNA in miR-146b-5p and -3p (5p and 3p) RT-PCR assays of synthetic miR-146b-5p and -3p RNA, respectively. A log2 scale is used for the X axis. E. Semi-quantification of the relative amounts of miR-146b-5p and -3p in RNA of 293T, BEAS-2B, MCF-7, and ML-2 (means, single experiments), and the three A549-derived cell-lines, (means and their standard errors for measurements from three different experiments) are shown.

Figure 2. Proliferation of the A549-derived cell-lines.

A. Cell density was indirectly assessed by a colorimetric assay for cell viability. Absorbance values at 450 nm with increasing hours of cell culture are shown. B. Effect on cell density following treatment with 0.5, 1.0 or 2.0 ug/ml cisplatin (Cis.) or doxorubicin (Dox.) for 2 days. Absorbance values at 450 nm obtained with a colorimetric cell viability assay are shown. C, D. Number and average areas of colonies seen in adherent (C) or non-adherent (D) culture on tissue culture plates or in soft agar, respectively. Two hundred (C) or 150 (D) cells were seeded per culture and colonies were quantified after two (C) or six (D) weeks. Means and their standard errors for triplicates (A, C, D) or quintuplicates (B), from one of three experiments with similar results for A, C and D, are shown.

Figure 3. Migration and invasion assays of the A549-derived cell-lines.

A. Migration of cells in an in vitro wound-healing assay is depicted as a reduction in the width of a scratch across a monolayer of cells after a day of culture. B. Migration of serum-starved cells towards 15% serum-containing medium through an 8 µm pore-sized polycarbonate membrane after 8 hours of culture. Migrated cells were fixed and stained with crystal violet, and indirectly quantified by measuring absorbance of extracts of cell stains at 595 nm. C. Like B, but with membranes coated with a murine basement membrane extract (BME) or rat collagen I, and culture for 16 hours. Migrated cells were imaged by red fluorescence microscopy at 5x magnification. Means and their standard errors for triplicates from one of three experiments with similar results are shown.

Figure 4. Lung colony formation by the A549-derived cell-lines, and miR-146b expressions in tumor stroma and epithelia.

A. Generation of pulmonary tumors by intravenous injection of the A549-derived cell-lines. A million cells per animal were injected in the tail veins of young female immunodeficient mice. After 12 weeks, animals were dissected and both of their lungs weighed. Five mice were used for each cell-line. B. Relative expression of let-7e, miR-30a-5p, and miR-146b-5p and -3p microRNAs in stromal and cancerous epithelial components of stage I non-small cell lung cancer as assessed using reverse transcription followed by PCR. Stroma and epithelia were microdissected using tumor sections from 12 cases of the disease. Levels are normalized to that of the RNU6B small nucleolar RNA. Means and their standard errors are shown. MicroRNA miR-146b-3p was detectable in both compartments of only 11 tumors. Only four tumors were used for let-7e quantification.

Figure 5. Expression of miR-146b-5p and -3p in a set of 77 cases of stage I non-small cell lung cancer.

A. Scatter-plots of microarray signal intensities, relative to that for a reference RNA, for miR-146b-5p or -3p, or their ratio, for RNA from resected tumor tissue from 37 cases with recurrence of the disease, and for 40 cases without recurrence during the ≥32 months of follow-up following surgery . Means and their standard errors are shown. B. Kaplan-Meier plots depicting post-resection recurrence-free survival among the case-groups with values of miR-146b-5p or -3p, or their ratio, lower (low, grey line) or higher (high) than the group median. Hazard ratios (HR) and time in months to recurrence following surgery are shown. Tick-marks along the lines indicate right-censored data-points.