miR-141 and miR-200c as markers of overall survival in early stage non-small cell lung cancer adenocarcinoma

Abstract

Background: Several treatments in non-small cell lung cancer (NSCLC) are histology-dependent, and the need for histology-related markers is increasing. MicroRNAs (miRNAs) are promising molecular markers in multiple cancers and show differences in expression depending on histological subtype. The miRNA family miR-200 has been associated with the regulation of epithelial-mesenchymal (EMT)/mesenchymal-epithelial transition (MET). EMT involves profound phenotypic changes that include the loss of cell-cell adhesion, the loss of cell polarity, and the acquisition of migratory and invasive properties that facilitates metastasis. A dual role for the miR-200 family in the prognosis of several tumors has been related to tumor cell origin. However, the prognostic role and function of miR-200 family in early-stage NSCLC adenocarcinoma and squamous cell carcinoma (SCC) have not been well established.

Methods: miRNA expression was determined using TaqMan assays in 155 tumors from resected NSCLC patients. Functional studies were conducted in three NSCLC cell lines: H23, A-549 and HCC-44.

Results: High miR-200c expression was associated with shorter overall survival (OS) in the entire cohort (p = 0.024). High miR-200c (p = 0.0004) and miR-141 (p = 0.009) expression correlated with shorter OS in adenocarcinoma - but not in SCC. In the multivariate analysis, a risk score based on miR-141 and miR-200c expression emerged as an independent prognostic factor for OS in the entire cohort (OR, 2.787; p = 0.033) and in adenocarcinoma patients (OR, 10.649; p = 0.002). Functional analyses showed that miR-200c, was related to mesenchymal-epithelial transition (MET) and affected cell migration and E-cadherin levels, while overexpression of miR-141 reduced KLF6 protein levels and produced an increase of secretion of VEGFA in vitro (H23, p = 0.04; A-549, p = 0.03; HCC-44, p = 0.02) and was associated with higher blood microvessel density in patient tumor samples (p<0.001).

Conclusion: High miR-141 and miR-200c expression are associated with shorter OS in NSCLC patients with adenocarcinoma through MET and angiogenesis.

Figure 1. OS analysis in the entire cohort.

OS according to (A) miR-200c expression levels in the entire cohort (N = 155); (B) miR-200c expression levels in stage I patients (N = 94); and (C) miR-141 expression levels in stage I patients (N = 94).

Figure 2. OS analysis by histological subtype.

Overall survival according to (A) miR-200c expression levels in patients with SCC (N = 70); (B) miR-141 expression levels in patients with SCC (N = 70); (C) miR-200c expression levels in patients with adenocarcinoma (N = 73); and (D) miR-141 expression levels in patients with adenocarcinoma (N = 73).

Figure 3. OS in 73 patients with adenocarcinoma according to the miR-141/miR-200c risk score.

Figure 4. Overexpression of miR-200c affects cell migration.

(A) After 36 or 48h of transfection with pre-miRNAs in the NSCLC cell lines, cell migration was measured by in vitro scratch assay. High levels of miR-200c reduced cell migration in comparison with control in all cell lines (H23, p = 0.005; A-549, p = 0.0085; HCC-44, p = 0.013), while high levels of miR-141 reduced cell migration only in A-549 (p = 0.04). (B) E-cadherin levels were evaluated by immunohistochemistry and increased levels were observed in cells transfected with pre-miR-200c in comparison with those transfected with pre-miR-141 or pre-miR-control in all three cell lines.

Figure 5. Overexpression of miR-141 negatively regulates KLF6, leading to increased VEGFA levels in vitro, and is related to higher microvessel density in patient samples.

(A) Immunoblotting of KLF6 in cells transfected with pre-miR-Negative Control or pre-miR-141/200c. miR-141 significantly reduced KLF6 protein level in the three cell lines. (B) After 48 h of transfection with pre-miRNAs in hypoxic conditions, VEGFA concentration in the culture supernatant was measured by ELISA. All results represent the mean ± SEM from 3 independent experiments. (C) A total of 29 adenocarcinoma tumor tissue sections were analyzed by immunohistochemistry with CD34+ as vessel marker; two representative cases with high/low levels of miR-141 are shown. (D) Significant differences in blood microvessel density between adenocarcinomas with high levels of miR-141 and those with low levels of miR-141 were observed (p<0.001).