MicroRNA-31 predicts the presence of lymph node metastases and survival in patients with lung adenocarcinoma

Abstract

Purpose: We conducted genome-wide miRNA-sequencing (miRNA-seq) in primary cancer tissue from patients of lung adenocarcinoma to identify markers for the presence of lymph node metastasis.

Experimental design: Markers for lymph node metastasis identified by sequencing were validated in a separate cohort using quantitative PCR. After additional validation in the The Cancer Genome Atlas (TCGA) dataset, functional characterization studies were conducted in vitro.

Results: MiR-31 was upregulated in lung adenocarcinoma tissues from patients with lymph node metastases compared with those without lymph node metastases. We confirmed miR-31 to be upregulated in lymph node-positive patients in a separate patient cohort (P = 0.009, t test), and to be expressed at higher levels in adenocarcinoma tissue than in matched normal adjacent lung tissues (P < 0.0001, paired t test). MiR-31 was then validated as a marker for lymph node metastasis in an external validation cohort of 233 lung adenocarcinoma cases of the TCGA (P = 0.031, t test). In vitro functional assays showed that miR-31 increases cell migration, invasion, and proliferation in an ERK1/2 signaling-dependent manner. Notably, miR-31 was a significant predictor of survival in a multivariate cox regression model even when controlling for cancer staging. Exploratory in silico analysis showed that low expression of miR-31 is associated with excellent survival for T2N0 patients.

Conclusions: We applied miRNA-seq to study microRNomes in lung adenocarcinoma tissue samples for the first time and potentially identified a miRNA predicting the presence of lymph node metastasis and survival outcomes in patients of lung adenocarcinoma.

Figure 1

microRNA deep sequencing reveals miR-31 expression is higher in primary lung adenocarcinoma tissue from patients with lymph node metastasis (N1+) than without (N0). B miR-31 expression in 21 pairs of matched normal adjacent tissues and lung ADC tissues (paired t-test, p=2.86 E-5). C miR-31 expression in an independent cohort of 23 N0 vs. 10 N1+ lung ADC patients (t-test, p=0.009). D miR-31 expression in 140 N0 patients vs. 93 N1+ patients from the TCGA lung ADC dataset (t-test, p=0.031). The middle bar represents the mean, upper and lower border of the box represents 25^th and 75^th percentiles, and the whiskers go down to the 5^th percentile and up to the 95^th percentile.

Figure 2

Ectopic expression of miR-31 increases migration/invasion capabilities of H23 cells. (A) Expression of miR-31 following infection with Lenti-miR vector containing miR-31 precursor was confirmed by TaqMan real-time PCR. (B) Cell invasion assay for miR-31 overexpressing H23 cells. (C) Cell migration assay for miR-31 overexpressing H23 cells using transwell membranes (upper panel, representative pictures of migration chambers; bottom panel, average counts from 10 random microscopic fields). The average counts were derived from ten random microscopic fields. (D) Cell proliferation assay for miR-31 knockdown H23 cells. Data are presented as mean ± S.D. *P<0.05.

Figure 3

(A) Reduction in ERK1/2 signaling in the H1573 miR-31 knockdown cell line. (B) ERK1/2 signaling activation in the H23 miR-31 overexpression cell line. (C) ERK1/2 signaling activation in the H2228 miR-31 overexpression cell line.

Figure 4

The effect of MEK inhibitor, AZD6244, on miR31 induced cell proliferation and migration. (A) pERK1/2 expression in H23/miR31 cells after treatment with AZD6244. Cell proliferation (B) and migration (C) assay for miR31 overexpressing H23 and control cells after treatment with AZD6244.