Serum microRNA Signature Is Capable of Early Diagnosis for Non-Small Cell Lung Cancer

Abstract

Despite decades of efforts, non-small-cell lung cancer (NSCLC) remains the leading cause of cancer mortality globally primarily due to the challenge in early detection of the cancer. Being an important player in cancer development, the dysregulated miRNAs have been shown promising values as non-invasive diagnostic and prognostic biomarkers for NSCLC. The aim of our study is to access the efficacy and reliability of a potential circulating miRNA panel in early diagnosis of NSCLC. We first selected eight candidate miRNAs, miR-146b, miR-205, miR-29c, miR-31, miR-30b, miR-337, miR-411, and miR-708, which have been shown frequently aberrant in primary NSCLC patients based on our previous studies and other reports. The serum level of each of these miRNAs was evaluated by quantitative real-time PCR (qRT-PCR) in training and testing sets. We found that 5 out of 8 miRNAs (miR-146b, miR-205, miR-29c, miR-30b, and miR-337) were significantly up-regulated in NSCLCs patients compared to healthy or cancer-free controls in both training and testing sets. Based on the logistic regression model, a 4-miRNAs set (miR-146b, miR-205, miR-29c and miR-30b) was picked out of the 5 miRNAs owing to its excellent diagnostic power for NSCLC patients in the training set (AUC=0.99, accuracy=95.00%), the testing set (AUC=0.93, accuracy=89.69%), and the training-testing combined set ( AUC=0.96, accuracy=92.00%). When pathological subtypes of NSCLC are compared, this 4-miRNA panel carried a relatively higher prediction power and higher sensitivity for adenocarcinoma (AC) (AUC=0.98, sensitivity=99.10%) than for squamous cell carcinoma (SCC) (AUC=0.93, sensitivity=90.32%). Additionally, this panel demonstrated a comparable diagnostic capacity for stage I (AUC=0.96) and stage II-III (AUC=0.95) of NSCLC, suggesting its role in reflecting the tumor load. Importantly, the high levels of miR-146b and miR-29c in serum were significantly associated with poor 5-year overall survival (OS) (both p=0.04). Further survival analysis showed that high level of miR-146b in serum is specifically correlated with poor survival rate in SCC patients (p=0.0035) but not in AC patients (p=0.83), consistent with our previous finding that the high tissue expression of miR-146b in lung cancer specimen is indicative of a poor prognosis for SCC patients. Altogether, our study demonstrated that the 4-miRNA panel is a novel, sensitive and non-invasive serum marker for the early diagnosis of NSCLC.

Keywords: early diagnosis; miRNA; non-small-cell lung cancer (NSCLC); prognosis; serum biomarker.

Figure 1

The expression levels of 6 serum miRNAs (miR-146b, miR-205, miR-29c, miR-30b, miR-337 and miR-411), in the training set (A) and testing set (B). Mann-Whitney tests were performed to ascertain statistical significance between the expression levels across groups. Except miR-411, other five miRNAs were significantly overexpressed (p<0.05) in serum from NSCLC patients compared to controls in training and testing sets.

Figure 2

Receiver-operator characteristic (ROC) curve analyses of 5 serum miRNAs. MiR-146b, miR-205, miR-29c, miR-30b, and miR-337, is differentially expressed in NSCLC patients compared to either healthy controls in the training set (TR, shown in solid line) or cancer-free controls in the testing set (TS, shown in dotted line).

Figure 3

Receiver-operator characteristic (ROC) curve analyses of the 4-miRNA panel (miR-146b, miR-205, miR-29c and miR-30b). (A) when distinguishing NSCLC patients from either healthy controls in the training set (TR, black-colored line) or NSCLC patients from cancer-free controls in the testing set (TS, red-colored line) and in the two sets combined (TR+TS, blue-colored line); (B): distinguishing NSCLC patients with different histological subtypes: adenocarcinoma (AC, purpled-colored line) or squamous cell carcinoma (SCC, light blue-colored line) from all cancer-free controls; (C): distinguishing NSCLC patients with different stages (Stage I: green-colored line; stage II-III: blue-colored line) from all cancer-free controls.

Figure 4

Kaplan-Meier analyses of 5-year overall survival curves for NSCLC patients stratified by high (dotted line) or low (solid line) serum expression levels of miR-146b (A) and miR-29c (B). The p-values were calculated using the log-rank test between patients with high- and low- (log2) fold changes.

Figure 5

KEGG pathway analysis of miR-146 targets genes.