Discovery and validation of extracellular vesicle-associated miRNAs as noninvasive detection biomarkers for early-stage non-small-cell lung cancer

Abstract

miRNAs in circulating extracellular vesicles (EVs) are promising biomarkers for cancer. However, their diagnostic ability for early-stage non-small-cell lung cancer (NSCLC) is not well known. In this study, the circulating EV miRNAs profiling was initially performed in 36 untreated NSCLC patients and 36 healthy controls by TaqMan Low Density Array (TLDA). Subsequently, we performed quantitative reverse-transcription PCR assay (RT-qPCR) validation in several independent cohorts that included 159 NSCLC patients, 120 age/sex-matched healthy controls and 31 benign nodule patients enrolled from three different clinical centres. In addition, 38 cases of NSCLC were analysed before and after surgery. We demonstrated that miR-520c-3p and miR-1274b were significantly and steadily increased in NSCLC patients in comparison with healthy controls and benign nodule patients (P < 0.001) and decreased markedly after tumour resection (P < 0.001). The areas under the curve (AUCs) of the ROC curve of the two-miRNA panel were 0.857 (95% CI, 0813-0.901; P < 0.0001) and 0.845 (95% CI, 0.793-0.896; P < 0.0001) for NSCLC and NSCLC stage I, respectively. Furthermore, the panel was able to differentiate NSCLC from benign nodules with an AUC of 0.823 (95% CI, 0.730-0.915; P < 0.0001). Furthermore, logistic regression analysis revealed the two-miRNA panel as an independent risk factor for NSCLC (OR = 16.128, P < 0.0001). In conclusion, miR-520c-3p and miR-1274b have biomarker potential for early diagnosis of NSCLC in multiple centres.

Keywords: biomarker; early diagnosis; circulating miRNAs; extracellular vesicles; non-small-cell lung cancer.

Fig. 1

Characterization of EVs derived from the serum and plasma of NSCLC patients and controls. (A) The shape and structure of serum and plasma EVs isolated by EXOquick kit under TEM. The red arrow represents EVs with typical characteristics (scale bars are 200 nm). (B) The size of EVs derived from control groups and NSCLC groups was analysed by NTA. (C) Western blots of EVs membrane markers, including Alix, CD63, TSG101, CD9 and one negative marker ALB.

Fig. 2

Heat map describing the markedly upregulated EV miRNAs from TLDA. EVs were derived from 3 pooled serum samples of NSCLC patients and 3 pooled serum samples of controls. Each pool was consisted of 12 individual samples. Heatmap of pairwise correlation values of the 6 samples. 37 miRNAs were upregulated and 33 were downregulated for more than 2‐fold.

Fig. 3

qPCR data on miRNA expression levels of selected miRNAs of serum and plasma in the screening set(n_NSCLC = 19, n_control = 19, A–J. The expression of miR‐15b (A), miR‐34a (B), miR‐720 (C), miR‐193b (D), miR‐212 (E), miR‐203 (F), miR‐206 (G), miR‐31 (H), miR‐1274b (I) and miR‐520c‐3p (J) was normalized by cel‐miR‐39. miR‐520c‐3p and miR‐1274b uniformly consistently elevated in EVs from both plasma and serum. Horizontal bars represent the mean value. p‐values were measured by Mann–Whitney independent t‐test. *P < 0.05.

Fig. 4

Expression levels of miR‐520c‐3p and miR‐1274b in the three sets from different hospitals (A–F). The relative expression of miR‐520c‐3p and miR‐1274b in training set (n _NSCLC = 41, n _control = 41), validation set (n _NSCLC = 30, n _control = 29) and testing set (n _NSCLC = 31, n _control = 31) calculated by Delta Ct method. Horizontal bars represent the mean. The two miRNAs in the three independent sets were uniformly significantly increased in the NSCLC patients. P‐values were measured by Mann–Whitney independent t‐test. ***P < 0.001.

Fig. 5

Expression levels of miR‐520c‐3p (A) and miR‐1274b (B) between normal controls, benign nodules patients and NSCLC patients. The relative expression of miR‐520c‐3p (A) and miR‐1274b (B) was detected in healthy groups (n = 120), benign nodules patients (n = 31) and NSCLC patients (n = 159). The level of two miRNAs was elevated significantly in NSCLC patients, but there is no difference between healthy groups and benign nodules patients. P‐values were measured by Mann–Whitney independent t‐test. ***P < 0.001.

Fig. 6

Diagnostic values of miRNAs by qPCR. The ROC and AUC were used to determine the sensitivity and specificity of select miRNAs. (A) ROC curve for the two‐miRNA panel to distinguish 159 NSCLC cases from 120 normal controls in all sets (black line, AUC = 0.857), and ROC curve for the two‐miRNA panel to distinguish 159 malignant lesion patients from 31 benign nodule patients (blue line, AUC = 0.823). (B) ROC curve for the two‐miRNA panel to distinguish 96 clinical stage I cases from 120 controls in all sets (purple line, AUC = 0.845) and ROC curve for the two‐miRNA panel to distinguish 61 stage II–IV cases from 120 controls (orange line, AUC = 0.881).

Fig. 7

Expression levels of miR‐520c‐3p and miR‐1274b before and after operations in 38 NSCLC patients. The relative expression of miR‐520c‐3p (A) and miR‐1274b (B) were tested before and after operations in NSCLC patients (n = 38). The expression levels of the two miRNAs were markedly decreased after surgery. P‐values were measured by paired t‐test. ***P < 0.001.

Fig. 8

Expression levels of miR‐520c‐3p and miR‐1274b in serum EVs and EV‐free fraction between normal controls and NSCLC patients. The concentrations of miR‐520c‐3p and miR‐1274b in serum EVs and EV‐free fraction were detected in healthy groups (n = 12), and NSCLC patients (n = 12). EV and EV‐free fraction samples were obtained by ultracentrifugation. P‐values were measured by Mann–Whitney independent t‐test. *P < 0.05, **P < 0.01.

Fig. 9

GO/KEGG enrichment analysis result with a fold change > 2. GO/KEGG enrichment of mRNAs targeted by the two miRNAs. BP biological process, CC cellular component, MF molecular function. (A) A bubble plot of enriched KEGG pathways (target genes of miR‐520c‐3p). (B) A bubble plot of enriched KEGG pathways (target genes of miR‐1274b). (C) A bubble plot of KEGG pathways (co‐target genes of miR‐520c‐3p and miR‐1274b). (D) A bar plot of GO terms (target genes of miR‐520c‐3p). (E) A bar plot of GO terms (target genes of miR‐1274b). (F) A bar plot of GO terms (co‐target genes of miR‐520c‐3p and miR‐1274b).