Identification of plasma microRNA-21 as a biomarker for early detection and chemosensitivity of non-small cell lung cancer

Abstract

Studies have shown cell-free microRNA (miRNA) circulating in the serum and plasma with specific expression in cancer, indicating the potential of using miRNAs as biomarkers for cancer diagnosis and therapy. This study was to investigate whether plasma miRNA-21 (miR-21) can be used as a biomarker for the early detection of non-small cell lung cancer (NSCLC) and to explore its association with clinicopathologic features and sensitivity to platinum-based chemotherapy. We used real-time RT-PCR to investigate the expression of miR-21 in the plasma of 63 NSCLC patients and 30 healthy controls and correlated the findings with early diagnosis, pathologic parameters, and treatment. Thirty-five patients (stages IIIB and IV) were evaluable for chemotherapeutic responses: 11 had partial response (PR); 24 had stable and progressive disease (SD+ PD). Plasma miR-21 was significantly higher in NSCLC patients than in age- and sex-matched controls (P < 0.001). miR-21 was related to TNM stage (P < 0.001), but not related to age, sex, smoking status, histological classification, lymph node status, and metastasis (all P > 0.05). This marker yielded a receiver operating characteristic (ROC) curve area of 0.775 (95% CI: 0.681- 0.868) with 76.2% sensitivity and 70.0% specificity. Importantly, miR-21 plasma levels in PR samples were several folds lower than that in SD plus PD samples (P = 0.049), and were close to that in healthy controls (P = 0.130). Plasma miR-21 can serve as a circulating tumor biomarker for the early diagnosis of NSCLC and is related to the sensitivity to platinum-base chemotherapy.

Figure 1.. The amplification and melting curves of miR-16 and miR-21.

A, amplification curve of miR-16; B, melting curve of miR-16; C, amplification curve of miR-21; D, melting curve of miR-21. Quantitative fluorescence amplification curves show the relationship between fluorescence and the number of cycles. The cycle threshold (Ct) is defined as the number of cycles required for the fluorescent signal to cross the threshold. Ct levels are inversely proportionate to the amount of target nucleic acid in the sample: the lower the Ct level, the greater the amount of target nucleic acid in the sample. Ct ≤ 29 indicates strong positive reactions indicative of abundant target nucleic acid in the sample. Melting curve is the quality control of amplification. It is used to analyze the homogeneity of PCR products. The results show that the Ct value of miR-16 is about 21, the Ct value of miR-21 is about 25, and the Tm is about 75°C. Impure or abnormal broaden peaks do not appear in the figure, indicating, that there is no contamination, primer dimmers, or nonspecific amplification.

Figure 2.. The correlation between the expression of miR-21 and clinical parameters of non–small cell lung cancer (NSCLC) analyzed by Mann-Whitney U test.

The expression of miR-21 is significantly higher in NSCLC than in normal control (P = 0.001), higher in stage T3–4 NSCLC than in stage T1–2 NSCLC (P = 0.036), and higher in stage III–IV NSCLC than in stage I–II NSCLC (P = 0.043).

Figure 3.. Diagnostic value of plasma miR-21 for NSCLC analyzed with receiver operating characteristics (ROC) curve.

Plasma miR-21 yielded an (area under the ROC curve (AUC) of 0.775 (95% CI: 0.681 – 0.868) with 76.2% sensitivity and 70.0% specificity. The cut-off value was 1.308.

Figure 4.. Plasma level of miR-21 in patients with partial remission (PR) or progressive disease (PD) and stable disease (SD).

Expression levels of the miR-21 (Log10 scale at Y-axis) are normalized to miR-16. Statistically significant differences were determined using the Mann-Whitney U test. Plasma level of miR-21 is significantly higher in PD plus SD samples than in PR samples (P < 0.05) and healthy control samples (P < 0.001), and is similar between PR samples and control samples. The quantity of miR-21 in plasma may be a potential biomarker for predicting platinum-based chemo-sensitivity of NSCLC.