Diagnostic and prognostic value of serum miR-145 and vascular endothelial growth factor in non-small cell lung cancer.

Previous studies have reported the diagnostic and prognostic value of serum microRNA (miR)-145 and vascular endothelial growth factor (VEGF) levels in various types of cancer; however, their clinical use in non-small cell lung cancer (NSCLC) remains unclear. The present study included 215 patients, 106 with NSCLC and 109 with other lung diseases (OLDs). miR-145 expression levels were determined using reverse transcription-quantitative PCR (RT-qPCR) and VEGF levels were measured using an ELISA. The diagnostic performance was assessed using a receiver operating characteristic curve and area under the curve (AUC) analysis. A Kaplan-Meier survival curve and Cox regression analysis were employed to evaluate the prognostic significance of the markers. The biological function of miR-145 was examined in A549 and H1792 cell lines. The effects of miR-145 on cell proliferation of NSCLC cells were evaluated by flow cytometry, and the expression levels of miR-145 and cell cycle-related genes were determined by RT-qPCR. The results revealed that miR-145 and VEGF exhibited fair diagnostic performance [AUC, 0.61 (95% CI, 0.55-0.68) and AUC, 0.64 (95% CI, 0.57-0.71), respectively]. Combining age and smoking status with miR-145 and VEGF provided the best model for differentiating patients with NSCLC from those with OLDs (AUC, 0.76; 95% CI, 0.69-0.83). Furthermore, low serum miR-145 levels were associated with poor overall survival [hazard ratio (HR), 0.48; 95% CI, 0.27-0.85], whereas high VEGF levels were not associated with poor overall survival (HR, 1.47; 95% CI, 0.81-2.68). In addition, the results of the in vitro experiments indicated that miR-145 decreased cell proliferation via the induction of cell cycle arrest. In conclusion, the findings of the present study suggested that combining miR-145 and VEGF levels with clinical risk factors may be a potential diagnostic scheme for NSCLC. In addition, serum miR-145 may be used as a prognostic marker. These results indicated that miR-145 may function as a tumor suppressor in NSCLC.

Serum miR-339-3p as a potential diagnostic marker for non-small cell lung cancer.

Objective: MicroRNA (miRNA), a short noncoding RNA, is claimed to be a potential blood-based biomarker. We aimed to identify and evaluate miRNAs as diagnostic biomarkers for non-small cell lung cancer (NSCLC). Methods: Profiles of 745 miRNAs were screened in the serum of 8 patients with NSCLC and 8 age- and sex-matched controls using TaqMan low-density arrays (TLDAs) and validated in 25 patients with NSCLC and 30 with other lung diseases (OLs) as well as in 19 healthy persons (HPs). The diagnostic performance of the candidate miRNAs was assessed in 117 cases of NSCLC and 113 OLs using quantitative real-time polymerase chain reaction (qRT-PCR). Differences in miRNA expression between patients with NSCLC and controls were assessed using the Mann-Whitney U test. The area under receiver operating characteristic (ROC) curve (AUC) was obtained based on the logistic regression model. Results: Ten miRNAs were found to be differentially expressed between patients with NSCLC and controls, including miR-769, miR-339-3p, miR-339-5p, miR-519a, miR-1238, miR-99a#, miR-134, miR-604, miR-539, and miR-342. The expression of miR-339-3p was significantly higher in patients with NSCLC than in those with OLs (P < 0.001) and HPs (P = 0.020). ROC analysis revealed an miR-339-3p expression AUC of 0.616 [95% confidence interval (CI): 0.561-0.702]. The diagnostic prediction was increased (AUC = 0.706, 95% CI: 0.649-0.779) in the model combining miR-339-3p expression and other known risk factors (i.e., age, smoking status, and drinking status). Conclusions: MiR-339-3p was significantly upregulated in patients with NSCLC compared with participants without cancer, suggesting a diagnostic prediction value for high-risk individuals. Therefore, miR-339-3p expression could be a potential blood-based biomarker for NSCLC.

MicroRNA Isolation by Trizol-Based Method and Its Stability in Stored Serum and cDNA Derivatives.

MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression at the post-transcriptional level. Since aberrant expression of miRNAs has been proposed as usage for blood-based biomarkers, hence reliable techniques for miRNA isolation as well as stability of miRNAs in various stored conditions needs to be explored. This present study aimed to investigate the efficacy of the Trizol-based isolation technique and the stability of miRNAs in stored serum and cDNA derivatives. Total RNA, including miRNAs, was isolated from human serum and a comparison of the efficiency of the Trizol®LS reagent isolation method against the miRNeasy®mini kit was conducted. Expression of RNU6, miR-145, and miR-20a was determined by quantitative real-time polymerase chain reaction (qRT-PCR). We showed that Trizol®LS isolation yielded significantly lower RNA concentrations than that of the miRNeasy®mini kit by approximately 35%. Purity of the isolated RNAs by both methods was similar. RNU6, miR-145, and miR-20a degraded at room temperature, but all genes were stable at 4ºC, -20ºC and -80ºC for a 72-hrs period, in both serum and cDNA storage conditions. In the stored cDNA derivatives, we observed the stability of RNU6, miR-145, and miR-20a for 3 months at -20ºC, and all genes also resisted 4 repeated freeze-thaw cycles at -20ºC. In conclusion, the Trizol-based method is efficient as well as economical to use for quantification of circulating miRNAs. In addition, we proposed that the storage of miRNA-derived cDNAs may be an alternative choice to avoid the stability effect.