Expression levels and co‑targets of miRNA‑126‑3p and miRNA‑126‑5p in lung adenocarcinoma tissues: Αn exploration with RT‑qPCR, microarray and bioinformatic analyses

Abstract

Lung adenocarcinoma (LUAD) is the most common histological subtype of lung cancer. Previous studies have found that many microRNAs (miRNAs), including miRNA‑126‑3p, may play a critical role in the development of LUAD. However, no study of LUAD has researched the synergistic effects and co‑targets of both miRNA‑126‑3p and miRNA‑126‑5p. The present study used real‑time quantitative polymerase chain reaction (RT‑qPCR) to explore the expression values of miRNA‑126‑3p and miRNA‑126‑5p in 101 LUAD and 101 normal lung tissues. Ten relevant microarray datasets were screened to further validate the expression levels of miRNA‑126‑3p and ‑5p in LUAD. Twelve prediction tools were employed to obtain potential targets of miRNA‑126‑3p and miRNA‑126‑5p. The results showed that both miRNA‑126‑3p and ‑5p were expressed significantly lower in LUAD. A significant positive correlation was also present between miRNA‑126‑3p and ‑5p expression in LUAD. In addition, lower expression of miRNA‑126‑3p and ‑5p was indicative of vascular invasion, lymph node metastasis (LNM), and a later tumor/node/metastasis (TNM) stage of LUAD. The authors obtained 167 targets of miRNA‑126‑3p and 212 targets of miRNA‑126‑5p; 44 targets were co‑targets of both. Eight co‑target genes (IGF2BP1, TRPM8, DUSP4, SOX11, PLOD2, LIN28A, LIN28B and SLC7A11) were initially identified as key genes in LUAD. The results of the present study indicated that the co‑regulation of miRNA‑126‑3p and miRNA‑126‑5p plays a key role in the development of LUAD, which also suggests a fail‑proof mode between miRNA‑3p and miRNA‑126‑5p.

Figure 1.

Diagnostic value of miRNA-126-3p and miRNA-126--5p for LUAD according to RT-qPCR. (A) Expression of miRNA-126-3p in 101 LUAD tissues and 101 adjacent normal lung tissues (3.511±2.118 vs. 6.674±3.362, P<0.001). (B) Expression of miRNA-126-5p in 101 LUAD tissues and 101 adjacent normal lung tissues (4.271±2.501 vs. 6.314±3.289, P<0.001). (C) ROC curve of miRNA-126-3p for LUAD based on the 101 LUAD tissues and 101 adjacent normal lung tissues (AUC=0.793, P<0.001). (D) ROC curve of miRNA-126-5p for LUAD based on the 101 LUAD tissues and 101 adjacent normal lung tissues (AUC=0.689, P<0.001). (E) Combined ROC curve of miRNA-126-3p and −5p for LUAD based on the 101 LUAD tissues and 101 adjacent normal lung tissues (AUC=0.821, P<0.001). LUAD, lung adenocarcinoma; AUC, area under the receiver operating characteristic (ROC) curve.

Figure 2.

Correlation between miRNA-126-3p and miRNA-126-5p and clinical pathological parameters based on RT-qPCR data. (A) Expression of miRNA-126-3p in early (I–II) and late (III–IV) TNM stages of LUAD (4.140±2.583 vs. 3.025±1.527, P<0.001). (B) ROC curve of miRNA-126-3p for TNM stages of LUAD (AUC=0.608, P=0.039). (C) Expression of miRNA-126-5p in samples with vascular invasion and samples without vascular invasion (3.045±1.544 vs. 4.814±2.657, P<0.001). (D) ROC cure of miRNA-126-5p for vascular invasion of LUAD (AUC=0.693, P=0.002). (E) Expression of miRNA-126-5p in samples with LNM and samples without LNM (3.671±2.090 vs. 5.142±2.713, P=0.002). (F) ROC of miRNA-126-5p for LNM of LUAD (AUC=0.672, P=0.003). (G) Expression of miRNA-126-5p in early TNM stage (I–II) and later TNM stage (III–IV) of LUAD (4.923±2.700 vs. 3.769±2.233, P=0.024). (H) ROC of miRNA-126-5p for TNM stage of LUAD (AUC=0.625, P=0.032). LUAD, lung adenocarcinoma; AUC, area under the receiver operating characteristic (ROC) curve.

Figure 3.

Forest plots of miRNA-126-3p and miRNA-126-5p based on microarray datasets. (A) Forest plot of miRNA-126-3p expression in LUAD according to 10 relevant microarray datasets and the present study. (B) Forest plot of miRNA-126-5p expression in LUAD according to 8 relevant microarray datasets and the present study. LUAD, lung adenocarcinoma; CI, confidence interval; SMD, standard mean difference.

Figure 4.

Sensitivity and specificity of the microarray datasets and the present study. Sensitivity and specificity of the 10 relevant microarray datasets and the present study according to the expression of (A) miRNA-126-3p and (B) miR-126-5p. CI, confidence interval.

Figure 5.

SROC of miRNA-126-3p and miRNA-126-5p for LUAD based on the microarrays and the present study. (A) SROC curve of miRNA-126-3p for LUAD according to the 10 relevant microarray datasets and the present study (AUC=0.97). (B) SROC curve of miRNA-126-5p for LUAD according to 8 relevant microarray datasets and the present study (AUC=0.90). LUAD, lung adenocarcinoma; SROC, summary ROC; AUC, area under the receiver operating characteristic (ROC) curve.

Figure 6.

Sensitivity analysis and publication bias of the selected microarray datasets. (A) Funnel plot of the 10 microarray datasets related to miRNA-126-3p and the present study. (B) Funnel plot of 8 microarray datasets related to miRNA-126-5p and the present study. (C) Sensitivity analysis based on the 10 microarray datasets related to miRNA-126-3p and the present study. (D) Sensitivity analysis based on 8 microarray datasets related to miRNA-126-5p and the present study. SMD, standard mean difference.

Figure 7.

Correlation between miRNA-126-3p and miRNA-126-5p based on GEO data. LUAD, lung adenocarcinoma.

Figure 8.

The process of screening the targets. LUAD, lung adenocarcinoma.

Figure 9.

PPI network based on 335 target genes of miRNA-126-3p and miRNA-126-5p. The blue nodes represent the target genes of miRNA-126-3p; the green nodes represent the target genes of miRNA-126-5p; the red nodes represent the co-regulated target genes of both miRNA-126-3p and miRNA-126-5p. The lines between the nodes represent the associations between the different target genes. PPI, protein-protein interaction.

Figure 10.

Expression levels of the eight key co-targets of miRNA-126-3p and miRNA-126-5p. The bars in the histogram with oblique lines represent LUAD tissues, and the blank bars represent normal lung tissues. All eight genes were expressed significantly higher in the LUAD tissues (***P<0.001). LUAD, lung adenocarcinoma.