Serum MicroRNA Expression Profiling Revealing Potential Diagnostic Biomarkers for Lung Adenocarcinoma
Lung cancer remains the most lethal disease worldwide, with non-small cell lung cancer (NSCLC) accounting for 80% to 85% of cases. Among NSCLC subtypes, lung adenocarcinoma (LA) is the most common. Early detection of LA is crucial for improving patient outcomes, as surgical resection in early-stage patients can yield a 5-year survival rate of 70% to 90%. However, approximately 75% of patients are diagnosed at advanced stages (III/IV) due to the lack of early symptoms. Current diagnostic tools, such as low-dose computed tomography (LDCT), are limited by radiation exposure, high misdiagnosis rates, and cost. Protein biomarkers like carcinoembryonic antigen (CEA) and cytokeratin-19-fragment (CYFRA21-1) also suffer from limited sensitivity and specificity. Therefore, there is an urgent need for non-invasive biomarkers with high diagnostic accuracy for LA.
MicroRNAs (miRNAs) are small, highly conserved non-coding RNAs that regulate gene expression by inhibiting translation or degrading messenger RNA targets. Circulating miRNAs have emerged as promising non-invasive biomarkers for various cancers, including NSCLC. However, inconsistencies in previous studies highlight the need for further research. This study aimed to identify serum miRNAs as potential diagnostic biomarkers for LA through a four-stage investigation.
The study was conducted between 2016 and 2017, enrolling 170 LA patients and 170 healthy controls (NCs). The experimental design included four phases: screening, training, testing, and external validation. In the screening phase, miRNA profiles were obtained from pooled serum samples using the Exiqon miRNA qPCR panel. Thirty-five dysregulated miRNAs were identified and further evaluated in the training and testing phases using quantitative real-time polymerase chain reaction (qRT-PCR). The diagnostic value of the identified miRNAs was validated in an external cohort.
In the screening phase, 35 miRNAs were selected as candidates based on their differential expression in pooled serum samples. The training phase involved qRT-PCR analysis of 24 LA patients and 24 NCs, narrowing the candidates to nine miRNAs. In the testing phase, these nine miRNAs were evaluated in 110 LA patients and 110 NCs, revealing four miRNAs (miR-133a-3p, miR-584-5p, miR-10b-5p, and miR-221-3p) that were significantly overexpressed in LA patients. These findings were further validated in an external cohort of 36 LA patients and 36 NCs.
The diagnostic accuracy of the four-miRNA panel was assessed using receiver operating characteristic (ROC) curves. The areas under the ROC curve (AUC) for the training, testing, and external validation stages were 0.734, 0.803, and 0.894, respectively. The combined AUC for the four-miRNA panel was 0.765, with a sensitivity of 66.5% and specificity of 76.5%. These results indicate that the four-miRNA panel has strong diagnostic potential for LA.
To explore the origin of the identified miRNAs, their expression levels were examined in 24 paired LA tumor tissues and adjacent normal tissues. Only miR-221-3p showed significant up-regulation in tumor tissues, suggesting it may play a direct role in LA pathogenesis. The other three miRNAs (miR-133a-3p, miR-584-5p, and miR-10b-5p) did not exhibit significant differences in tissue samples, indicating they may be selectively released into the serum rather than being directly involved in tumorigenesis.
The study also investigated the expression of the four miRNAs in serum-derived exosomes. Exosomes are nano-sized vesicles that protect miRNAs from degradation and are secreted by most cell types, including cancer cells. Among the four miRNAs, only miR-10b-5p was significantly up-regulated in exosomes from LA patients. This finding aligns with previous studies suggesting that miR-10b-5p plays a role in promoting tumor progression and metastasis.
Additionally, the study explored the relationship between miRNA expression and epidermal growth factor receptor (EGFR) mutation status, a critical factor in LA treatment. The expression levels of miR-133a-3p, miR-584-5p, and miR-10b-5p were significantly elevated in LA patients with EGFR mutations compared to NCs. The AUC for the three-miRNA panel in distinguishing EGFR-mutant LA patients from NCs was 0.855, with a sensitivity of 77.8% and specificity of 92.31%. These results suggest that these miRNAs may serve as supplementary biomarkers for identifying LA patients with EGFR mutations, who are likely to benefit from targeted therapy.
Bioinformatics analysis was conducted to identify potential target genes and pathways associated with the four miRNAs. The analysis revealed that miR-10b-5p and miR-221-3p are closely linked to cancer-related pathways, such as the p53 signaling pathway. These findings provide insights into the molecular mechanisms underlying the roles of these miRNAs in LA.
The study has several limitations. First, it focused on only two types of EGFR mutations (exon 19 deletions and exon 21 mutations), excluding other sensitizing mutations like T790M and ALK rearrangements. Second, the study population was limited to Chinese individuals, and further research is needed to validate the findings in diverse ethnic groups. Third, the sample size was relatively small, and larger prospective studies are required to confirm the results. Finally, the relationship between tissue miRNA expression and EGFR status was not explored, warranting further investigation.
In conclusion, this study identified a four-miRNA signature in serum that can distinguish LA patients from healthy controls with high diagnostic accuracy. The miRNAs miR-133a-3p, miR-584-5p, miR-10b-5p, and miR-221-3p were significantly overexpressed in LA patients and demonstrated strong diagnostic potential. The findings also highlight the potential roles of these miRNAs in LA pathogenesis, particularly in relation to EGFR mutation status. Further research is needed to explore the functional mechanisms of these miRNAs and their clinical applications in LA diagnosis and treatment.
doi.org/10.1097/CM9.0000000000001100
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