• prognosis
• biomarkers
• NSCLC
• microRNA

Lung cancer is the most common cause of cancer-related death worldwide and non small cell lung cancer (NSCLC) accounts for about 80% of all the lung cancers. Despite potentially curative surgery and tumor markers greatly improved diagnosis, about 40% of the patients will relapse within 5 years.
Recently, a central role in cancer development has been established for microRNA. microRNA are a group of highly evolutionarily conserved non coding RNA that modulate protein expression by regulating translational efficiency or cleavage of their target mRNA. miRNA have been shown to play a key role in the regulation of known oncogenes and tumor suppressor genes and essential cellular processes, including cell proliferation, growth, differentiation, apoptosis, and angiogenesis. Moreover, aberrant microRNA expression has been shown to be associated with many types of cancers, including NSCLC.
The aim of this study was to evaluate the expression profile of microRNA Let-7g and miR-21 in a series of 80 patients with NSCLC and to correlate it with the main clinico-pathological features, mutational status of EGFR and K-Ras genes, and prognosis.
In order to evaluate the involvement of the selected microRNA in NSCLC, total RNA from 80 NSCLC patients and 27 normal lung tissues was profiled by Real-time PCR using specific TaqMan® microRNA assays for the mature form of Let-7g and miR-21.
Our data showed that the miR-21 expression was significantly higher in tumor tissues than in normal lung tissues (p<0.0001), while no significant alterations were observed for the expression of Let-7g.
To evaluate whether microRNA expression was associated with the main clinico-pathological features of the NSCLC patients, tumors were classified as low- and high-expressor tumors on the basis of the median value of the fold change. No statistical association was observed between Let-7g or miR-21 expression and the main clinico-pathological characteristics of the patients, including age at the diagnosis, gender, histology, tumor size, or smoking habit. However, although no statistically significant both Let-7g and miR-21 microRNAs were down-regulated in squamous cell carcinomas respect adenocarcinomas. Moreover, we showed a significant relationship (p=0.046) between the low Let-7g expression and the presence of positive lymph nodes at the diagnosis, sustaining an important role of Let-7g as tumor suppressor microRNA.
In order to identify the genes whose expression might be regulated by Let-7g and miR-21, we performed a bioinformatics analysis by publicly available algorithms. This analytical approach led to the identification of 24 putative target genes for Let-7g, such as HMGA2, N-RAS, ERCC6 or MAP3K3 genes, and 26 target genes for miR-21, such as PDCD4, CCL1, WWP1, MSH2 or MALT1 genes.
In our study, genomic profiling of NSCLC patients showed that EGFR and K-Ras mutations were mutually exclusive and mutation frequency was 29.5% and 20.5%, respectively. Interestingly, among the NSCLC patients high level of miR-21 expression was significantly correlated with the K-Ras status (p=0.0003), with higher expression of this microRNA in the K-Ras mutant patients than wild type ones (Fold change: 14.237±1.638 vs. 7.316±0.792).
Finally, we performed a survival analysis in order to establish whether the expression of Let-7g and miR-21 could have any impact on prognosis of the NSCLC patients treated with the tyrosine-kinase inhibitors. Survival analysis by Kaplan-Meier method showed a highly significant correlation between disease free survival or overall survival and the performance status of the NSCLC patients, the response to the treatment with the tyrosine-kinase inhibitors, and the different histotype of NSCLC, but failed to show any significant correlation with Let-7g or miR-21 expression or the main clinico-pathological features. Interestingly, when we further restricted the observation window to 30 months from diagnosis, we found that NSCLC patients with a high Let-7g and miR-21 expression showed a highly significant lower survival rate both in terms of disease free survival and overall survival.
In conclusion, our data indicate that Let-7g and miR-21 profiling in conjunction with the determination of K-Ras mutational status may be considered as a useful diagnostic and prognostic biomarker in NSCLC allowing to identify the patients at high risk of disease recurrence and poor outcome.