Mirella Giordano

P2X7 mRNA expression in non‑small cell lung cancer: MicroRNA regulation and prognostic value

The human P2X7 receptor is significant and exhibits several functions in neoplasia. At present, little is known with regard to its regulation. P2X7 expression may be regulated post-transcriptionally and putative microRNA (miRNA) binding sites are considered to be involved. The aim of this study was to determine whether miRNAs (miR-21, let-7 g and miR-205) regulate P2X7 mRNA stability. In addition, the impact of P2X7 expression in patients with non-small cell lung cancer (NSCLC) was investigated. P2X7 mRNA and mature Let-7 g, miR-21, and miR-205 expression levels were quantified in 96 NSCLC cases using quantitative reverse transcription polymerase chain reaction. In all samples, epidermal growth factor receptor and K-Ras mutational analysis was also performed. Samples with low P2X7 expression were found to exhibit a higher fold change in miR-21 expression when compared with samples exhibiting high P2X7 expression. Significantly higher miR-21 expression was observed in the tumors of NSCLC patients with a K-Ras mutation when compared with patients who had K-Ras wild-type tumors (P=0.003). Additionally, to evaluate the association between P2X7 expression and prognosis in NSCLC patients, survival analysis was performed using the Kaplan-Meier method. A significant difference in the progression-free survival and overall survival in the NSCLC patients with high P2X7 expression was identified, when compared with that of patients with low expression (P=0.03 and P=0.02, respetively). Therefore, we hypothesized that high levels of miR-21 expression in NSCLC patients with K-Ras mutations may be regulated by a complex circuit, including P2X7 downregulation and together these processes may promote tumor progression.

Role of gene expression profiling in defining indeterminate thyroid nodules in addition to BRAF analysis.

Fine‐needle aspiration (FNA) is routinely used in the preoperative evaluation of thyroid nodules. However, 15% to 30% of aspirations yield indeterminate cytologic findings. Because the assessment of BRAF mutations seems to improve the diagnostic accuracy, this study evaluated BRAF mutations with Sanger sequencing and real‐time methods in 650 consecutive thyroid aspirates. In addition, the expression of a large number of genes involved in basement membrane remodeling, extracellular matrix proteolysis, and cell adhesion was studied in both benign and malignant nodules to identify new diagnostic tools. In this prospective series, despite the use of a very sensitive BRAF mutational testing method, the frequency of a BRAF alteration being identified in indeterminate FNA samples was 3 of 68. Expression analysis revealed several genes that were differentially expressed between benign and malignant nodules (transforming growth factor, cadherin 1, collagen α1, catenin α1, integrin α3, and fibronectin 1 [FN1]), between follicular adenomas and follicular variant of papillary thyroid carcinoma (FN1, laminin γ1, integrin β2, connective tissue growth factor, catenin δ1, and integrin αV), and between BRAF–wild‐type and BRAF‐mutated papillary thyroid carcinomas (TIMP metallopeptidase inhibitor 1; catenin α1; secreted phosphoprotein 1; FN1; ADAM metallopeptidase with thrombospondin type 1 motif, 1; and selectin L). These data were partially confirmed with real‐time polymerase chain reaction analysis and immunohistochemistry. When the cost/benefit ratio of the procedures was taken into account, BRAF mutational testing failed to increase diagnostic accuracy in cytologically indeterminate nodules. However, the additional analysis of the expression of specific molecular markers could have possible utility as a diagnostic tool, although further evidence based on a large series of samples is needed before definitive conclusions can be drawn. Cancer Cytopathol 2016;124:340–9.

P2X7 protein expression and polymorphism in non-small cell lung cancer (NSCLC)

BackgroundP2X7, a purinergic receptor, plays important roles in inflammatory diseases, but recently its expression has been found in several tumors, suggesting a potential role as a cancer cell biomarker. Moreover, the relative amount of P2X7 varies among human individuals due to numerous single nucleotide polymorphisms resulting in either a loss- or gain-of-function; the P2X7 gene is highly polymorphic, and polymorphisms in the promoter or coding region may modify its expression or function. A polymorphism in exon 13 of the P2X7 receptor gene at the +1513 position (Glu496Ala substitution, corresponding to SNP rs3751143) has been shown to eradicate the function of this receptor and has been correlated with histological variants and clinical parameters in thyroid cancer. Until now, no data regarding P2X7 expression and polymorphisms in lung cancer have been published; based on these premises, we decided to evaluate the impact of the P2X7 expression and polymorphisms in ninety-seven cases of non-small cell lung cancer (NSCLC).ResultsNo significant difference in the genotype frequency of the A1513C polymorphism was found between the two histological variants of NSCLC, adenocarcinoma and squamous cell carcinoma, and no statistically significant associations were observed between P2X7 protein expression and the main clinico-pathological characteristics of the NSCLC patients.ConclusionsBased on our results, P2X7 expression and polymorphisms seem to have no potential impact in patients with non-small cell lung cancer; however, further studies will surely provide deeper insights regarding the role of this receptor at the clinical level in NSCLC.

miR-19a and miR-20a and Tissue Factor Expression in Activated Human Peripheral Blood Mononuclear Cells

Background and Aims To investigate the behaviour of miR-19a and miR-20a, two microRNAs involved in posttranscriptional modulation of TF expression in peripheral blood mononuclear cells (PBMCs) exposed to high glucose (HG) and lipopolysaccharide (LPS), and to evaluate the involvement of angiotensin II in that process. Methods TF Procoagulant Activity (PCA, one-stage clotting assay), antigen (Ag, ELISA), and miR-19a and miR-20a levels (specific TaqMan® MicroRNA Assays) were evaluated in PBMCs exposed to high glucose (HG, 50 mM), LPS (100 ng/mL), and Olmesartan (OLM, 10−6 M), an angiotensin II type 1 receptor antagonist. Results HG increased TF expression and decreased both miRs as compared to control glucose conditions (11.1 mM). In HG-activated PBMCs, LPS stimulated TF expression and downregulated miR-20a, an effect reverted by OLM (10−6 M); miR-19a expression was unchanged by LPS in both CG and HG conditions. Conclusions miR-19a and miR-20a are inhibited by inflammatory stimuli active on TF expression and their response differs by the stimulus under investigation; angiotensin II may participate in that mechanism.

Prognostic role of TPL2 in early‑stage non‑small cell lung cancer

Non-small cell lung cancer (NSCLC) accounts for ~70% of all lung cancer-associated mortalities worldwide. The serine/threonine protein kinase tumor progression locus 2 [TPL2/MAP3 kinase 8 (MAP3K8)] may impact oncogenic events; however the role of TPL2 in lung carcinogenesis remains unclear. The present study was focused on the potential prognostic role of TPL2 in 101 patients with early-stage NSCLC. Since TPL2 is a potential target of miR-21, the association between TPL2 and miR-21 expression was also examined. TPL2 and miR-21 mRNA expression was quantified using reverse transcription quantitative polymerase chain reaction (RT-qPCR). TPL2 protein levels were evaluated by immunohistochemistry (IHC). The present study identified that the mRNA expression of TPL2 was low in 52/101 (51%) cases and high in 49/101 (49%) cases. IHC analysis of TPL2 protein expression often demonstrated identical mRNA results. No statistically significant associations were observed between the mRNA expression of TPL2 and the predominant clinicopathological characteristics of the patients with NSCLC, as well as identifying no association between TPL2 and miR-21. TPL2 mRNA expression was significantly higher in patients with NSCLC with good prognosis (disease-free interval P=0.009; overall survival P=0.024), when compared with those of poor prognosis. Focusing on the difference in mRNA expression of TPL2 among the adenocarcinomas in affected patients, TPL2 was more highly expressed in lepidic adenocarcinomas compared with in the other subtypes (P=0.012). The present study is the first examination, to the best of our knowledge, of TPL2 in early-stage NSCLC in relation to miR-21, and in different adenocarcinoma subtypes. Future studies must clarify the mechanism by which TPL2 is involved in lung carcinogenesis due to its important translational implications.

Expression profiling and microRNA regulation of the LKB1 pathway in young and aged lung adenocarcinoma patients

Lung cancer in young patients appears to have distinct clinicopathological features. The present study focused on the role of the serine/threonine kinase liver kinase B1 (LKB1), a known tumor suppressor gene, and its miRNA regulation in lung adenocarcinoma, particularly in young versus elderly patients. A total of 88 patients with lung adenocarcinoma were retrospectively analysed. A simultaneous quantification was performed of the expression of LKB1 mRNA and 15 microRNAs (miRNA/miRs; miRs −93, −96, −34a, −34c, −214, −33a, −30b, −145, −182, −30c, −183, −29b, −29c, −153 and −138) involved in the LKB1 pathway, as well as of 5 identified target mRNAs [cyclin D1 (CCND1), catenin β-1 (CTNNB1), lysyl oxidase (LOX), yes-associated protein 1 (YAP1) and survivin], using NanoString technology. KRAS mutations were investigated by pyrosequencing analysis. Patients ≤50 years were defined as a younger group, while patients >50 years old as an older group (n=44/group). No difference between the two groups was identified in terms of survival times analysed using the Kaplan-Meier method or KRAS mutations. Subsequently, the LKB1 signalling pathway was focused on, as a target for therapy in lung adenocarcinoma, and assessed with regards to clinicopathological features; we found that LOX levels in adenocarcinoma patients were significantly associated with histological subtype (P=0.03), stage (P<0.0001) and prognosis (P=0.02 for disease-free interval and P=0.005 for overall survival), but not with age. Furthermore, the miRNA target prediction model indicated that miR-93 and miR-30b appeared to have functional binding sites and downregulate the gene expression of LKB1 and LOX, respectively. In conclusion, young patients appeared have similar survival rates to elderly patients. The assessment of LKB1, its downstream genes and its regulation by miRNAs may have an impact on future research on lung adenocarcinoma in young and elderly patients. Further investigations will be necessary to elucidate the potential of this pathway as a novel target for therapy.