MicroRNAs MiR-218, MiR-125b, and Let-7g Predict Prognosis in Patients with Oral Cavity Squamous Cell Carcinoma

<div><p>MicroRNAs (miRNAs) have a major impact on regulatory networks in human carcinogenesis. In this study, we sought to investigate the prognostic significance of miRNAs in patients with oral cavity squamous cell carcinoma (OSCC). In a discovery phase, RNA was extracted from 58 OSCC tumor samples and paired normal tissues. MiRNAs expression was evaluated with TaqMan Array Card and TaqMan MicroRNA assays. The prognostic significance of the miRNA signature identified in the discovery phase was validated by qRT-PCR in a replication set consisting of 141 formalin-fixed, paraffin-embedded (FFPE) samples. We identified a miRNA regulatory network centered on the three hub genes (<i>SP1</i>, <i>MYC</i>, and <i>TP53</i>) that predicted distinct clinical endpoints. Three miRNAs (miR-218, miR-125b, and let-7g) and their downstream response genes had a concordant prognostic significance on disease-free survival and disease-specific survival rates. In addition, patients with a reduced expression of miR-218, miR-125b, and let-7g have a higher risk of poor outcomes in presence of specific risk factors (p-stage III-IV, pT3-4, or pN+). Our findings indicate that specific miRNAs have prognostic significance in OSCC patients and may improve prognostic stratification over traditional risk factors.</p></div

Multivariable analysis of 5-year control and survival rates in OSCC patients (<i>n</i> = 58).

<p>Abbreviations: HR, hazard ratio; CI, confidence interval; ns, not significant.</p><p>* Indicates risk factors significantly associated with outcomes in univariate analysis.</p

<b>The mean vector and variance component assignment of the emission probability models at the initiation step.</b>

<p>The assignments of M1, M5 and M9 are extended to all the other models.</p

Kaplan-Meier survival plots of OSCC patients according to traditional risk factors and miRNAs expression levels.

<p>(<b>A</b>) In the subgroup of patients with pT3-4 disease, subjects with high and low miR-125b expression had significantly different local control rates (92% vs. 50%); (<b>B</b>) the subgroup with low miR-218 expression showed a higher rate of distant metastases in patients with pN+ disease (86% vs. 57%); (<b>C</b>) a high let-7g expression was associated with a lower risk of tumor relapse in patients with advanced pathological stage (46% vs.71%); (<b>D</b>) an increased expression of let-7g predicted a better disease-specific survival in the subgroup of patients with pT3-4 disease (76% vs. 40%).</p

The shaded region applied to filter unreliable CNV prediction.

<p>The shaded region applied to filter unreliable CNV prediction.</p

<b>Comparison of concordant events across the three algorithms using the benchmark events published by Kidd et al (2008).</b>

1<p>Concordance Rate = number of concordant events/number of predicted events. The concordant events refer to the predicted segments that overlap with the benchmark events for least one SNP loci.</p

A toy example of simulation.

<p>The two male samples in the figure both provide their X chromosomes with their original amplifications or deletions. The chromosome from sample 1 carries amplifications from SNP1 to SNP3 and deletions at SNP6 and SNP7. The chromosome from sample 2 carries amplification at SNP3 and deletions at SNP5 and SNP6.</p

Prognostic miRNA modulators centered on the three hub genes <i>SP1</i>, <i>MYC</i>, and <i>TP53</i>.

<p>The downstream genes were grouped into outcome-specific clusters according to their prognostic significance (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102403#pone.0102403.s005" target="_blank">Tables S4</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102403#pone.0102403.s007" target="_blank">S6</a>). The associations between the upstream miRNAs and the downstream gene clusters were identified using sparse partial least square regression. Both the upstream miRNAs and the downstream responsive gene clusters showed a concordant prognostic impact on disease-free survival and disease-specific survival. The solid lines indicate the experimentally-confirmed physical interactions. The dotted lines show the results of the functional analysis performed using the DAVID package. <i>ABCA1</i>, <i>DDIT3</i>, <i>NDUFS8</i>, and <i>NDUFB9</i> regulate cell growth and proliferation. <i>TNFSF10</i> and <i>TNFRSF12A</i> are involved in the cell's apoptotic machinery. The remaining genes encode for molecules regulating cell adhesion or glycosaminoglycan metabolism. The miRNA modulators identified in this predicted poor outcomes in OSCC. Hopefully, our findings may lead to the development of novel prognostic models integrating molecular signatures and traditional risk factors for improving the prognostic stratification and the treatment modalities of OSCC patients.</p

A haplotype tree with only two loci.

<p>A haplotype tree with only two loci.</p

Centers of the eleven emission probability models.

<p>The green, blue and red circles represent the observed samples with genotype AA, AB and BB according to the genotypes identified by Birdseed. The stars are the centers of the three initial groups. All the other centers are assigned according to the grids passing through the stars. M0 and M10 are extended from M1 and M9 so that distance between centers of M1 and M0 is equal to the distance between centers of M1 and M4, and the distance between centers of M9 and M10 is equal to the distance between centers of M9 and M8.</p