A Quest for miRNA Bio-Marker: A Track Back Approach from Gingivo Buccal Cancer to Two Different Types of Precancers

<div><p>Deregulation of miRNA expression may contribute to tumorigenesis and other patho-physiology associated with cancer. Using TLDA, expression of 762 miRNAs was checked in 18 pairs of gingivo buccal cancer-adjacent control tissues. Expression of significantly deregulated miRNAs was further validated in cancer and examined in two types of precancer (leukoplakia and lichen planus) tissues by primer-specific TaqMan assays. Biological implications of these miRNAs were assessed bioinformatically. Expression of <i>hsa-miR-1293, hsa-miR-31, hsa-miR-31*</i> and <i>hsa-miR-7</i> were significantly up-regulated and those of <i>hsa-miR-206, hsa-miR-204</i> and <i>hsa-miR-133a</i> were significantly down-regulated in all cancer samples. Expression of only <i>hsa-miR</i>-31 was significantly up-regulated in leukoplakia but none in lichen planus samples. Analysis of expression heterogeneity divided 18 cancer samples into clusters of 13 and 5 samples and revealed that expression of 30 miRNAs (including the above-mentioned 7 miRNAs), was significantly deregulated in the cluster of 13 samples. From database mining and pathway analysis it was observed that these miRNAs can significantly target many of the genes present in different cancer related pathways such as “proteoglycans in cancer”, <i>PI3K-AKT</i> etc. which play important roles in expression of different molecular features of cancer. Expression of <i>hsa-miR-31</i> was significantly up-regulated in both cancer and leukoplakia tissues and, thus, may be one of the molecular markers of leukoplakia which may progress to gingivo-buccal cancer.</p></div

A Quest for miRNA Bio-Marker: A Track Back Approach from Gingivo Buccal Cancer to Two Different Types of Precancers - Figure 3

<p><b>A: Manhattan plot of p-values for 520 miRNAs from the cluster of 13 samples.</b> The plot of relative location of 520 miRNAs (along the horizontal axis) across the human chromosome and their corresponding –log₁₀ transformed p-value (along the vertical axis). Benjamini-Hochberg corrected P-value cut off was 0.00298 (Horizontal line in the middle of figure). <b>B: Heat map diagram of ΔΔCt values of 30 miRNAs</b>. Expression of these miRNAs was significantly deregulated in the cluster of 13 samples. Each row represents a miRNA and each column represents a sample. Sky-blue colored cells stand for failed assay (i.e.no data in those cells). Red and green colors signify up- and down-regulation of expression, respectively. Heat map was constructed using Heatmap 2 of R's “gplot” package. <b>C: Highly correlated expression of miR-411* and miR-411</b>.</p

Reported targets associated with 7 miRNAs significantly deregulated in 18 cancer samples.

@<p>: Up-regulation of expression of miRNAs.</p>$<p>: Down regulation of expression of miRNAs.</p>∧<p>Benjamini-Hochberg corrected p-value cut off at 5% level: 6.5E⁻⁰⁴.</p><p><b>ΔΔCt</b> =  ΔCt _{of a gene in cancer tissue} - ΔCt _{of that gene in control tissue}.</p>#<p>; Expression of <i>has-miR-1</i> is not significantly deregulated and shown for comparison only.</p><p>MN- Mouth Neoplasm, HN- Head and Neck Cancer, SCC- Squamous Cell Carcinoma, LN- Laryngeal Neoplasm, EN- Esophageal Neoplasm, OLP-Oral Leukoplakia, <i>GCN1L1</i>- general control of amino-acid synthesis 1-like 1.</p

Demography of 18 leukoplakia patients.

<p><b>*</b>All except two (LK5 & LK9) patients had tobacco habits.</p

Demography of 18 cancer patients and their tumor differentiation status.

<p><b>*</b>All patients had tobacco habits;</p>@<p>Mx: metastasis status not known.</p

Demography of 12 lichen planus patients.

<p>*2 out of 12 (Patient LP1 and LP6) patients had tobacco habits.</p