Oncogenic microRNAs as biomarkers of oral tumorigenesis and minimal residual disease.

OBJECTIVES: Classical diagnostic methods are not sensitive enough in detecting oral lesions that may progress to cancer and in assessing minimal residual disease (MRD) in oral surgical margins. Altered expression of microRNAs (miRNAs) contributes to human cancer, including oral cancer. Although there are many studies on microRNAs in oral cancer, there is no reported study comparing the expression of microRNAs during oral tumor progression and in oral surgical margins. MATERIALS AND METHODS: This study analyzed the expression of 72 miRNAs that were reported (till June 2011) to be differentially expressed in oral cancer, during phases of oral cancer progression and in oral surgical margins. RESULTS: Of the 72 miRNAs analyzed, four (hsa-miR-125a, hsa-miR-184, hsa-miR16 and hsa-miR-96) showed a common pattern of expression in both sets of tissues. We further analyzed the downstream target genes of hsa-miR-16 BCL2 and CCND1. The in silico network analysis of these four microRNAs and their target genes revealed presence of genes involved in tumor progression and transcription factors. CONCLUSIONS: The findings suggest that the combinatorial regulation by these miRNAs and their target transcription factors might play a substantial role in oral tumorigenesis. Here we report for the first time that a decreased expression of hsa-miR-125a, hsa-miR-184 and hsa-miR-16 and an increased expression of hsa-miR-96 could be useful in predicting oral tumorigenesis and importantly in the detection of MRD and decision-making process for postoperative treatment modalities.

C-T variant in a miRNA target site of BCL2 is associated with increased risk of human papilloma virus related cervical cancer--an in silico approach.

MicroRNAs control gene expression at the posttranscriptional level by base-pairing to the 3'-UTR of their target mRNAs, thus leading to mRNA degradation of protein fabrication. We hypothesize, SNPs within miRNAs and their targets could be of significance to an individual's risk of developing cancer. We analyzed in silico SNP information on cervical cancer associated aberrant alleles and further investigated this in a case-control study by examining eleven SNPs from different genes. It was observed that a C to T polymorphism in putative miRNA target site of BCL2 was significantly conspicuous for the aberrant SNP allele in cancer tissues as compared to controls. This study provides evidence that SNPs in miRNA-binding sites may play an important role in increasing risk of cancer. The results also paves way for future studies to validate these results in other well-characterized populations as well as to explore the biological significance of these particular SNPs.

Identification and analysis of novel microRNAs from fragile sites of human cervical cancer: computational and experimental approach.

Accurate identification of mature miRNAs is an important requirement for exploring the post-transcriptional regulatory mechanism of organisms. In this work we present a novel computational tool 'Mpred' which first identifies pre-miRNAs and then predicts its mature miRNAs. We first use our method to learn with high accuracy characteristic features of human miRNA precursors from miRbase registry and then apply to sequences from fragile site regions related to cervical cancer in search of novel miRNA genes. The study identified 13 putative miRNA-like sequences and most of them were not related to each other and do not share homology with annotated sequences. Finally, four of the top scoring predictions were verified experimentally using quantitative RT-PCR validation. Expression profile studies revealed that four novel miRs were present in cervical tissues and these data compiled here provide a regulatory framework of miRNA genes that may have roles in tumorigenesis.

Overexpression of COX-2 gene in oral cancer is independent of stage of disease and degree of differentiation.

The incidence of oral cancer is high in certain parts of the world including Southeast Asia. Smokeless tobacco and areca nut chewing is proposed as a possible factor. Cyclooxygenase 2 (COX-2) receptors are present on neoplastic cells and are proposed to participate in initiation, transformation, progression and metastasis of cancer. In a prospective case-controlled study, 42 cases of squamous cell carcinoma of the oral cavity, 13 cases of oral premalignant lesions, and oral mucosa from 32 normal subjects were evaluated for COX-2 gene expression using reverse transcriptase polymerase chain reaction. The mean age of the patients with oral cancer was 60.2 years. The majority of cancer patients were males while the majority of controls were females. A significantly higher expression of COX-2 was found in cancer patients compared to both normal controls (p=0.0001) and patients with premalignant lesions (0.015). The expression in premalignant lesions was higher compared to healthy subjects (p=0.05). COX-2 expression in oral cancer was found to be independent of grade of tumor and stage of disease. These results show up-regulation of the COX-2 gene in oral cancer and precancer. This suggests a role for COX2 receptors in oral cancer carcinogenesis, and provides the foundation for a large randomized trial to determine the role COX2 inhibitors may play in prevention of oral carcinogenesis.

NF-kappaB and COX-2 during oral tumorigenesis and in assessment of minimal residual disease in surgical margins.

Oral cancer is a major health problem in many parts of the world including India. The molecular mechanisms involved in oral tumorigenesis are not completely understood. Although surgery continues to be the most common treatment modality for this cancer, survival rates of oral cancer patients have still not significantly improved over the last few decades. Classical diagnostic methods are still not sensitive enough in detecting completeness of surgery and assessing minimal residual disease. This study investigated the role of NF-kappaB and COX-2 both in oral cancer progression and assessment of minimal residual disease. Expression of NF-kappaB proteins and its inhibitory protein IkappaB-alpha was evaluated using immunohistochemistry, ELISA and EMSA, while RT-PCR was used to detect COX-2 expression. Cytoplasmic expression as well as nuclear translocation of NF-kappaB proteins increased with histological progression of oral cancer (from normal to leukoplakia to cancer). A similar pattern of expression was observed for COX-2 also. NF-kappaB proteins, both cytoplasmic and nuclear, had a significant negative correlation from tumor to surgical margin to extra margin; COX-2 paralleled the expression of NF-kappaB proteins. Our results thus point to NF-kappaB and COX-2 as participants in oral tumor progression and also to the validation of these two molecular markers in assessing minimal residual disease.

Expression of activated transcription factor nuclear factor-kappaB in periodontally diseased tissues.

BACKGROUND: Chronic periodontitis is an inflammatory disease found mainly in adults. Little is known about molecular level changes associated with host response in this condition. Nuclear factor-kappaB (NF-kappaB) is a transcription factor implicated in immune and inflammatory responses. NF-kappaB activation has also been reported to be associated with many chronic inflammatory diseases. The purpose of this paper was to compare the nuclear and cytoplasmic expression of NF-kappaB transcription factor (p50/p65) and cytoplasmic expression of IkappaB in periodontal tissues of periodontitis patients and controls. METHODS: Twenty patients with chronic periodontitis and 20 healthy controls were included in the study. Gingival tissues taken during extraction were processed for immunohistochemical staining and evaluation. RESULTS: Nuclear (activated) p50 was found in 90% of periodontal patient tissues compared to only 30% of healthy tissues. A more significant result was obtained with p65 (75% versus 5%). Intense cytoplasmic immunoreactivity was also observed in periodontitis tissues. IkappaB, the inhibitor of NF-kappaB, was expressed only in 5% of periodontally diseased tissues. CONCLUSION: Activation of NF-kappaB (p50/p65) is significant in periodontally diseased tissues, suggesting the potential of inhibitors of NF-kappaB in managing periodontitis.