miRNAs In Oral Squamous Cell Carcinoma Research Articles

A new perspective on diagnostic strategies concerning the potential of saliva-based miRNA signatures in oral cancer.

Oral cancer, the most prevalent cancer worldwide, is far more likely to occur after the age of forty-five, according to the World Health Organization. Although many biomarkers have been discovered over the years using non-invasive saliva samples, biopsies, and human blood, these biomarkers have not been incorporated into standard clinical practice. Investigating the function of microRNAs (miRNAs) in the diagnosis, aetiology, prognosis, and treatment of oral cancer has drawn more attention in recent years. Though salivary microRNA can act as a window into the molecular environment of the tumour, there are challenges due to the heterogeneity of oral squamous cell carcinoma (OSCC), diversity in sample collection, processing techniques, and storage conditions. The up and downregulation of miRNAs has been found to have a profound role in OSCC as it regulates tumour stages by targeting many genes. As a result, the regulatory functions of miRNAs in OSCC underscore their significance in the field of cancer biology. Salivary miRNAs are useful diagnostic and prognostic indicators because their abnormal expression profiles shed light on tumour behaviour and patient prognosis. In addition to their diagnostic and prognostic value, miRNAs hold promise as therapeutic targets for oral cancer intervention. The current review sheds light on the challenges and potentials of microRNA studies that could lead to a better understanding of oral cancer prognosis, diagnosis, and therapeutic intervention. Furthermore, the clinical translation of OSCC biomarkers requires cooperation between investigators, physicians, regulatory bodies, and business partners. There is much potential for improving early identification, tracking therapy response, and forecasting outcomes in OSCC patients by including saliva-based miRNAs as biomarkers.

The Potential MicroRNA Diagnostic Biomarkers in Oral Squamous Cell Carcinoma of the Tongue.

Oral squamous cell carcinoma (OSCC) of the tongue is a common type of head and neck malignancy with a poor prognosis, underscoring the urgency for early detection. MicroRNAs (miRNAs) have remarkable stability and are easily measurable. Thus, miRNAs may be a promising biomarker candidate among biomarkers in cancer diagnosis. Biomarkers have the potential to facilitate personalized medicine approaches by guiding treatment decisions and optimizing therapy regimens for individual patients. Utilizing data from The Cancer Genome Atlas, we identified 13 differentially expressed upregulated miRNAs in OSCC of the tongue. Differentially expressed miRNAs were analyzed by enrichment analysis to reveal underlying biological processes, pathways, or functions. Furthermore, we identified miRNAs associated with the progression of OSCC of the tongue, utilizing receiver operating characteristic analysis to evaluate their potential as diagnostic biomarkers. A total of 13 upregulated miRNAs were identified as differentially expressed in OSCC of the tongue. Five of these miRNAs had high diagnostic power. In particular, miR-196b has the potential to serve as one of the most effective diagnostic biomarkers. Then, functional enrichment analysis for the target gene of miR-196b was performed, and a protein-protein interaction network was constructed. This study assessed an effective approach for identifying miRNAs as early diagnostic markers for OSCC of the tongue.

Establishment and assessment of an oral squamous cell carcinoma N7-methylguanosine methyltransferase associated microRNA prognostic model.

Background: N7-methylguanosine (m7G) methyltransferases and microRNAs (miRNAs) are closely associated with tumor progression. However, the role of m7G methyltransferase-related miRNAs as prognostic markers in oral squamous cell carcinoma (OSCC) has not been studied. This study aimed to explore the m7G methyltransferase-related miRNAs in OSCC, establish a prognostic model based on m7G methyltransferase-related miRNAs, investigate their correlation with immune cell infiltration, and assess their potential prognostic value. Methods: Transcriptional and clinical data of patients with OSCC were obtained from The Cancer Genome Atlas (TCGA) database. TargetScan and miRWalk were used to predict m7G methyltransferase-related miRNAs. Subsequently, differentially expressed m7G methyltransferase-related miRNAs in TCGA-OSCC were selected. Cox and least absolute shrinkage and selection operator (LASSO) regression analyses were used to build an m7G methyltransferase-related miRNA risk prognostic model for TCGA-OSCC. Patients were stratified into high- and low-risk groups. The predictive and diagnostic accuracies of the risk prognostic model were further validated using Kaplan-Meier survival analysis, receiver operating characteristic (ROC) curve analysis, independent prognosis analysis, and nomogram plots. Finally, quantitative real-time polymerase chain reaction (qPCR) was used to validate the expression levels of m7G methyltransferase-related miRNAs in postoperative cancer and adjacent normal tissues from 60 patients with OSCC. Results: Through Cox and LASSO regression analysis, six candidate miRNAs (hsa-miR-338-3p, hsa-miR-1251-3p, hsa-miR-3129-5p, hsa-miR-4633-3p, hsa-miR-216a-3p, and hsa-miR-6503-3p) most relevant to the prognosis of patients with OSCC were identified to construct an m7G methyltransferase-related miRNA risk prognostic model. In this model, the overall survival (OS) of the high-risk group was significantly shorter than that of the low-risk group (P < 0.001). The model effectively predicted prognosis and served as an independent prognostic indicator for patients with OSCC. Compared with the low-risk group, the high-risk group exhibited a significantly increased capacity for immune cell infiltration (P < 0.05), while the activation and initiation abilities of immune cells were decreased. Finally, six m7G methyltransferase-related miRNAs were validated in OSCC tissue samples. Conclusion: The risk prognostic model based on six m7G methyltransferase-related miRNAs can predict the OS rate of patients with OSCC and has the potential to guide individualized treatment. This prognostic model is closely associated with immune cell infiltration in patients with OSCC.

Cold Atmospheric Plasma Jet Irradiation Decreases the Survival and the Expression of Oncogenic miRNAs of Oral Carcinoma Cells

Despite recent advancements, therapies against advanced oral squamous cell carcinoma (OSCC) remain ineffective, resulting in unsatisfactory therapeutic outcomes. Cold atmospheric plasma (CAP) offers a promising approach in the treatment of malignant neoplasms. Although the effects of CAP in abrogating OSCC have been explored, the exact mechanisms driving CAP-induced cancer cell death and the changes in microRNA (miRNA) expression are not fully understood. We fabricated and calibrated an argon-CAP device to explore the effects of CAP irradiation on the growth and expression of oncogenic miRNAs in OSCC. The analysis revealed that, in OSCC cell lines following CAP irradiation, there was a significant reduction in viability; a downregulation of miR-21, miR-31, miR-134, miR-146a, and miR-211 expression; and an inactivation of the v-akt murine thymoma viral oncogene homolog (AKT) and extracellular signal-regulated kinase (ERK) signals. Pretreatment with blockers of apoptosis, autophagy, and ferroptosis synergistically reduced CAP-induced cell death, indicating a combined induction of variable death pathways via CAP. Combined treatments using death inhibitors and miRNA mimics, alongside the activation of AKT and ERK following the exogenous expression, counteracted the cell mortality associated with CAP. The CAP-induced downregulation of miR-21, miR-31, miR-187, and miR-211 expression was rescued through survival signaling. Additionally, CAP irradiation notably inhibited the growth of SAS OSCC cell xenografts on nude mice. The reduced expression of oncogenic miRNAs in vivo aligned with in vitro findings. In conclusion, our study provides new lines of evidence demonstrating that CAP irradiation diminishes OSCC cell viability by abrogating survival signals and oncogenic miRNA expression.

A novel panel of clinically relevant miRNAs signature accurately differentiates oral cancer from normal mucosa.

Although a considerable body of knowledge has been accumulated regarding the early diagnosis and treatment of oral squamous cell carcinoma (OSCC), its survival rates have not improved over the last decades. Thus, deciphering the molecular mechanisms governing oral cancer will support the development of even better diagnostic and therapeutic strategies. Previous studies have linked aberrantly expressed microRNAs (miRNAs) with the development of OSCC. We combined bioinformatical and molecular methods to identify miRNAs with possible clinical significance as biomarkers in OSCC. A set of 10 miRNAs were selected via an in silico approach by analysing the 3'untranslated regions (3'UTRs) of cancer-related mRNAs such as FLRT2, NTRK3, and SLC8A1, TFCP2L1 and etc. RT-qPCR was used to compare the expression of in silico identified miRNAs in OSCC and normal tissues (n=32). Among the screened miRNAs, miR-21-5p (p < 0.0001), miR-93-5p (p < 0.0197), miR-146b-5p (p <0.0012), miR-155-5p (p < 0.0001), miR-182-5p (p < 0.0001) were significantly overexpressed, whereas miR-133b (p < 0.05) was significantly downregulated in OSCC tissues, a scenario confirmed in two additional OSCC validation cohorts: Regina Elena National Cancer Institute (IRE cohort, N=74) and The Cancer Genome Atlas Data Portal (TCGA cohort, N=354). Initial stage tumors (T1, T2) expressed significantly higher levels of miR-133b (p < 0.0004) compared to more advanced ones (T3, T4). Also, we identified miR-93-5p (p < 0.0003), miR-133b (p < 0.0017) and miR-155-5p (p < 0.0004) as correlated with HPV-induced OSCC. The high expression of these 6 miRNAs as a signature predicted shorter disease-free survival (DFS) and could efficiently distinguish OSCC cases from healthy controls with areas under the curve (AUC) of 0.91 with sensitivity and specificity of 0.98 and 0.6, respectively. Further target identification analysis revealed enrichment of genes involved in FOXO, longevity, glycan biosynthesis and p53 cancer-related signaling pathways. Also, the selected targets were underexpressed in OSCC tissues and showed clinical significance related to overall survival (OS) and DFS. Our results demonstrate that a novel panel consisting of miR-21-5p, miR-93-5p, miR-133b, miR-146b-5p, miR-155-5p and miR-182-5p could be used as OSCC-specific molecular signature with diagnostic and prognostic significance related to OS and DFS.

Evaluation of the current MicroRNAs expression levels as potential biomarkers in Oral Squamous Cell Carcinoma.

As the most common malignancy, oral squamous cell carcinoma (OSCC) is typically fatal. The survival of patients with oral cancer has not improved, and tumor recurrence remains high. During tumorigenesis, microRNAs (miRNAs) regulate gene expression. Patients' life expectancy can be determined by prognostic survival biomarkers, which can focus therapy on specific targets. This study evaluated five miRNAs associated with OSCC for their prognostic impact. It was determined through microarray analysis and quantitative reverse transcription polymerase chain reaction that there was a significant difference in the expression of miRNAs between OSCC patients and control patients in plasma. We used the unpaired t-tests and the Mann-Whitney test to conduct the statistical analysis. Based on the study's results, five miRNAs have been found to have significantly different expression levels in the plasma of patients with OSCC; in particular, miR-31 was found to have a significantly higher expression level in OSCC patients' plasma as compared with healthy controls. Aside from that, there was a significant reduction in the expression of miR-100, miR-199a, miR-203, and mir345 in the plasma of OSCC patients (P < 0.05). To better understand the importance of miRNAs in OSCC, various OSCC cases were analyzed. Detecting miRNAs in plasma may be a useful diagnostic tool for oral squamous cell carcinoma.

In-silico, interactomic based screening and identification of differentially expressed miRNAs in oral submucous fibrosis and oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the most common type of head and neck cancer, and its progression follows mostly the transition from oral potentially malignant disorders (OPMDs). Oral submucous fibrosis (OSMF) is a critical OPMD that transforms to OSCC in around 5% of cases; it's detected at later stages when it turns to invasive cancer. microRNA's (miRNAs) are essential regulators of many tumorigenic processes with aberrant expression profiles observed in many cancers. miRNAs are studied and identified as possible diagnostic markers in many cancers. Therefore, they may also serve as risk stratification biomarkers in OSCC transformed from OSMF. The present study performed a three-phase screening for differentially expressed microRNAs in OSCC and OSMF datasets from publicly available databases. The first phase includes screening dysregulated miRNAs in OSCC from cancer-specific miRNA Databases such as miRCancer, miRDB, and miRWalk, and fourteen common miRNAs obtained from these databases. Secondly, the OSCC differentially expressed miRNAs obtained from NCBI-GEO microarray datasets. Eleven out of earlier fourteen miRNAs had aberrant expressions with Fold change >1.5 and P-value <0.05. Further, out of the above eleven miRNA's five, i.e., miR-375, miR-451, miR-221, miR-133a, and miR-133b, were shared between the OSMF and OSCC differentially expressed miRNAs. Differentially expressed miRNAs for OSMF obtained from miRWalk, a disease-specific miRNAs database for predicted and validated datasets. The target gene search for these five miRNAs was performed using TargetScan, followed by the interactomic analysis to study the functional importance of these miRNA targets in tumorigenesis. These miRNA targets were extensively enriched mechanisms such as epithelial-mesenchymal transition, cell-cell adhesion, cell cycle regulation, which are already studied as critical pathways involved in OSMF tumorigenesis. Therefore, these five screened miRNAs may serve as risk stratification biomarkers with further validation in larger categorical datasets and clinical samples.

MiR-31-NUMB Cascade Modulates Monocarboxylate Transporters to Increase Oncogenicity and Lactate Production of Oral Carcinoma Cells.

Oral squamous cell carcinoma (OSCC) is among the leading causes of cancer-associated death worldwide. miR-31 is an oncogenic miRNA in OSCC. NUMB is an adaptor protein capable of suppressing malignant transformation. Disruption of the miR-31-NUMB regulatory axis has been demonstrated in malignancies. Mitochondrial dysfunction and adaptation to glycolytic respiration are frequent events in malignancies. Monocarboxylate transporters (MCTs) function to facilitate lactate flux in highly glycolytic cells. Upregulation of MCT1 and MCT4 has been shown to be a prognostic factor of OSCC. Here, we reported that miR-31-NUMB can modulate glycolysis in OSCC. Using the CRISPR/Cas9 gene editing strategy, we identified increases in oncogenic phenotypes, MCT1 and MCT4 expression, lactate production, and glycolytic respiration in NUMB-deleted OSCC subclones. Transfection of the Numb1 or Numb4 isoform reversed the oncogenic induction elicited by NUMB deletion. This study also showed, for the first time, that NUMB4 binds MCT1 and MCT4 and that this binding increases their ubiquitination, which may decrease their abundance in cell lysates. The disruptions in oncogenicity and metabolism associated with miR-31 deletion and NUMB deletion were partially rescued by MCT1/MCT4 expression or knockdown. This study demonstrated that NUMB is a novel binding partner of MCT1 and MCT4 and that the miR-31-NUMB-MCT1/MCT4 regulatory cascade is present in oral carcinoma.

Imprints of miRNA- An Update

Introduction: Micro Ribonucleic acid (miRNA) is the novel fraction of non-coding RNA, which is considered as the biological measure of genetic state of a cell. It plays a mantle regulatory role post transcriptionally, involving both biological and pathologic cellular processes. miRNAs possess unique properties like, abundant tissue expression which allows for reproducible isolation and quantification. Methods: Literature search was carried out by using different combinations of keywords on Pub Med and Cochrane database to retrieve the data for the literature review. Discussion: Distinct expression profiles of miRNA in OSCC (Oral Squamous Cell Carcinoma) offers the use of specific miRNAs signature for early-stage diagnosis, prediction and prognosis. It plays a critical role in the initiation and progression of cancer functioning either as an oncogene or as tumor suppressor. At present, it has a huge impact on understanding the aberrant gene modifications in OSCC and its future effects on prognosis and therapeutic relevance needs to be looked upon.Conclusion: Distinct phases of miRNA from its origin, properties, functionality, detection, cancer association to future trends are updated in this review.

MicroRNA‑199a‑5p functions as a tumor suppressor in oral squamous cell carcinoma via targeting the IKKβ/NF‑κB signaling pathway.

MicroRNAs (miRNAs) have been shown to have a significant role in the progression of several types of cancer, including oral squamous cell carcinoma (OSCC). However, the biological function and regulatory mechanisms of miRNAs in OSCC remain to be fully elucidated. The aim of the present study was to investigate the role of miRNAs in OSCC and the relevant mechanism. Using a microarray, it was found that miRNA (miR)-199a-5p was one of the most downregulated miRNAs in OSCC tissues. A low expression of miR-199a-5p was closely associated with tumor differentiation, lymph node metastasis, tumor-node-metastasis stage, and overall survival rate. Functionally, the overexpression of miR-199a-5p suppressed cell proliferation, induced G0/G1 cell cycle arrest, and promoted the apoptosis of Tca8113 and SCC-4 cells. Subsequently, inhibitor of nuclear factor-κB (NF-κB) kinase β (IKKβ), an important regulator of NF-κB activation, was identified as a direct target of miR-199-5p. An inverse correlation was found between miR-199a-5p and IKKβ in tumor tissues. Further investigations revealed that the overexpression of IKKβ efficiently abrogated the influences caused by the overexpression of miR-199a-5p. It was also found that the miR-199a-5p-mediated anticancer effects were dependent on the inhibition of NF-κB activation. These findings indicate that miR-199a-5p functions as a tumor suppressor through regulation of the NF-κB pathway by targeting IKKβ in OSCC.

Loss of miR-16 contributes to tumor progression by activation of tousled-like kinase 1 in oral squamous cell carcinoma

ABSTRACTA different expression signature of miRNA in oral squamous cell carcinoma (OSCC) has been validated. MicroRNA-16 (miR-16) as one of the distinctly dysregulated miRNAs in OSCC, its functional role in progression of OSCC remains not fully clear. Herein, miR-16 expression was significantly lower in OSCC tissues compared to that in adjacent normal tissues (n = 131). A lower level of miR-16 was found to be associated with poor prognosis on a cohort of 131 patients with OSCC, and on an extensive public data (457) from TCGA database. Additionally, expression of TLK1 was significantly higher in OSCC tissues compared to that in adjacent normal tissues, which is negatively correlated with miR-16 expression in OSCC. Bioinformatics analyses exhibited that TLK1 is a potential downstream effector of miR-16 by directly targeting the 3′-untranslated regions (3′-UTR) of mRNA. Forced expression of miR-16 in OSCC cell lines inhibits cell proliferation in vitro, and tumor growth in vivo by inhibition of TLK1. Mechanistically, downregulation of TLK1 by miR-16 enhances higher level of DNA damage leading to a significant increase of G2/M arrest in SCC9 cells. And, overexpression of TLK1 substantially reduces DNA damage and G2/M arrest by activation of TLK1-dependent cell cycle checkpoint response. To conclude, miR-16 is downregulated in OSCC and serves as tumor suppressor in OSCC progression by targeting TLK1, which has potential to be the novel therapeutic targets and diagnostic biomarkers for OSCC.

Integrated miRNA-mRNA spatial signature for oral squamous cell carcinoma: a prospective profiling study of Narrow Band Imaging guided resection

Oral squamous cell carcinoma (OSCC) is a common malignancy for which there is poor prognosis and limited therapeutic options. The objective was to identify mRNA targets of dysregulated miRNAs in OSCC using integrated analysis and understand molecular abnormality in surgical margins. We used biopsies along the spatial axis from normal tissue defined by narrow band imaging (NBI) through conventional white light (WL) margins to tumour from 18 patients undergoing surgical resection for OSCC. Overall 119 miRNA and 4794 mRNA were differentially expressed along the adjacent normal tissue to tumour axis. Analysis of miRNA profiles demonstrated the NBI margins were molecularly distinct from both the tumour and WL margin. Integrated analysis identified 193 miRNA-mRNA interactions correlated to the spatial axis of NBI-WL-T. We used cross-validation analysis to derive a spatial interactome signature of OSCC comprising 100 putative miRNA-mRNA interactions between 40 miRNA and 96 mRNA. Bioinformatic analysis suggests that miRNA dysregulation in OSCC may contribute to activation of the oncostatin M, BDNF and TGF-β pathways. Our data demonstrates that surgical margins defined by NBI leave less potentially malignant residual tissue. The miRNA-mRNA interactome provides insight into dysregulated miRNA signalling in OSCC and supports molecular definition of tumour margins.

MiR-494-3p Induces Cellular Senescence and Enhances Radiosensitivity in Human Oral Squamous Carcinoma Cells.

Oral squamous cell carcinoma (OSCC) is the most common malignancy of head and neck. Although radiotherapy is used for OSCC treatment, the occurrence of radioresistant cancer cells limits its efficiency. MicroRNAs (miRNAs) are non-coding RNAs with lengths of 18–25 base pairs and known to be involved in carcinogenesis. We previously demonstrated that by targeting B lymphoma Mo-MLV insertion region 1 homolog (Bmi1), miR-494-3p functions as a putative tumor suppressor miRNA in OSCC. In this study, we further discovered that miR-494-3p could enhance the radiosensitivity of SAS OSCC cells and induce cellular senescence. The overexpression of miR-494-3p in SAS cells increased the population of senescence-associated β-galactosidase positive cells, the expression of p16INK4a and retinoblastoma 1 (RB1), as well as downregulated Bmi1. The knockdown of Bmi1 by lentiviral-mediated delivery of specific short hairpin RNAs (shRNAs) also enhanced the radiosensitivity of SAS cells and the activation of the senescence pathway. Furthermore, the inverse correlation between Bmi1 and miR-494-3p expression was observed among OSCC tissues. Results suggest that miR-494-3p could increase the radiosensitivity of OSCC cells through the induction of cellular senescence caused by the downregulation of Bmi1.

Clinical Diagnostic Implications of Body Fluid MiRNA in Oral Squamous Cell Carcinoma: A Meta-Analysis.

Oral cancer, predominantly oral squamous cell carcinoma (OSCC), is one of the most leading causes of cancers worldwide. Due to a low 5-year survival rate, highly effective methods for the early detection of OSCC are totally needed. MicroRNAs (miRNAs), as promising biomarkers, can bring insights into tumorigenesis of oral cancers. However, studies on the accuracy of miRNAs detection in OSCC have inconsistent conclusions, leading us to conduct this meta-analysis. The aim of this study was to systematically review the articles investigating the diagnostic value of miRNAs in OSCC.The PubMed, Embase, Chinese National Knowledge Infrastructure (CNKI), Web of Science were searched (updated to June 11th, 2015) to identify all articles evaluating the diagnostic yield of miRNAs for OSCC. The pooled sensitivity, specificity, and other diagnostic parameters were used to assess the performance of miRNAs assays on OSCC detection. Statistical analysis was conducted by employing the R software.The present meta-analysis comprised 23 studies from 10 articles, including 598 OSCC patients and 320 healthy individuals, available for analysis. The summary receiver operator characteristic (SROC) curve was plotted. Meanwhile, the pooled diagnostic parameters and the area under curve (AUC) were calculated based on all included studies. The pooled diagnostic parameters calculated from all 23 studies were as follows: pooled sensitivity of 0.759 (95% CI: 0.701–0.809), pooled specificity of 0.773 (95% CI: 0.713–0.823) and AUC of 0.832, which indicates a relatively high diagnostic accuracy of miRNAs in differentiating OSCC patients from healthy controls. Meanwhile, In addition, subgroup analyses were conducted to access the heterogeneity between studies, which is based on specimen (serum/plasma/blood/saliva/ tissue) and ethnicity (Asian/Caucasian).In summary, our meta-analysis suggests that miRNAs might be used in noninvasive screening tests for OSCC, which needs further large-scale studies to be validated.

MiR-138 suppresses the proliferation of oral squamous cell carcinoma cells by targeting Yes-associated protein 1.

Aberrant microRNA expression has been suggested to be an important event in the pathologies of various types of cancer. MicroRNA-138 (miR-138) has been reported to be frequently downregulated in various types of human cancer, including oral squamous cell carcinoma (OSCC). However, the precise molecular mechanism of miR-138 underlying OSCC remains largely unknown. The aim of the present study was to investigate the expression of miR-138 in OSCC tumor tissues and several OSCC cell lines and validated its interaction with the 3'-untranslated region (3'-UTR) of Yes-associated protein 1 (YAP1). The results showed that, miR-138 was significantly downregulated in OSCC tumor tissues and cell lines. Overexpression of miR-138 inhibited cell proliferation of OSCC cells whereas the downregulation of miR-138 promoted cell proliferation. A direct interaction between miR-138 and 3'-UTR of YAP1 was validated by dual-luciferase reporter assay. Moreover, overexpression of miR-138 in OSCC cells significantly decreased the expression of YAP1 and downregulation of miR-138 inhibited the expression of YAP1. Specifically, the inhibitory effect of miR-138 on the proliferation of OSCC cells was eliminated by transfection with YAP1 overexpression vectors that did not harbor any specific miR-138 binding specific sequences in 3'-UTR. In addition, the miR-138‑overexpressing OSCC cells exhibited a low growth rate in the xenograft tumor assay with a decreased expression of YAP1 in tumor tissues. The results suggest that miR-138 is a tumor suppressor miRNA in OSCC through targeting YAP1, which serves as a promising therapeutic target for the treatment of OSCC.

Altered levels of miR-21, miR-125b-2*, miR-138, miR-155, miR-184, and miR-205 in oral squamous cell carcinoma and association with clinicopathological characteristics.

Previous studies have described the aberrantly expressed microRNAs (miRNAs) in oral squamous cell carcinoma (OSCC), and we reasoned that studying frequently deregulated candidate miRNAs in OSCC of Indian ethnicity could aid in better understanding of the genetic/environmental impact on the expression statuses of these miRNAs. Therefore, we evaluated the differential expression of six selected miRNAs namely hsa-miR-21, hsa-miR-125b2*, hsa-miR-138, hsa-miR-155, hsa-miR-184, and hsa-miR-205 in OSCC specimens of Indian ethnicity. Two-step Reverse transcriptase quantitative PCR using inventoried TaqMan single miRNA assays was employed to study the expression of the selected miRNAs in 42 OSCC tumors and eight adjacent normal specimens. The expression levels of the miRNAs were tested for any association with clinicopathological parameters. miR-21 was significantly elevated while miR-125b-2* was significantly downregulated in tumors compared to controls (P < 0.01 and P < 0.05 respectively). miR-138 and miR-184 were observed to be predominantly downregulated in the tumor samples. High levels of miR-155 were associated with the habit of chewing tobacco/betel quid. Our results corroborate the previous findings on the overexpression of mir-21 and downregulation of miR-138 in OSCC. As the expression of miR-184 is controversial in tongue/oral cancer, the downregulation may be specific to tumor anatomical localization. On the other hand, to the best of our knowledge, this is the first report to show the association of miR-155 with tobacco chewing and the downregulation of miR-125b-2* in OSCC. Computational predictions suggest that miR-125b-2* may have a role in alternative splicing.

Abstract 1487: Identification of oral carcinoma miRNA signature and control OSCC tumorigenesis through Wnt/β-catenin signaling

Abstract Aberrant microRNAs (miRNAs) expressions are often happened in cancer. In order to identify miRNAs altered in oral carcinogenesis, we investigated miRNAs and cDNA expression profiles in 40 pair-wise oral squamous cell carcinoma (OSCC) specimens. Eighty-four miRNAs were differentially expressed in the OSCC specimens compared to the matched tissue. Interestingly, 19 down-regulated miRNAs are clustered in DLK-1/Meg3 imprinted domain of chromosome 14q32.2 region. Loss of 14q32.2-miRNAs expression also raised the 14q32.2-miRNA targets expression in OSCC. We identified one target protein, Wnt-7b, was highly expressed in OSCC and regulated by miR-329 and -410. Wnt-7b increased the Wnt/β-catenin signaling activity and promoted OSCC tumor progression. Furthermore we also identify the regulation mechanism that down-regulated 14q32.2 miRNAs. Hyper-methylation in meg-3 differential methylated region (DMR) was confirmed by bisulfate sequencing. The hyper-methylation of meg-3 DMR was also induced by betel nuts extract exposure. In summary, we identified the expression signature of aberrant miRNAs in OSCC and provide a novel molecular insight how betel quid contribute to oral carcinogenesis through the epigenetic silencing of tumor suppressor miRNAs targeting the Wnt/β-catenin signaling pathway. Note: This abstract was not presented at the meeting. Citation Format: Wei-Min Chang, Yuan-Ming Hsu, Sung-Tau Chou, Yi-Shing Shieh, Michael Hsiao, Jenn-Ren Hsiao, Jang-Yang Chang, Shine-Gwo Shiah. Identification of oral carcinoma miRNA signature and control OSCC tumorigenesis through Wnt/β-catenin signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1487. doi:10.1158/1538-7445.AM2014-1487

Reciprocal regulation of MicroRNA-99a and insulin-like growth factor I receptor signaling in oral squamous cell carcinoma cells

BackgroundMicroRNAs (miRNAs), small noncoding RNA molecules can function as oncogenes or tumor suppressors in tumorigenesis. Oral squamous cell carcinoma (OSCC) is one of the most prevalent cancers worldwide with a 5-year survival rate of approximately 50%.MethodsThe expression of microRNA-99a (miR-99a) in OSCC tissues and cell lines was investigated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The functions of miR-99a in migration/invasion and lung colonization were determined by transwell and tail vein injection assays, respectively. Specific targets of miR-99a were determined by software prediction, correlation with target protein expression, and luciferase reporter assay. The signaling pathways involved in regulation of miR-99a were investigated using the kinase inhibitors.ResultsWe observed reduced levels of miR-99a, identified as one of the most downregulated miRNA in OSCC and all tested OSCC cell lines compared to normal oral keratinocytes. Ectopic miR-99a expression in OSCC cells markedly reduced migration and invasion in vitro as well as lung colonization in vivo. When evaluating the specific targets of miR-99a, we found that ectopic miR-99a expression downregulates insulin-like growth factor 1 receptor (IGF1R) protein and that the expression of miR-99a correlates negatively with IGF1R protein in OSCC cells. Insertion of the 3′UTR of IGF1R mRNA into the 3′UTR of a reporter gene markedly reduced luciferase activity in OSCC cells expressing miR-99a, suggesting that miR-99a reduces luciferase activity by targeting the 3′UTR of IGF1R mRNA. When evaluating the mechanisms of miR-99a downregulation, we observed the upregulation of miR-99a expression in serum-starved conditions and its suppression in response to insulin-like growth factor (IGF1) stimulation. Inhibitors of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) kinase inhibited IGF1-induced suppression of miR-99a, suggesting the negative regulation of miR-99a expression by IGF1R signaling.ConclusionOverall, results indicate that miR-99a functions as a tumor metastasis suppressor in OSCC cells and mutually regulates IGF1R expression in a reciprocal regulation.

Integrative analysis in oral squamous cell carcinoma reveals DNA copy number-associated miRNAs dysregulating target genes.

To better understand possible mechanisms involved in the dysregulation of gene expression unique to oral squamous cell carcinoma (OSCC) metastasis, the investigators examined the differential expression of microRNAs (miRNAs) in OSCC metastasis and their functional impact on target gene expression. Observational assessment of DNA copy number, miRNA, and RNA expression in primary and metastatic OSCC. University of Washington Medical Center and affiliated hospitals. Tumor samples were taken from patients with primary incident OSCC; cells were laser-capture microdissected from 17 nonmetastatic primary tumors and 20 metastatic lymph nodes. DNA copy number aberrations and gene expression profiles were previously determined using Affymetrix 250K Nsp I SNP arrays and HU133 plus 2.0 expression arrays. miRNAs were interrogated with Exiqon's Ready-to-Use PCR Panels assessing the expression of 368 human miRNAs. Investigators found 31 miRNAs differentially expressed between metastatic and nonmetastatic samples (false discovery rate <0.4; 26 overexpressed and 5 underexpressed in metastatic samples). Expression of 7 of these miRNAs was significantly associated with their DNA copy numbers, and expressions of 8 of these miRNAs were significantly associated with their target genes. Among these unique miRNAs, miR-140-3p, miR-29c, and miR-29a were differentially expressed in metastasis versus nonmetastatic samples and had a strong positive correlation with their DNA copy numbers and a negative correlation with the expression of their target genes. Results suggest that DNA copy number aberration may play a role in the dysregulation of some differentially expressed miRNAs in OSCC metastasis, warranting further investigation.

Abstract 3052: MicroRNA as a potential diagnostic marker for oral squamous cell carcinoma

Abstract MicroRNAs (miRNAs), a group of small non-coding RNAs, regulate post transcriptional gene expression by binding to their target messenger RNAs resulting in their degradation or reduced translation. Evidence suggests that miRNAs are involved in the pathogenesis of cancer and could potentially be used as diagnostic or prognostic markers. MicroRNAs have been studied extensively in solid tumors such as breast, lung and colon cancers; however, there are few studies of miRNA in oral squamous cell carcinoma (OSCC). The purpose of this study was to investigate the potential role of miRNA in OSCC. We selected tumors from 40 patients with OSCC (10 HPV-positive oral cavity tumors, 10 HPV-negative oral cavity tumors, 10 HPV-positive oropharyngeal tumors, and 10 HPV-negative oropharyngeal tumors) and normal oral tissues from 10 patients without cancer (all HPV-negative) who were treated at three University of Washington affiliated medical centers between December 2003 and April 2007 and were enrolled in the study with informed consent. The tissues were obtained at the time of surgery and DNA and RNA were simultaneously extracted using TRIzol method (Invitrogen, Carlsbad, CA). RNA was tested using miRCURY LNATM microRNA v.11.0 (Exiqon, Vedbaek, Denmark). This array covers 1,282 mature human miRNAs and 80 viral miRNAs. Using TIGR software and significance analysis of microarrays (SAM) and a cut point of 2-fold change, we found 30 miRNAs (12 up-regulated and 18 down-regulated) differentially expressed between OSCC and controls. We found no difference in miRNA expression between HPV-positive and HPV-negative tumors, both overall and among oropharyngeal tumors. To confirm the Exiqon array results, we conducted qRT-PCR of miRNAs that had &amp;gt;4-fold change (miR-223: 6.2-fold; miR-21: 4.5-fold); the correlation between miRNA expression as measured by Exiqon array and qRT-PCR was 0.63 (p&amp;lt;0.001) for miR-223 and 0.64 (p&amp;lt;0.001) for miR-21. In linear regression analyses adjusting for age and sex in an independent set of 121 OSCC cancers and 30 normal oral tissue from controls, the difference in mean Ct value between cases and controls was 2.7 (CI, 1.6 to 3.8), p&amp;lt;0.001 and 2.9 (CI, 2.4 to 3.4), p&amp;lt;0.001 for miR-223 and miR-21, respectively. To evaluate whether expression of miR-223 and miR-21 were associated with overall and OSCC-specific survival, we performed Cox proportional hazards regression adjusted for age and sex on 161 cases with a median of 36 months of follow-up. We observed no difference in overall survival associated with increasing level of miR-223 expression (HR=1.0; CI, 0.9-1.1) or miR21 expression (HR=1.0; CI, 0.8-1.2). Results were similar for OSCC-specific survival. In conclusion, our results show that a number of miRNAs are differentially expressed between OSCC and normal oral tissue. These deregulated miRNAs may serve as novel diagnostic markers for OSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3052.