Study of MicroRNA (miR-221-3p, miR-133a-3p, and miR-9-5p) Expressions in Oral Submucous Fibrosis and Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the common types of cancer. Its progression follows a transition from oral potentially malignant disorders (OPMDs) such as oral submucous fibrosis (OSMF). Epigenetic modifiers, especially microRNAs (miRNAs), have an appreciable role in the regulation of various carcinogenic pathways which are being used as biomarkers. miRNAs may also be helpful in the differentiation of oral submucous fibrosis from oral squamous cell carcinoma. Three miRNAs, miR-221-3p, miR133a-3p, and miR-9-5p, were found differentially expressed in many cancers in the literature search supported by our preliminary database search-based screening. The literature and our functional enrichment analysis in an earlier study have reported these miRNAs to regulate carcinogenesis at various steps. In the present study, the expression of these miRNAs was examined in 34 histopathologically confirmed OSCC, 30 OSMF, and 29 control (healthy volunteers) human samples. There was a significant downregulation of miRNA-133a-3p in OSCC compared to OSMF and controls, whereas there was up-regulation in oral submucous fibrosis compared to controls. There was no significant difference in the expression of miR-221-3p between OSCC and OSMF, but an upregulation in OSCC compared to controls. miR-9-5p was also found upregulated in both OSCC and OSMF. Further, miR-133a-3p expression was negatively correlated with age, smoking, drinking status, and AJCC staging, whereas miR-9-5p expression was only positively associated with tobacco/ areca nut chewing. The ROC plots, logistic regression model generated, and the correlation between the expression of miR-9-5p and miR-133a-3p in blood and tissue suggests that these could be used as risk stratification biomarkers.

Global and promoter specific hypermethylation of tumor suppressor genes P16, SOCS1, and SHP1 in oral squamous cell carcinoma and oral submucous fibrosis.

ABSTRACTS: Aberrant methylation pattern leads to altered gene expression, that is, involved in the transformation of various cancers, including oral squamous cell carcinoma (OSCC). In the present study, an attempt has been made to examine the association of global and promoter-specific methylation of tumor suppressor genes in patients with OSCC and oral submucous fibrosis (OSMF). Promoter-specific methylation of tumor suppressor genes P16, SOCS1, and SHP1 had been studied earlier for their aberrant methylation patterns in other cancers; however, these studies were mainly conducted in-vitro or in animal models, and as such, only a few studies are available on human samples. In the present study evaluation of promoter-specific methylation of genes P16, SOCS1, and SHP1 in 76 patients' blood and tissue samples was done and compared with methylation of 35 healthy control samples using qPCR. Further, these samples were analyzed for global methylation patterns using ELISA. The results have shown a significant decreasing trend of promoter methylation (OSCC > OSMF > Controls); the methylation indices (MI) were significantly higher in OSCC than in the controls. The median MI of three genes for OSCC were P16MI (0.96), SHP1MI (0.79), and SOCS1 (0.80). Similarly, median MIs for OSMF were P16MI (0.18), SHP1 MI (0.19), and SOCS1 MI (0.5) against controls with MI (0) for each of the three genes. The global methylation %mC values were 1.9, 0.5, and 0.1, respectively. The values of MI and %mC were found to correlate with various risk factors such as tobacco, smoking, and alcohol consumption, which are positively involved in OSMF pathogenesis followed by oral cancer progression. Further, the methylation trend in tissue was reflected in blood samples, proving a window for methylation load to be used as a lesser invasive biomarker. The sensitivity and specificity of methylation load were also found reasonable. Therefore, the current study suggests that there may be a role of global and promoter-specific methylation load in the transition of OSMF to OSCC.

Differential Expression of Zinc-Dependent HDAC Subtypes and their Involvement in Unique Pathways Associated with Carcinogenesis.

OBJECTIVE: The present study aims to identify the effect of ZnHDACs expression on the survival of the patients. Further, reveal the unique and common genes associated with each ZnHDACs and their associated pathways. METHODS: The patient data was obtained from the Cancer Genome Atlas Program (TCGA) database and was analyzed using cBioportal and Gene Expression Profiling Interactive Analysis 2(GEPIA2) online tools. Protein-protein interactions and functional interactomic analysis were done using STRING, DAVID, and KEGG pathway databases. RESULTS: HDAC1, 2, 8, 11 were over-expressed and, HDAC4, 5, 6, 7, and 10 were down-regulated in all the cancer types, but there are few exceptional expression patterns such as HDAC7 and HDAC10 overexpression in HNSC, HDAC3 down-regulation in LUAD, and PRAD. The unique genes interacting with each ZnHDACs provided a better understanding of ZnHDAC's putative role in carcinogenesis. The present study reported that JARID2, stem cell regulation gene uniquely interacts with HDAC1, BPTF-CHRAC-BAZIA axis, enzymes for chromatin modeling selectively interacting with only HDAC2, HDAC3 in H2A acetylation via DMAP1 and YEATS4. HDAC6 associated unique genes regulate protein stability, HDAC7 in subnuclear localization and splicing, HDAC8 in telomere maintenance, HDAC9 in chromosomal rearrangements, and HDAC11 in maintaining histone core and folding. CONCLUSION: The unique genes and pathways associated with a particular ZnHDACs could provide a wide window for interrogating these genes for obtaining putative drug targets.

In-Silico Analysis of Differentially Expressed Genes and Their Regulating microRNA Involved in Lymph Node Metastasis in Invasive Breast Carcinoma.

Axillary nodal metastasis is related to poor prognosis in breast cancer (BC). Key candidate genes in BC lymph node metastasis have been identified from Gene Expression Omnibus datasets and explored through functional enrichment database for annotation, visualization and integrated discovery (DAVID) , protein-protein interaction by Search Tool for the Retrieval of Interacting Genes and proteins (STRING), network visualization (Cytoscape), survival analysis (GEPIA, KM Plotter), and target prediction (miRNet). A total of 102 overlapping differentially expressed genes were found. In-silico survival and expression analyses revealed six candidate hub genes, Desmocollin 3 (DSC3), KRT5, KRT6B, KRT17, KRT81, and SERPINB5, to be significantly associated with nodal metastasis and overall survival, and 83 MicroRNA (miRNAs), which may be potential diagnostic markers and therapeutic targets in BC patients.

Identification of unique subtype-specific interaction features in Class II zinc-dependent HDAC subtype binding pockets: A computational study.

Zinc-dependent HDAC subtypes (ZnHDACs) exhibit differential expression in various cancer types and significantly contribute to oncogenic cell transformation, and hence are interesting anticancer drug targets. The approved pan HDAC inhibitors (PHIs) lack subtype specificity and inhibit all ZnHDACs, causing severe sideeffects. Considering the distinct tissue distribution and roles of individual ZnHDACs in specific cancer types, it is crucial to rationally design subtype-specific inhibitors (SSIs) for enhanced efficacy and reduced side-effects. There are numerous approaches already conducted for designing SSIs, especially Class I ZnHDACs, whereas Class II and III ZnHDACs are relatively unexplored and equally important in disease pathogenesis. This study attempts to decipher the specificity rendering interaction features of six different ZnHDACs by robust analyses of reported experimental data employing sophisticated computational methods like homology modelling, docking, pharmacophore analysis, and molecular dynamic (MD) simulations. Experimentally validated SSIs (activity<1000 nM) of different ZnHDACs and 8 approved PHIs were docked to 40 MD generated conformations of each ZnHDACs followed by MM-GBSA binding energy estimations. Sequences, structures, physicochemical properties, and interaction patterns of the binding sites obtained from docking were exhaustively compared to identify unique subtype-specific interaction features for each Class II ZnHDACs. To further validate the stabilities of these features, 20 ns MD simulations were performed on 12 complexes (each Class II ZnHDACs bound to one SSI and one PHI) in explicit water models. Distinct pharmacophoric patterns were observed in the binding pockets of each subtype despite high sequence similarities. Presence of amides, ketone, hydroxyl, carboxyl groups, and moieties occupying additional sub-pockets and interacting with Zn 2+, etc., in the SSIs affect the orientations of the binding site residues (BSRs) owing to subtype-specific protein- ligand interactions. Stable and unique residue interactions specific for a HDAC subtype are, e.g. E329 for HDAC4, S904 for HDAC5, W496 S563 I569 for HDAC6, M793 for HDAC9, and E302 for HDAC10. Such unique interaction features and pharmacophoric patterns can be utilized for subtype-specific ZnHDAC inhibitor design.