Mayakannan Manikandan

Insights on the functional impact of microRNAs present in autism-associated copy number variants.

Autism spectrum disorder is a complex neurodevelopmental disorder that appears during the first three years of infancy and lasts throughout a person’s life. Recently a large category of genomic structural variants, denoted as copy number variants (CNVs), were established to be a major contributor of the pathophysiology of autism. To date almost all studies have focussed only on the genes present in the CNV loci, but the impact of non-coding regulatory microRNAs (miRNAs) present in these regions remain largely unexplored. Hence we attempted to elucidate the biological and functional significance of miRNAs present in autism-associated CNV loci and their target genes by using a series of computational tools. We demonstrate that nearly 11% of the CNV loci harbor miRNAs and a few of these miRNAs were previously reported to be associated with autism. A systematic analysis of the CNV-miRNAs based on their interactions with the target genes enabled the identification of top 10 miRNAs namely hsa-miR-590-3p, hsa-miR-944, hsa-miR-570, hsa-miR-34a, hsa-miR-124, hsa-miR-548f, hsa-miR-429, hsa-miR-200b, hsa-miR-195 and hsa-miR-497 as hub molecules. Further, the CNV-miRNAs formed a regulatory loop with transcription factors and their downstream target genes, and annotation of these target genes indicated their functional involvement in neurodevelopment and synapse. Moreover, miRNAs present in deleted and duplicated CNV loci may explain the difference in dosage of the crucial genes controlled by them. These CNV-miRNAs can also impair the global processing and biogenesis of all miRNAs by targeting key molecules in the miRNA pathway. To our knowledge, this is the first report to highlight the significance of CNV-microRNAs and their target genes to contribute towards the genetic heterogeneity and phenotypic variability of autism.

Oral squamous cell carcinoma: microRNA expression profiling and integrative analyses for elucidation of tumourigenesis mechanism

BackgroundThe advantages and utility of microRNAs (miRNAs) as diagnostic and prognostic cancer markers is at the vanguard in recent years. In this study, we attempted to identify and validate the differential expression of miRNAs in oral squamous cell carcinoma (OSCC), to correlate their expression with the clinico-pathological profile of tumours and to identify the signaling pathways through which the aberrantly expressed miRNAs effect tumourigenesis.MethodsmiRCURY LNA™ array with probes specific to 1168 miRNAs and TaqMan assays specific for 10 miRNAs was employed to evaluate and validate miRNA expression in a discovery cohort (n = 29) and validation cohort (n = 61) of primary OSCC tissue specimens, respectively. A computational pipeline with sequential integration of data from miRTarBase, CytoScape, UniProtKB and DIANA-miRPath was utilized to map the target genes of deregulated miRNAs and associated molecular pathways.ResultsMicroarray profiling identified 46 miRNAs that were differentially expressed in OSCC. Unsupervised clustering demonstrated a high degree of molecular heterogeneity across the tumour samples as the clusters did not represent any of their clinico-pathological characteristics. The differential expression of 10 miRNAs were validated by RT-qPCR (let-7a, let-7d, let-7f and miR-16 were downregulated while miR-29b, miR-142-3p, miR-144, miR-203, and miR-223 were upregulated in OSCC; the expression of miR-1275 was variable in tumours, with high levels associated to regional lymph node invasion; additionally, miR-223 exhibited an association with advanced tumour stage/size). In silico analyses of the experimentally confirmed target genes of miRNAs revamp the relationship of upregulated miRNAs with tumour suppressor genes and of downregulated miRNAs with oncogenes. Further, the differentially expressed miRNAs may play a role by simultaneously activating genes of PI3K/Akt signaling on one hand and by repressing genes of p53 signaling pathway on the other.ConclusionsThe identified differentially expressed miRNAs and signaling pathways deregulated in OSCC have implications for the development of novel therapeutic strategies. To the best of our knowledge, this is the first report to show the association of miR-1275 with nodal invasion and the upregulation of miR-144 in 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

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Mining the 3′UTR of Autism-implicated Genes for SNPs Perturbing MicroRNA Regulation

Autism spectrum disorder (ASD) refers to a group of childhood neurodevelopmental disorders with polygenic etiology. The expression of many genes implicated in ASD is tightly regulated by various factors including microRNAs (miRNAs), a class of noncoding RNAs ∼22 nucleotides in length that function to suppress translation by pairing with ‘miRNA recognition elements’ (MREs) present in the 3′untranslated region (3′UTR) of target mRNAs. This emphasizes the role played by miRNAs in regulating neurogenesis, brain development and differentiation and hence any perturbations in this regulatory mechanism might affect these processes as well. Recently, single nucleotide polymorphisms (SNPs) present within 3′UTRs of mRNAs have been shown to modulate existing MREs or even create new MREs. Therefore, we hypothesized that SNPs perturbing miRNA-mediated gene regulation might lead to aberrant expression of autism-implicated genes, thus resulting in disease predisposition or pathogenesis in at least a subpopulation of ASD individuals. We developed a systematic computational pipeline that integrates data from well-established databases. By following a stringent selection criterion, we identified 9 MRE-modulating SNPs and another 12 MRE-creating SNPs in the 3′UTR of autism-implicated genes. These high-confidence candidate SNPs may play roles in ASD and hence would be valuable for further functional validation.

Expression profiling of long non-coding RNA identifies linc-RoR as a prognostic biomarker in oral cancer:

Oral squamous cell carcinoma is the most aggressive cancer that is associated with high recurrence, metastasis, and poor treatment outcome. Dysregulation of long non-coding RNAs has been shown to promote tumor growth and metastasis in several cancers. In this study, we investigated the expression of 11 selected long non-coding RNAs that are associated with cell proliferation, metastasis, and tumor suppression in oral squamous cell carcinomas and normal tissues by quantitative real-time polymerase chain reaction. Out of the 11 long non-coding RNAs profiled, 9 were significantly overexpressed in tumors with tobacco chewing history. Moreover, the long non-coding RNA profile was similar to the head and neck cancer datasets of The Cancer Genome Atlas database. Linc-RoR, a regulator of reprogramming, implicated in tumorigenesis was found to be overexpressed in undifferentiated tumors and showed strong association with tumor recurrence and poor therapeutic response. In oral squamous cell carcinomas, for the first time, we observed linc-RoR overexpression, downregulation of miR-145-5p, and overexpression of c-Myc, Klf4, Oct4, and Sox2, suggesting the existence of linc-RoR-mediated competing endogenous RNA network in undifferentiated tumors. Taken together, this study demonstrated the association of linc-RoR overexpression in undifferentiated oral tumors and its prognostic value to predict the therapeutic response.

Haploinsufficiency of Tumor Suppressor Genes is Driven by the Cumulative Effect of microRNAs, microRNA Binding Site Polymorphisms and microRNA Polymorphisms: An In silico Approach.

Haploinsufficiency of tumor suppressor genes, wherein the reduced production and activity of proteins results in the inability of the cell to maintain normal cellular function, is one among the various causes of cancer. However the precise molecular mechanisms underlying this condition remain unclear. Here we hypothesize that single nucleotide polymorphisms (SNPs) in the 3′untranslated region (UTR) of mRNAs and microRNA seed sequence (miR-SNPs) may cause haploinsufficiency at the level of proteins through altered binding specificity of microRNAs (miRNAs). Bioinformatics analysis of haploinsufficient genes for variations in their 3′UTR showed that the occurrence of SNPs result in the creation of new binding sites for miRNAs, thereby bringing the respective mRNA variant under the control of more miRNAs. In addition, 19 miR-SNPs were found to result in non-specific binding of microRNAs to tumor suppressors. Networking analysis suggests that the haploinsufficient tumor suppressor genes strongly interact with one another, and any subtle alterations in this network will contribute to tumorigenesis.

Expression and integrity of dermatopontin in chronic cutaneous wounds: a crucial factor in impaired wound healing

Chronic cutaneous wound (CCW) is a major health care burden wherein the healing process is slow or rather static resulting in anatomical and functional restriction of the damaged tissue. Dysregulated expression and degradation of matrix proteins, growth factors and cytokines contribute to the disrupted and uncoordinated healing process of CCW. Therefore, therapeutic approaches for effective management of CCW should be focused towards identifying and manipulating the molecular defects, such as reduced bioavailability of the pro-healing molecules and elevated activity of proteases. This study essentially deals with assessing the expression and integrity of an extracellular matrix protein, Dermatopontin (DPT), in CCW using real-time quantitative reverse transcriptase PCR and immunological techniques. The results indicate that, despite DPT’s high mRNA expression, the protein levels are markedly reduced in both CCW tissue and its exudate. To elucidate the cause for this contradiction in mRNA and protein levels, the stability of DPT is analyzed in the presence of wound exudates and various proteases that are naturally elevated in CCW. DPT was observed to be degraded at higher rates when incubated with certain recombinant proteases or chronic wound exudate. In conclusion, the susceptibility of DPT protein to specific proteases present at high levels in the wound milieu resulted in the degradation of DPT, thus leading to impaired healing response in CCW.

Dysregulation of miR-200 family microRNAs and epithelial-mesenchymal transition markers in oral squamous cell carcinoma

MicroRNAs (miRNAs) are reported to function as a major component in the cellular signaling circuit, which regulates epithelial-mesenchymal transition (EMT). Dysregulation of the microRNA-200 (miR-200) family and EMT-associated genes enables tumor metastasis and resistance to therapy. The present study profiled miR-200 family members miR-200a, miR-200b, miR-200c, miR-141 and miR-429, and also several EMT-regulatory genes including zinc finger E-box-binding homeobox (ZEB)1, ZEB2, epithelial cadherin and vimentin in 40 oral primary tumors in order to understand their role(s) in oral squamous cell carcinoma (OSCC). The reverse transcription-quantitative polymerase chain reaction was used to analyze each sample. Results demonstrated a significant downregulation of miR-200 family members in tumors with a history of tobacco chewing/smoking (P<0.0006, P=0.0467, P=0.0014, P=0.0087 and P=0.0230, respectively) and undifferentiated pathology (miR-200a, P=0.0067; miR-200c, P=0.0248). EMT markers ZEB2 (P=0.0451) and vimentin (P=0.0071) were significantly upregulated in the oral tumors. Furthermore, ZEB2 antisense RNA1 was overexpressed in 50% of OSCC samples (P=0.0075). EMT-regulatory genes did not exhibit any association with clinical outcome. The present study also analyzed the expression of EMT-regulatory genes in 523 head and neck squamous cell carcinoma (HNSCC) samples from The Cancer Genome Atlas (TCGA) database, and the association with treatment outcome. Analysis of TCGA datasets also demonstrated no significant association in the expression of EMT markers with disease recurrence and treatment outcome. The results of the present study revealed dysregulation of miR-200 family miRNAs and EMT-regulatory genes in OSCC without any significant effect on treatment outcome.

Absence of the TP53 Poly-A Signal Sequence Variant rs78378222 in Oral, Cervical and Breast Cancers in South India

With recent advancements in genomics, rare genetic variants attract the attention of cancer researchers as they represent one among the many diverse mechanisms that determine cancer risk and outcome. One such rare variant is the Single Nucleotide Polymorphism rs78378222 that alters the polyadenylation signal (AATAAA to AATACA) of TP53. Initially identified in Caucasian population, the variant ‘C’ allele was associated with an increased risk for prostate cancer, brain cancer and colorectal adenoma but not with colorectal cancer, breast cancer and melanoma (Stacey et al., 2011). Further, the risk allele impaired the proper termination of TP53 resulting in ‘cancer promoting’ longer mRNA transcripts that hindered p53 expression and its downstream functions in apoptosis (Stacey et al., 2011; Li et al., 2013). Simultaneously it was reported that rs78378222 increased the risk of esophageal cancer in Han Chinese population (Zhou et al., 2012). A similar study based on Caucasian population, showed that the rare variant was associated with risk for glioma and also significantly improved survival in patients (Egan et al., 2012). However, the association to survival was not observed in glioma patients of Northern European ancestry although it increased glioma risk (Enciso-Mora et al., 2013). A recent study showed that the heterozygous ‘AC’ genotype was not associated with melanoma and lung cancer, but had a protective role against squamous cell carcinoma of head and neck in non-Hispanic whites (Guan et al., 2013). Given the multitude of effects of rs78378222 in cancer patients of different ethnicity and on account of paucity of information on ‘C’ allele frequency in Indian population, we genotyped 439 genomic DNA samples from oral (108), cervical (96) and breast (235) cancer patients of Indian ethnicity. Blood samples were collected from Government Royapettah Hospital & Government Kasturba Gandhi Hospital for Women and Children, Chennai and clinical information from subjects was undertaken with informed consent and relevant ethical review board approval was accorded. We adopted the PCR-RFLP methodology described by Zhou et al (2012), with a single base mismatch in reverse primer creating a Hind III recognition site when the wild type A allele is present (Figure 1). The RFLP results were confirmed by direct sequencing of 10% of case/control samples chosen at random (Figure 2). We found that the ‘C’ allele was totally absent in cancer samples. We also genotyped 504 healthy controls LETTER to the EDITOR

Complete Mitochondrial Genome Analysis and Clinical Documentation of a Five-Generational Indian Family with Mitochondrial 1555A>G Mutation and Postlingual Hearing Loss

Hearing loss is the most common sensory disorder and is genetically heterogeneous. Apart from nuclear gene mutations, a number of inherited mitochondrial mutations have also been implicated. The m.1555A>G mutation in the mitochondrial MT‐RNR1 gene is reported as the most common mutation causing nonsyndromic hearing loss in various ethnic populations. We report here for the first time the clinical, genetic and molecular characterisation of a single large five‐generational Tamil‐speaking South Indian family with maternally inherited nonsyndromic postlingual hearing loss. Molecular analysis led to identification of m.1555A>G in 28 maternal relatives with variable degree of phenotypic expression. The penetrance of hearing loss among the maternal relatives in this family was 55%. Sequence analysis of the complete mitochondrial genome in 36 members of this pedigree identified 25 known variants and one novel variant co‐transmitted along with m.1555A>G mutation. The mtDNA haplotype analysis revealed that the maternal relatives carry the R*T2 haplotype similar to Europeans and South Asians. Sequencing of the coding exon of GJB2 nuclear gene did not show any pathogenic mutations. The results suggest that other nuclear or environmental modifying factors could have played a role in the differential expression of mutation m.1555A>G in postlingual hearing loss in this family.