Generating high-fidelity cochlear organoids from human pluripotent stem cells.

Mechanosensitive hair cells in the cochlea are responsible for hearing but are vulnerable to damage by genetic mutations and environmental insults. The paucity of human cochlear tissues makes it difficult to study cochlear hair cells. Organoids offer a compelling platform to study scarce tissues in vitro; however, derivation of cochlear cell types has proven non-trivial. Here, using 3D cultures of human pluripotent stem cells, we sought to replicate key differentiation cues of cochlear specification. We found that timed modulations of Sonic Hedgehog and WNT signaling promote ventral gene expression in otic progenitors. Ventralized otic progenitors subsequently give rise to elaborately patterned epithelia containing hair cells with morphology, marker expression, and functional properties consistent with both outer and inner hair cells in the cochlea. These results suggest that early morphogenic cues are sufficient to drive cochlear induction and establish an unprecedented system to model the human auditory organ.

Mitochondrial calcium uniporter is essential for hearing and hair cell preservation in congenic FVB/NJ mice.

Mitochondrial Ca2+ regulates a wide range of cell processes, including morphogenesis, metabolism, excitotoxicity, and survival. In cochlear hair cells, the activation of mechano-electrical transduction and voltage-gated Ca2+ channels result in a large influx of Ca2+. The intracellular rise in Ca2+ is partly balanced by the mitochondria which rapidly uptakes Ca2+ via a highly selective channel comprised of the main pore-forming subunit, the mitochondrial Ca2+ uniporter (MCU), and associated regulatory proteins. MCU thus contributes to Ca2+ buffering, ensuring cytosolic homeostasis, and is posited to have a critical role in hair cell function and hearing. To test this hypothesis, Ca2+ homeostasis in hair cells and cochlear function were investigated in FVB/NJ mice carrying the knockout allele of Mcu (Mcu+/- or Mcu-/-). The Mcu knockout allele, which originated in C57BL/6 strain cosegregated along with Cdh23ahl allele to the FVB/NJ strain, due to the close proximity of these genes. Neither Mcu+/- nor Mcu-/- genotypes affected cochlear development, morphology, or Ca2+ homeostasis of auditory hair cells in the first two postnatal weeks. However, Mcu-/- mice displayed high-frequency hearing impairment as early as 3 weeks postnatal, which then progressed to profound hearing loss at all frequencies in about 6 months. In Mcu+/- mice, significantly elevated ABR thresholds were observed at 6 months and 9 months of age only at 32 kHz frequency. In three-month-old Mcu-/- mice, up to 18% of the outer hair cells and occasionally some inner hair cells were missing in the mid-cochlear region. In conclusion, mitochondrial Ca2+ uniporter is not required for the development of cochlea in mice, but is essential for hearing and hair cell preservation in congenic FVB/NJ mice.

Whole genome analysis of water buffalo and global cattle breeds highlights convergent signatures of domestication.

More people globally depend on the water buffalo than any other domesticated species, and as the most closely related domesticated species to cattle they can provide important insights into the shared evolutionary basis of domestication. Here, we sequence the genomes of 79 water buffalo across seven breeds and compare patterns of between breed selective sweeps with those seen for 294 cattle genomes representing 13 global breeds. The genomic regions under selection between cattle breeds significantly overlap regions linked to stature in human genetic studies, with a disproportionate number of these loci also shown to be under selection between water buffalo breeds. Investigation of potential functional variants in the water buffalo genome identifies a rare example of convergent domestication down to the same mutation having independently occurred and been selected for across domesticated species. Cross-species comparisons of recent selective sweeps can consequently help identify and refine important loci linked to domestication.

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.

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.

Analysis of APOBEC3A/3B germline deletion polymorphism in breast, cervical and oral cancers from South India and its impact on miRNA regulation.

Breast cancer and cervical cancer are the leading causes of death in women worldwide as well as in India, whilst oral cancer is the top most common cancer among Asian especially in Indian men in terms of both incidence and mortality rate. Genetic factors determining the predisposition to cancer are being explored to identify the signature genetic variations associated with these cancers. Recently, a germline deletion polymorphism in APOBEC3 gene cluster which completely deletes APOBEC3B coding region has been studied for its association with cancer risk. We screened the germline deletion polymorphism in 409 cancer patients (224 breast cancer, 88 cervical cancer and 97 oral cancer samples), 478 controls and 239 cervical cancer tissue DNAs of South Indian origin. The results suggest that the APOBEC3A/3B deletion polymorphism is not significantly associated with cancer risk in our study population (OR 0.739, 95 % CI, p value 0.91457). Considering the viral restriction property of APOBEC3s, we also screened cervical cancer tissue DNAs for the human papilloma virus infection. We observed a gradual increase in the frequency of HPV16 infection from AA/BB cases (66.86 %) to AA/-- cases (71.43) which signifies the impact of this deletion polymorphism in HPV infection. In addition, we performed in silico analysis to understand the effect of this polymorphism on miRNA regulation of the APOBEC3A/3B fusion transcript. Only 8 APOBEC3B targeting miRNAs were observed to regulate the fusion transcript of which miR-34b-3p and miR-138-5p were found to be frequently downregulated in cancers suggesting miRNA-mediated deregulation of APOBEC3A expression in cancer patients harbouring this particular deletion polymorphism.

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

BACKGROUND: The 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. METHODS: miRCURY 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. RESULTS: Microarray 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. CONCLUSIONS: The 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.

TERT promoter hot spot mutations are frequent in Indian cervical and oral squamous cell carcinomas.

Squamous cell carcinoma (SCC) of the uterine cervix and oral cavity are most common cancers in India. Telomerase reverse transcriptase (TERT) overexpression is one of the hallmarks for cancer, and activation through promoter mutation C228T and C250T has been reported in variety of tumors and often shown to be associated with aggressive tumors. In the present study, we analyzed these two hot spot mutations in 181 primary tumors of the uterine cervix and oral cavity by direct DNA sequencing and correlated with patient's clinicopathological characteristics. We found relatively high frequency of TERT hot spot mutations in both cervical [21.4 % (30/140)] and oral [31.7 % (13/41)] squamous cell carcinomas. In cervical cancer, TERT promoter mutations were more prevalent (25 %) in human papilloma virus (HPV)-negative cases compared to HPV-positive cases (20.6 %), and both TERT promoter mutation and HPV infection were more commonly observed in advanced stage tumors (77 %). Similarly, the poor and moderately differentiated tumors of the uterine cervix had both the TERT hot spot mutations and HPV (16 and 18) at higher frequency (95.7 %). Interestingly, we observed eight homozygous mutations (six 228TT and two 250TT) only in cervical tumors, and all of them were found to be positive for high-risk HPV. To the best of our knowledge, this is the first study from India reporting high prevalence of TERT promoter mutations in primary tumors of the uterine cervix and oral cavity. Our results suggest that TERT reactivation through promoter mutation either alone or in association with the HPV oncogenes (E6 and E7) could play an important role in the carcinogenesis of cervical and oral cancers.

Down Regulation of miR-34a and miR-143 May Indirectly Inhibit p53 in Oral Squamous Cell Carcinoma: a Pilot Study.

BACKGROUND: Aberrant microRNA expression has been associated with the pathogenesis of a variety of human malignancies including oral squamous cell carcinoma (SCC). In this study, we examined primary oral SCCs for the expression of 6 candidate miRNAs, of which five (miR-34a, miR-143, miR-373, miR-380-5p, and miR- 504) regulate the tumor suppressor TP53 and one (miR-99a) is involved in AKT/mTOR signaling. MATERIALS AND METHODS: Tumor tissues (punch biopsies) were collected from 52 oral cancer patients and as a control, 8 independent adjacent normal tissue samples were also obtained. After RNA isolation, we assessed the mature miRNA levels of the 6 selected candidates against RNU44 and RNU48 as endogenous controls, using specific TaqMan miRNA assays. RESULTS: miR-34a, miR-99a, miR-143 and miR-380-5p were significantly down-regulated in tumors compared to controls. Moreover, high levels of miR-34a were associated with alcohol consumption while those of miR-99a and miR-143 were associated with advanced tumor size. No significant difference was observed in the levels of miR-504 between the tumors and controls whereas miR-373 was below the detection level in all but two tumor samples. CONCLUSIONS: Low levels of miR-380-5p and miR-504 that directly target the 3'UTR of TP53 suggest that p53 may not be repressed by these two miRNAs in OSCC. On the other hand, low levels of miR-34a or miR-143 may relieve MDM4 and SIRT1 or MDM2 respectively, which will sequester p53 indicating an indirect mode of p53 suppression in oral tumors.

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.

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.

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.

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.

Single nucleotide polymorphisms in microRNA binding sites of oncogenes: implications in cancer and pharmacogenomics.

Cancer, a complex genetic disease involving uncontrolled cell proliferation, is caused by inactivation of tumor suppressor genes and activation of oncogenes. A vast majority of these cancer causing genes are known targets of microRNAs (miRNAs) that bind to complementary sequences in 3' untranslated regions (UTR) of messenger RNAs and repress them from translation. Single Nucleotide Polymorphisms (SNPs) occurring naturally in such miRNA binding regions can alter the miRNA:mRNA interaction and can significantly affect gene expression. We hypothesized that 3'UTR SNPs in miRNA binding sites of proto-oncogenes could abrogate their post-transcriptional regulation, resulting in overexpression of oncogenic proteins, tumor initiation, progression, and modulation of drug response in cancer patients. Therefore, we developed a systematic computational pipeline that integrates data from well-established databases, followed stringent selection criteria and identified a panel of 30 high-confidence SNPs that may impair miRNA target sites in the 3' UTR of 54 mRNA transcripts of 24 proto-oncogenes. Further, 8 SNPs amidst them had the potential to determine therapeutic outcome in cancer patients. Functional annotation suggested that altogether these SNPs occur in proto-oncogenes enriched for kinase activities. We provide detailed in silico evidence for the functional effect of these candidate SNPs in various types of cancer.

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.

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.