Understanding of molecular basis of histological graded horn cancer by transcriptome profiling.

Horn cancer is most devastating and prominent cancer in Indian zebu cattle that affects socio-economic condition of small-scale farmers who depends on their cattle for farm work. Development in the field for genomics through next generation sequencing and bioinformatics advancement have helped to identify genes which have a role in horn cancer development. Histopathological examination of cancerous tissues of horn revealed myxomatous changes, well, moderate and poorly differentiated squamous cell carcinoma. Differential gene expression analysis showed 40, 11, 66 and 29 upregulated genes and 10, 14, 08 and 07 down-regulated genes in myxomatous, well, moderate and poorly differentiated squamous cell carcinoma as compared to normal. Significant differentially expressed genes are related to cell development, cell proliferation, cell-cell communication, cell signaling and angiogenesis which are linked to Akt pathway, mTOR pathway and Wnt pathway. Activity of these genes and related pathways have already been established about their role in development of cancer. Among the candidate genes; keratin family, keratin family related gene, chemokine signaling and cytokines signaling associated genes could be a prominent target for the development of stage specific prognosis marker after further detailed study at large sample population level. CSTA, PTN, SPP1 genes have upregulation in all stages of cancer and they have enrolled as biomarkers for horn cancer.

Molecular portrait of squamous cell carcinoma of the bovine horn evaluated by high-throughput targeted exome sequencing: a preliminary report.

BACKGROUND: Squamous Cell Carcinoma of horn, also known as horn cancer, is a prevailing type of cancer in cattles especially Bos indicus. It is one of the most prevalent disease in Indian bullocks often resulting in death and huge economic losses to farmers. Here, we have reported the use of targeted exome sequencing to identify variants present in horn cancer affected horn mucosa tissue and blood of the same animal to identify some of the prevalent markers of horn cancer. RESULTS: We have observed higher number of variants present in tissue as compared to blood as well as among cancer samples compared to samples from normal animals. Eighty six and 1437 cancer-specific variants were identified among the predicted variants in blood and tissue samples, respectively. Total 25 missense variants were observed distributed over 18 genes. KRT8 gene coding for Keratin8, one of the key constituents of horn, displayed 5 missense variants. Additionally, three other genes involved in apoptosis pathway and two genes involved in antigen presentation and processing also contained variants. CONCLUSIONS: Several genes involved in various apoptotic pathways were found to contain non-synonymous mutations. Keratin8 coding for Keratin, a chief constituent of horn was observed to have the highest number of mutations. In all, we present a preliminary report of mutations observed in horn cancer.

Transcriptome analysis of Clarias magur brain and gonads suggests neuro-endocrine inhibition of milt release from captive GnRH-induced males.

Transcriptome analysis of Clarias magur brain and gonads at preparatory, mature, 6 and 16 h post-GnRH injection (hpi) stages yielded 9.5 GB data with 39,738 contigs. Sequences of 45 reproductive genes were identified for the first time in C. magur along with unique and differentially expressed genes. The expression of 20 genes was validated by qRT-PCR. Upregulation of Cyp11A1, Cyp17A1 and FTZF1 genes in the 16hpi testis accompanied by the 17ß-HSD3 expression indicates testosterone (T) synthesis in response to LH surge, while reduced expression of CYP11B1 suggests a high T: 11-KT ratio. It is evident by the gene expression analysis that the inhibitory neurotransmitter GABA, altered T: 11-KT, increased testicular bile acids, and oxytocin-like neuropeptide in the male brain, appear to be involved in arresting the pulsatile motion of testicular smooth muscles. The work generates important leads for an effective induced breeding strategy for silurid catfish.

Expression profiling revealed keratins and interleukins as potential biomarkers in squamous cell carcinoma of horn in Indian bullocks (Bos indicus).

Horn cancer is most prevalent in Bos indicus and poorly defined genetic landscape makes disease diagnosis and treatment difficult. In this study, RNA-Seq and data analysis using CLC Genomics Workbench was employed to identify biomarkers associated with horn cancer. As a result, a total of 149 genes were found significant differentially expressed in horn cancer samples compared to horn normal samples. The study revealed 'keratins' and 'interleukins' as apex groups of significant differentially expressed genes (DEGs). Functional analysis showed that the upregulated keratins support metastasis of tumor via cell proliferation, migration, and affecting cell stability, while downregulated interleukins along with other associated chemokine receptors deprive the immune response to tumor posing clear path for metastasis of horn cancer. Combi-action of both the group facilitates the tumor microenvironment to reproduce tumorigenesis. Analysis of pathways enriched in DEGs and exemplified protein-protein interaction network indicated actual role of DEGs in horn cancer at a fine level. Important effect of deregulated expression of keratin and interleukin genes in horn cancer enrolling their candidacy as potential biomarkers for horn cancer prognosis. This study appraises the possibility to mitigate horn cancer at fine resolution to extract attainable identification of prognostic molecular portraits.

Transcriptomic analysis to affirm the regulatory role of long non-coding RNA in horn cancer of Indian zebu cattle breed Kankrej (Bos indicus).

Long non-coding RNA (lncRNA) was previously considered as a non-functional transcript, which now established as part of regulatory elements of biological events such as chromosome structure, remodeling, and regulation of gene expression. The study presented here showed the role of lncRNA through differential expression analysis on cancer-related coding genes in horn squamous cell carcinoma of Indian zebu cattle. A total of 10,360 candidate lncRNAs were identified and further analyzed for its coding potential ability using three tools (CPC, CPAT, and PLEK) that provide 8862 common lncRNAs. Pfam analysis of these common lncRNAs gave 8612 potential candidates for lncRNA differential expression analysis. Differential expression analysis showed a total of 59 significantly differentially expressed genes and 19 lncRNAs. Pearson's correlation analysis was used to identify co-expressed mRNA-lncRNAs to established relation of the regulatory role of lncRNAs in horn cancer. We established a positive relation of seven upregulated (XLOC_000016, XLOC_002198, XLOC_002851, XLOC_ 007383, XLOC_010701, XLOC_010272, and XLOC_011517) and one downregulated (XLOC_011302) lncRNAs with eleven genes that are related to keratin family protein, keratin-associated protein family, cornifelin, corneodesmosin, serpin family protein, and metallothionein that have well-established role in squamous cell carcinoma through cellular communication, cell growth, cell invasion, and cell migration. These biological events were found to be related to the MAPK pathway of cell cycle regulation indicating the role of lncRNAs in manipulating cell cycle regulation during horn squamous cell carcinomas that will be useful in identifying molecular portraits related to the development of horn cancer.

Identification and quantification of novel RNA isoforms in horn cancer of Bos indicus by comprehensive RNA-Seq.

Horn cancer (HC) is a squamous cell carcinoma of horn, commonly observed in Bos indicus of the Asian countries. To elucidate the complexity of alternative splicing present in the HC, high-throughput sequencing and analysis of HC and matching horn normal (HN) tissue were carried out. A total of 535,067 and 849,077 reads were analysed after stringent quality filtering for HN and HC, respectively. Cufflinks pipeline for transcriptome analysis revealed 4786 novel splice isoforms comprising 2432 exclusively in HC, 2055 exclusively in HN and 298 in both the conditions. Based on pathway clustering and in silico verification, 102 novel splice isoforms were selected and further analysed with respect to change in protein sequence using Blastp. Finally, fourteen novel splicing events supported both by Cufflinks and UCSC genome browser were selected and confirmed expression by RT-qPCR. Future studies targeted at in-depth characterization of these potential candidate splice isoforms might be helpful in the development of relevant biomarkers for early diagnosis of HC. The results reported in this study refine the available information on transcriptome repertoire of bovine species and boost the research in the line of development of relevant biomarkers for early diagnosis of HC.

In silico analysis of consequences of non-synonymous SNPs of Slc11a2 gene in Indian bovines.

The aim of our study was to analyze the consequences of non-synonymous SNPs in Slc11a2 gene using bioinformatic tools. There is a current need of efficient bioinformatic tools for in-depth analysis of data generated by the next generation sequencing technologies. SNPs are known to play an imperative role in understanding the genetic basis of many genetic diseases. Slc11a2 is one of the major metal transporter families in mammals and plays a critical role in host defenses. In this study, we performed a comprehensive analysis of the impact of all non-synonymous SNPs in this gene using multiple tools like SIFT, PROVEAN, I-Mutant and PANTHER. Among the total 124 SNPs obtained from amplicon sequencing of Slc11a2 gene by Ion Torrent PGM involving 10 individuals of Gir cattle and Murrah buffalo each, we found 22 non-synonymous. Comparing the prediction of these 4 methods, 5 nsSNPs (G369R, Y374C, A377V, Q385H and N492S) were identified as deleterious. In addition, while tested out for polar interactions with other amino acids in the protein, from above 5, Y374C, Q385H and N492S showed a change in interaction pattern and further confirmed by an increase in total energy after energy minimizations in case of mutant protein compared to the native.

In silico analysis of functional nsSNPs in human TRPC6 gene associated with steroid resistant nephrotic syndrome.

The aim of the present study is to identify functional non-synonymous SNPs of TRPC6 gene using various in silico approaches. These SNPs are believed to have a direct impact on protein stability through conformation changes. Transient receptor potential cation channel-6 (TRPC6) is one of the proteins that plays a key role causing focal segmental glomerulosclerosis (FSGS) associated with the steroid-resistant nephritic syndrome (SRNS). Data of TRPC6 was collected from dbSNP and further used to investigate a damaging effect using SIFT, PolyPhen, PROVEAN, and PANTHER. The comparative analysis predicted that two functional SNPs "rs35857503 at position N157T and rs36111323 at position A404V" showed a damaging effect (score of 0.096-1.00).We modeled the 3D structure of TRPC6 using a SWISS-MODEL workspace and validated it via PROCHECK to get a Ramachandran plot (83.0% residues in the most favored region, 12.7% in additionally allowed regions, 2.3% in a generously allowed region and 2.0% were in a disallowed region). QMEAN (0.311) and MUSTER (10.06) scores were under acceptable limits. Putative functional SNPs that may possibly undergo post-translation modifications were also identified in TRPC6 protein. It was found that mutation at N157T can lead to alteration in glycation whereas mutation at A404V was present at a ligand binding site. Additionally, I-Mutant showed a decrease in stability for these nsSNPs upon mutation, thus suggesting that the N157T and A404V variants of TRPC6 could directly or indirectly destabilize the amino acid interactions causing functional deviations of protein to some extent.

Transcriptome analysis and SNP identification in SCC of horn in (Bos indicus) Indian cattle.

Single Nucleotide Polymorphisms (SNPs) have become the marker of choice for genome wide association studies. In order to provide the best genome coverage for the analysis of disease, production and performance traits, a large number of relatively evenly distributed SNPs are needed. The main objective of present work was to identify large numbers of gene-associated SNPs using high-throughput sequencing in squamous cell carcinoma of horn. RNA-seq analysis was conducted on 2 tissues viz. Horn Cancer (HC) and Horn Normal (HN) in Kankrej breed of cattle. A total of 909,362 reads with average read length of 405 bp for HC and 583,491 reads with average read length of 411 bp for HN were obtained. We found 9532 and 7065 SNPs as well as 1771 and 1172 Indels in HC and HN, respectively, from which, 7889 SNPs and 1736 Indels were uniquely present in HC, 5886 SNPs and 1146 Indels were uniquely present in HN and reported first time in Bos indicus, whereas the rest are already reported in Bos taurus dbSNP database. The gene-associated SNPs and Indels were high in upregulated genes of HC as compared to HN. Analysis of differentially expressed genes was identified, these genes are involved in regulation of cell proliferation, apoptosis, gene transcription, cell survival and metabolism through various metabolic pathways. The result of transcriptome expression profiling was validated using Real Time quantitative PCR in nine randomly selected genes. We identified numbers aberrant signaling pathways responsible for carcinogenesis in HC which are also commonly altered in squamous cell carcinoma (SCC) of lung in human being. We conclude that a large number of altered genes and dysfunction of multiple pathways are involved in the development of Horn Cancer. The present findings contribute to theoretical information for further screening of genes and identification of markers for early diagnosis of HC as well as SNPs identified in this report provide a much needed resource for genetic studies in B. indicus and shall contribute to the development of a high density SNP array. Validation and testing of these SNPs using SNP arrays will form the material basis for gene associated SNPs in HC.

Identification of novel exons and transcripts by comprehensive RNA-Seq of horn cancer transcriptome in Bos indicus.

Horn cancer (HC) is a widely prevalent cancer amongst Bos indicus cattle and frequently associated with economic losses due to reduction in draught and milk production capacity. To identify novel transcripts and characterize HC transcriptome, we employed high throughput RNA-Seq analysis of HC and horn normal (HN) tissue. Total of 584,450 and 920,450 high quality reads were analyzed for HN and HC tissue, respectively, resulting in the identification of 16,803 unique genes comprising of 14,754 annotated genes and 2049 novel transcripts. We found 251 significant differentially expressed genes in HC transcriptome, which includes 52 putative novel transcripts. We validated presence of 23 out of 28 novel transcripts by RT-PCR and confirmed differential expression of 12 novel transcripts through RT-qPCR on 4 samples each of HC and HN. Functional analysis revealed that HC is characterized by deregulation of genes involved in epithelial cell differentiation and complement cascade. Our study will serve as a step further in detailed characterization of HC transcriptome and provide firm base to explore and mitigate HC at finer resolution.

RNA-Seq reveals differentially expressed isoforms and novel splice variants in buccal mucosal cancer.

Buccal mucosal cancer (BMC) is a multifactorial disease with poorly defined genetic profile and prognosis due to late detection stage and unavailability of reliable prognostic markers. To identify aberrant transcriptional events, we employed high throughput RNA-Seq analysis of BMC and normal tissue. Comparative transcriptome analysis with Cufflinks revealed 260 up and 328 down regulated genes whereas, 350 up and 397 down regulated isoforms by at least two folds over buccal normal in BMC. Study revealed 46 splice variants in normal and 106 in cancer, out of which 10 variants were validated with end point RT-PCR. Expression of two isoforms of CD74 was validated using RT-qPCR and found in accordance with RNA-Seq. Further extensive follow up analysis of modulator genes, isoforms and splice variants found in this study, might be useful in deep understanding of pathological changes in BMC and development of prospective intervention strategies.

Identification of novel splice variants in horn cancer by RNA-Seq analysis in Zebu cattle.

Horn cancer accounts for nearly 83% of total tumors found in Indian Zebu cattle, which results in chronic suffering and causes heavy economic losses. Alternative splicing has been frequently implicated in the various types of cancer progression. Utilizing the transcriptome sequence generated by next generation sequencing, we analyzed the transcript data for the presence of alternative splicing using BLAT program and identified 27 alternatively spliced genes, of which 12 spliced variants appeared to be the novel spliced candidates. Protein prediction of these novel spliced variants revealed that splice variation has caused either truncation of protein, insertion/deletion of stretch of amino acids or formation of unique carboxy terminus. The RT-PCR analysis confirmed the expression of 8 of the 12 novel spliced variants observed by transcriptome sequencing. Additionally, altered splicing/expression of these novel candidates between cancer and normal tissues revealed by qPCR suggests their potential involvement in the development of horn cancer.

Identification of novel transcripts deregulated in buccal cancer by RNA-seq.

The differential transcriptome analysis provides better understanding of molecular pathways leading to cancer, which in turn allows designing the effective strategies for diagnosis, therapeutic intervention and prediction of therapeutic outcome. This study describes the transcriptome analysis of buccal cancer and normal tissue by CLC Genomics Workbench from the data generated by Roche's 454 sequencing platform, which identified total of 1797 and 2655 genes uniquely expressed in normal and cancer tissues, respectively with 2466 genes expressed in both tissues. Among the genes expressed in both tissues, 1842 were up-regulated whereas 624 were down-regulated in cancer tissue. Besides transcripts known to be involved in cancer, this study led to the identification of novel transcripts, with significantly altered expression in buccal cancer tissue, providing potential targets for diagnosis and cancer therapeutics. The functional categorization by the KEGG pathway and gene ontology analysis revealed enrichment of differentially expressed transcripts to various pathways leading to cancer, including the p53 signaling pathway. Moreover, the gene ontology analysis unfolded suppression of transcripts involved in actin mediated cell contraction process. The down-regulation of four of these transcripts MYL1, ACTA1, TCAP and DESMIN in buccal cancer were further supported by quantitative PCR signifying its possible implication in the cancer progression.

A preliminary sketch of horn cancer transcriptome in Indian zebu cattle.

Horn cancer, a type of squamous cell carcinoma, in zebu cattle is an expensive affair in Indian agriculture sector, which accounts for 83.34% of total tumors found. In general, cancer tissue confirms considerably different expression patterns when compared to a normal stage. This includes not only up/down regulation, but also, the aberrant gene expression, the presence of different non-coding RNAs (ncRNAs), pseudogenes expression and genes involved in unusual pathways. We employed Roche 454 next generation sequencing platform to sequence Bos indicus cancerous and normal horn tissue transcripts. This resulted into a total of 909,345 high-confidence deep sequencing reads and detected a range of unusual transcriptional events including tumor associated genes. We also validated expression of two of the four tested genes in five other similar tissue samples by RT-qPCR. Further, seven cancer specific non-coding transcripts were accessed and a few of them have been suggested as cancer specific markers. This study for the first time provides primary transcriptome sketch of Bos indicus horn cancer tissue, and also demonstrates the suitability of the 454 sequencer for transcriptome analysis, which supports the concept of varied gene expression in cancerous condition.