Short Communication Identification of novel transcripts deregulated in buccal cancer by RNA-seq Manisha R. Sajnani a, 1 , Amrutlal K. Patel a, 1 , Vaibhav D. Bhatt a , Ajai K. Tripathi a , Viral B. Ahir a , Vangipuram Shankar b , Siddharth Shah b , Tejas M. Shah a , Prakash G. Koringa a , Subhash J. Jakhesara a , Chaitanya G. Joshi a, ⁎ a Department of Animal Biotechnology, College of Veterinary Science & Animal Husbandry, Anand Agricultural University, Anand-388 001, Gujarat, India b M. S. Patel Cancer Center, Pramukhswami Medical College, Karamsad, Anand-388 001, Gujarat, India abstract article info Article history: Accepted 18 July 2012 Available online 27 July 2012 Keywords: Buccal cancer RNA-seq Biomarkers RT-qPCR KEGG pathway Gene ontology The differential transcriptome analysis provides better understanding of molecular pathways leading to can- cer, which in turn allows designing the effective strategies for diagnosis, therapeutic intervention and predic- tion 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 var- ious 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 quan- titative PCR signifying its possible implication in the cancer progression. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Squamous cell carcinoma of the buccal mucosa is the commonest of the oral cavity cancers in men in the Indian subcontinent (Govekar et al., 2009). About 13,500 cases were reported during 1990–96 in the various Indian cancer registries, compared to 1272 cases registered worldwide during this six year period (Parkin et al., 2001). The high prevalence of oral cancer is largely attributable to smoking, betel chewing and the usage of tobacco in its various forms. The incidences of oral cancers are increasing worldwide with a global estimate of 300,000 cases now diagnosed every year (Rautava et al., 2007; Sharma et al., 2010). Recently, attempts have been made to iden- tify the potential therapeutic targets through large scale gene expres- sion profiling of various oral cancers including head and neck (Han et al., 2009), tongue (Estilo et al., 2009; Ye et al., 2008), and oral squamous cell carcinoma (Tuch et al., 2010). Tumors involving different sites within the oral cavity may vary in their expression profile, clinical presentations as well as outcomes (Govekar et al., 2009; Rautava et al., 2007; Ye et al., 2008). The development of cancer is a multistep process involving accu- mulation of a wide range of genetic and phenotypic alterations which in turn leads to the aberrant expression of genes regulating cell proliferation. Although microarray analyses have led to the iden- tification of genes that are significantly altered in oral cancers (Estilo et al., 2009; Han et al., 2009; Ye et al., 2008), it has limited sensitivity as well as being prone to cross hybridization between homologous DNA fragments (Tuch et al., 2010). The low-cost and rapid sequencing demand has led to the development of high throughput next generation sequencing (NGS) technologies such as Roche's 454 GS-FLX, Illumina's Solexa, Applied Biosystems' SOLiD, and Helicos HeliScope platforms for the qualitative and quantitative analyses of whole genomes as well as transcriptomes (Morozova et al., 2009). As an alternative to microar- ray, these technologies can be successfully employed to analyze the transcriptome, quantify gene expression levels, and identify novel splice variants and genetic alterations in the coding sequences (Morozova et al., 2009; Tuch et al., 2010). This has revolutionized our ability to characterize cancers at the molecular level by measuring gene expression and structural variation across the entire genome. Although, prevalence of buccal cancer is high in many parts of the world, studies describing detail cancer-specific molecular profile have not yet been reported. The present study reports the application of Gene 507 (2012) 152–158 Abbreviations: NGS, next-generation sequencing; RPKM, reads per kilobase of exon model per million mapped reads; bp, base pair; SOLiD, sequencing by oligonucleotide ligation and detection; RT-qPCR, real time quantitative reverse transcriptase polymer- ase chain reaction; KEGG, Kyoto encyclopedia of genes and genomes. ⁎ Corresponding author. Tel./fax: +91 2692 261201. E-mail address: cgjoshi@rediffmail.com (C.G. Joshi). 1 Authors have equal contributions. 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.07.036 Contents lists available at SciVerse ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene