Alteration of BRN3A expression influences the expression of genes involved in the regulation of important biological processes in the HPV induced cervical Cancer
Cervical cancer is the fourth most common cancer worldwide in females. This occurs primarily due to the infection of high-risk human papillomavirus (HPV) although in advanced stages it requires support from host cellular factors. BRN3A is one such host cellular factor that upregulates tumorigenic high-risk HPV expression. The expression of BRN3A in cervical cancer remains high where it enhances tumorigenicity. The effect of BRN3A on HPV-mediated cervical cancer and the underlying mechanism remains obscure. Thus, we have investigated the overall effect of BRN3A on genes associated with cancer-related different biological processes. In approach, we have altered the expression of BRN3A through overexpression and knockdown in cervical cancer cells following transcriptome profiling through next-generation RNA-sequencing. This study revealed a substantial change in the expression of several genes associated with cancer-promoting biological processes including viral processes, immune response, cell-death, cell-proliferation, and different signaling pathways, etc. Additionally, promoter analysis through in silico mode revealed that a total of 32.7% of genes possess BRN3A binding sites at their promoters. Physical interaction of BRN3A with IFITM1, OAS3, ISG15, BCL2L1, and HSP90AB1 genes was also confirmed. Thus, the present study identified molecular targets of BRN3A and provided new insight into the pathogenesis of cervical cancer. According to our knowledge, this is the first report on the effect on eukaryotic transcriptomes after over-expression and knocking down BRN3A. Overall design: Method: Total RNA was isolated from transfected SiHa cells in both conditions: (i) Over expression (OE) of BRN3A was done through expression vector pCDNA3.1; and (ii) knock down (KD) of BRN3A expression was done through siRNA oligos (5'-GCCAUAAUCUGCAACUUCAdTdT-3') in biological treplicates. Further, RNA profiles were generated by sequencing, in triplicate, using Illumina Hiseq2500 platform using 51bp pair-end reads in flowcell (Flowcell ID: C7K52ACXX).This resulted in a sequencing depth of 15-20 million reads. The resulting sequencing fastq files were mapped to the Human genome (hg19) using Tophat with Bowtie. The aligned SAM/BAM file for each was processed for guided transcript assembly using Cufflink 2.1.1 and gene counts were determined. Mapped reads were assembled using Cufflinks. Transcripts from all samples were subjected to merge with Cuffmerge to get final transcriptome assembly across samples. In order to ascertain differential expression of transcripts between different samples, Cuffdiff 2.1.1 was used (Trapnell et al., 2012).