GSE106638
RNA-Seq analysis to identify novel genes associated with Neuropilin-1 expression in the BT-474 breast cancer cell model
Background: Neuropilin-1 (NRP-1) is a non-tyrosine kinase glycoprotein receptor that elicits multiple functions depending on the ligand bound. It is known to promote cancer progression however; transcriptome-wide changes triggered by NRP-1 are not defined. In this study we aimed to identify novel molecules associated with NRP-1 expression using a global transcriptomic approach in a recombinant breast cancer cell model. Methods: NRP-1 was stably overexpressed in the BT-474 breast cancer cell line (BT-474 NRP-1) and subjected to next generation RNA sequencing using the Illumina HiSeq 4000 sequencing system. Two replicates each of the control BT-474 and BT474-NRP1 were utilized for sequencing. The count per million (CPM) method was utilized for filtering low counts/noise by NOISeq. The clean reads were mapped to reference genome using HISAT/ Bowtie2 tool. The Fragments Per Kilobase of transcript per Million mapped reads (FPKM) method was utilized to calculate the expression levels. False Detection Rate (FDR) = 0.001, absolute value of Log2 ratio = 2 and consistency between the two replicates were used as the default threshold to identify significant DEGs. Results: A total of 22,655,647 clean reads from 22,914,684 raw sequencing reads were generated upon sequencing. 22 upregulated and 61 downregulated genes were identified. Among the DEGs, 11 upregulated and 11 downregulated genes were shortlisted based on their known roles in cancer and/or EMT for confirmation with real-time qPCR. 2 upregulated genes TNC and TARP and 5 downregulated genes ACE, APOD, DDIT3, P2RX6 and ATF3 indicated consistently differential expression with qPCR. Conclusion: We report for the first time the global transcriptome-wide changes elicited by NRP-1 overexpression in a breast cancer cell model. Our study identified the novel association between several candidate molecules such as TNC, a known oncogene, and NRP-1 expression that may controbute to the tumourigenic role of NRP-1 in breast cancer. Overall design: Transcriptome-wide profile changes were determined on Neuropilin-1 overexpression in the BT-474 breast cancer cell line using Illumina HiSeq4000-based RNA sequencing