BioProject

Purpose: We investigated the function of overexpressed BCL2L14-ETV6 fusion variants in triple-negative breast cancer using high-throughput mRNA sequencing (RNA-Seq) analysis in BT20 human basal-like breast cancer cell line. More...

Purpose: We investigated the function of overexpressed BCL2L14-ETV6 fusion variants in triple-negative breast cancer using high-throughput mRNA sequencing (RNA-Seq) analysis in BT20 human basal-like breast cancer cell line. Methods: The BCL2L14-ETV6 fusion variants or wildtype ETV6 cDNA containing the full-length ORFs were subcloned into the lentiviral pLenti7.3 vector, and later transduced into the BT20 cell line. BT20 cells were cultured in EMEM (ATCC) with 10 % fetal bovine serum (Hyclone). Total RNA was extracted from cells in individual confluent 10 cm dish, using the standard procedure of Qiagen RNeasy kit. The NovaSeq 6000 library for DNA sequencing was prepared using TruSeq Stranded mRNA Library Prep Kit (Illumina) following the protocol provided by the manufacturer. The final libraries were normalized by quantification with LightCycler 480 II (Roche Applied Science, Indianapolis, IN, USA) and qualification with Bioanalyzer (Agilent, Palo Alto, CA, USA). The final loading concentration was adjusted to 10 pM following the NovaSeq 6000 loading protocol. The NovaSeq 6000 S2 Reagent Kit (Illumina) was used for paired-end reads (2×150 bp) sequencing reactions. Sequencing data was given as raw data with a Phred Q30 score of 80 or better. Results: Confirming the overexpression of BCL2L14-ETV6 fusion variants, the reads that match to partial of BCL2L14 and ETV6 mRNA transcripts were 32~256 fold higher in the stable BT20 cell lines expressing BCL2L14-ETV6 fusion, compared to the cells the express the vector control. We found some key genes in cell migration and invasion are up-regulated in these stable BT20 cells compared with the cells the express the vector control and the wildtype ETV6. Conclusions: BCL2L14-ETV6 fusions endow enhanced cell migration and invasion in breast cancer. Overall design: We generated the stable BT20 cell models by transduction of the lentivirus containing either the pLenti7.3 vector control expresssing the lacZ gene, wildtype ETV6 cDNA, or three BCL2L14-ETV6 fusion variants E2E3 (BCL2L14-Exon2_ETV6-Exon3), E4E3 (BCL2L14-Exon4_ETV6-Exon3) and E4E2 (BCL2L14-Exon4_ETV6-Exon2). Each group was triplicated in the RNAseq expriments. Less...