Detection of cis-SAGe chimeric mRNA in prostate cell lines and clinical tissues.

Abstract

<p><b>A</b>, distribution of 16 fusion RNAs in nuclear vs. cytoplasmic fractions. Traditional protein-coding gene <i>GAPDH</i> and a known long non-coding RNA MALAT1 were used as controls. The protein-coding potential of each fusion is marked below. N: not affecting protein coding, applies to fusions where junction sites fall in the UTR. O: out-of-frame, applies to fusions where the reading frame of the 5′ gene is different from that of the 3′ gene. I: in-frame, the reading frame of 5′ gene is the same as that of 3′ gene. <b>B,</b> qRT-PCR for <i>ADCK4-NUMBL</i> and the parental genes. LNCaP cells were transfected with siRNAs targeting the fusion RNA (si-AN1 and si-AN2). Levels of various transcripts were normalized to that in si-negative control (si-). <b>C,</b> cell motility was measured by wound healing assay. Cells were transfected with siRNAs targeting <i>ADCK4-NUMBL</i> and si-negative control. The changes of the wound size were normalized to that in the si-negative control group (n>3, p<0.0001) <b>D,</b> detection of the 16 cis-SAGe chimeras in RWPE-1 (benign prostate cell), LNCaP, and PC-3 cells by RT-PCR and followed by agarose electrophoresis. GAPDH as internal control. <b>E</b>, summary of the 16 cis-SAGe chimeric RNAs in 14 clinical prostate cancer and normal tissues. STAR software was used to align the chimeras onto the clinical RNA-seq data; samtools and IGV were used to map spanning reads across the fusion junction. Black indicates the absence of a fusion, while red indicates the detection of a fusion.</p