PO-374 LINC00152 long non-coding RNA promotes the proliferation of SW480 colon carcinoma cells through regulation of cell cycle and WNT signalling pathway

Abstract

IntroductionLong non-coding RNAs (lncRNAs) contribute to different cancers including colorectal cancer (CRC). Previous studies have shown altered LINC00152 expression in CRC, but the detailed mechanism of its effects during CRC development and progression is not well studied. We aimed to study the effects of LINC00152 silencing on the cell cycle regulation and whole transcriptome in colon carcinoma cells. We also analysed the DNA methylation alterations caused by LINC00152 knockdown.Material and methodsLINC00152 were silenced in SW480 colon carcinoma cells using Stealth siRNAs. Flow cytometric cell cycle analysis was performed using propidium-iodide DNA staining. Cyclin D1 protein expression was detected using flow cytometry after labelling with anti-cyclin D1 antibody. The effect of LINC00152 silencing to genome-wide gene expression was studied on Human Transcriptome Array 2.0 microarrays. DNA methylation alterations after LINC00152 knockdown were evaluated using Reduced Representation Bisulfite Sequencing (RRBS) method using NextSeq500 device.Results and discussionsSilencing of LINC00152 significantly suppressed cell growth compared to negative control cells (p<0.05). LINC00152 knockdown caused approximately two-fold increase in apoptosis (48 hour: si-NEG: 4%, si-LINC00152: 8%; 72 hour: si-NEG: 11%, si-LINC00152: 23%) (p<0.05). In parallel with the growth inhibitory effect, silencing of LINC00152 could reduce cyclin D1 expression already after 48 hours, however, significant decrease was detected after 72 hours in the LINC00152 siRNA-treated cells (si-NEG MFI=0.70 and si-LINC00152 MFI=0.56) without attenuation of phospho-S6 protein. Whole transcriptome microarray analysis of LINC00152 silenced cells revealed significant under-expression of genes with oncogenic and/or metastasis promoting function (e.g. STC1, YES1, HES1, KLK6, PORCN) and up-regulation of tumour suppressor genes (e.g. DKK1, PERP) (FDR p<0.05, absolute value of logFC >1). Using RRBS, DNA methylation alterations after LINC00152 silencing could be detected genome-widely including hypomethylation in SFRP2 and ALDH1A3 gene promoters.ConclusionOur results suggest that LINC00152 lncRNA can contribute to CRC pathogenesis by facilitating cell proliferation through up-regulation of several oncogenes/metastatic genes in WNT, Notch and TP53 pathways and of cyclin D1 cell cycle progression gene, furthermore, by affecting the promoter methylation status of certain CRC associated genes.