Orsolya Galamb1,2, Alexandra Kalmár1,2, Anna Sebestyén3, Titanilla Dankó3, Csilla Tolnai-Kriston3, Barnabás Wichmann1,2, Gábor Barna3, Zsolt Tulassay1, Péter Igaz1,2, Béla Molnár1
1 Molecular Medicine Research Group, Hungarian Academy of Sciences, Budapest
2 Semmelweis University, 2nd Department of Internal Medicine, Budapest
3 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest
Aims: Long non-coding RNAs (lncRNAs) contribute to different cancers including colorectal cancer (CRC). Altered LINC00152 expression in CRC was described, but the 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 analyzed the DNA methylation alterations caused by LINC00152 knockdown.
Method: LINC00152 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. 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.
Results: Silencing of LINC00152 significantly suppressed cell growth compared to negative control cells (p<0.05). LINC00152 knockdown caused approximately two-fold increase in apoptosis (48H: si-NEG:4%, si-LINC00152:8%; 72H: si-NEG:11%, si-LINC00152:23%) (p<0.05). 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 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 tumor 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.
Conclusion: Our 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.
New National Excellence Program of The Ministry of Human Capacities ÚNKP 17-4
Young Researcher Fellowship (ÚNKP-17-4-III-SE-51)
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