Abstract 1964: NcRNAs fingerprints in colon cancer in vitro model by EGCG, genistein and their combinatorial effects
Authors: Berindan Neagoe Ioana , Pileczki Valentina , Braicu Cornelia , Ivan Cristina , Zhang Xinna , Calin George Adrian 
Published: July 2016

INTRODUCTION

Epidemiological studies have associated green tea intake with reduced risk in cancer. Up to date, thirty-five clinical trials use green tea polyphenol epigallocathechin-3-O-gallate (EGCG) in cancer treatment. The mechanism responsible for apoptosis and the perturbation of the noncoding transcriptome after polyphenols are largely unknown. Our aim was to examine the combined effects of two compounds, EGCG and Genistein alone or in various combinations, on two colon cancer cell lines on the expression of noncodingRNAs.

MATERIAL AND METHODS

The experiments were performed on two colon cancer cell lines RKO (p53+ wild type) and HCT116 (Ras mutated and transforming growth factor beta 1 (TGF beta 1) and beta 2 (TGF beta 2) expression positive) In order to establish the IC50 dose we performed MTT assay. For future analysis, cells were treated for 48 h either with Genistein and EGCG with established IC50 doses for both cell lines or the following combinations: 30 μM EGCG and 10 μM Genistein and 30 μM Genistein and 10 μM EGCG. We performed genome-wide profiling with a non-codingRNA array composed by probes for about 1500 human miRNAs and about 3500 lncRNAs, and ncRNA microarray analyses were performed.

RESULTS

After 48h exposer, both compounds effectively reduced cell viability in a dose-dependent manner in RKO and HCT116 cells. In the combined treatment the most interesting results were obtained at 30 μM EGCG and 10 μM Genistein and 30 μM Genistein and 10 μM EGCG, in both cell lines. Were we observed a 80% decrease of cell viability in the first situation and more than 80% of the cells maintained their viability in thesecond one.

Bioinformatic analysis was performed using R (version 3.0.1) and Bioconductor (h&gt;p:// www.bioconductor.org/). The raw intensity for each probe is the median feature pixel intensity with the median background subtracted.

We found eighteen miRNAs and six lncRNAs commonly DE in HCT 116 cells and 56 miRNAs and 14 lncRNAs commonly DE in RKO cells by various treatments. Among the former genes we identified the oncogenic miR-18 and miR-222, as well as the long-non-codingRNAs CCAT2 that we previously identified as oncogenic in colon cancer, and MALAT-1. Furthermore, we recognized multiple ncRNA transcripts specifically disregulated by one specific type of treatment.

The expression of specific miRNAs was negatively correlated with some lncRNAs supporting a potential direct interaction by sequence complementarity, while for other miRNA-lncRNA pairs the expression was positively correlated suggesting common transcriptional mechanisms. We confirmed the array data by qRT- PCR and a restricted panel of confirmed ncRNAs was tested on the phenotypic effects in colon cancer cells.

CONCLUSION

This is the first study to prove that EGCG and /or genistein alone or in combination affect the expression of multiple types of noncodingRNAs in a correlated manner; their transcriptomic effects are more extensive as initially expected.

Citation Format: Ioana Berindan Neagoe, Valentina Pileczki, Cornelia Braicu, Cristina Ivan, Xinna Zhang, George Adrian Calin. NcRNAs fingerprints in colon cancer in vitro model by EGCG, genistein and their combinatorial effects. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1964.