Abstract 1172: The impact of siRNA targeting Colon Cancer Associated Transcript 1 (CCAT-1) on proliferation of HT-29 cells

Abstract

Abstract Introduction: Colon cancer Associated Transcript-1 (CCAT-1) is a non-coding RNA transcript highly expressed in colorectal cancer (CRC) but not in normal colonic tissue. The role of CCAT-1 in tumorigenesis is yet to be defined. Small interfering RNAs (siRNA) are a group of sequence-specific posttranscriptional gene silencing molecules. Recent advances in the design and delivery of targeting siRNA molecules allows efficient and highly specific gene silencing in mammalian systems. The most common application of siRNA gene-silencing in oncology is to identify loss-of-function phenotypes in genes that will result in decreased proliferation or death of cancer cells. Aim: To study the role of colon cancer associated transcript 1 (CCAT-1) in proliferation of human colon cancer cells using RNA interference. Methods: Small interfering RNAs (siRNA) targeting CCAT-1 were transfected into HT-29 colon cancer cells. At day one, 80×105 HT-29 cells per well were plated and suspended in RPMi 1640 medium. At day two, 50 pmol CCAT-1 siRNA and 0.5 µl lipofectamine 2000 were added to each well. CCAT-1 expression was studied by real-time PCR in order to evaluate gene silencing. Cell proliferation was studied by MTT incorporation assay. Twenty four hrs after transfection, 50 µl of MTT were added to the cells media and incubated for 4 h at 37° C. The purple Formazan product, converted MTT, was dissolved by the addition of 150µl/well DMSO and OD readings were obtained at 550 nm. Results:By examining AV average OD readings at 550 nm, untreated HT-29 cells (10 wells), cells treated with lipofectamine 2000 (10 wells) and cells silenced by siRNA of CCAT-1 (10 wells) showed a significant decrease in proliferation: 2.4±0.64, 1.54±0.63, 0.8±0.14 respectively (p=0.002). Furthermore, comparing AV OD readings between 24 and 48 h in the silenced cells, showed a decrease of 38% in proliferation. This was compared to untransfected cells and cells treated with lipofectamine 2000 which showed an increase of 39% and 22% respectively. Twenty four hrs after transfection, real time PCR results showed a reduction in CCAT-1 RNA levels of 70% compared to controls and 48 hrs after transfection, CCAT-1 RNA levels were reduced to 55% of baseline. Conclusions: Our results may support a role for CCAT-1 in the regulation of colon cancer cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1172. doi:10.1158/1538-7445.AM2011-1172