Detection of Long Intervening Non-coding RNA - CCAT1 in Living Cells and Human Tissues by Forced Intercalation - Peptide Nucleic Acids (FIT-PNAs)

Background: Colorectal Cancer Associated Transcript-1 [CCAT1] is a 2628 nucleotide-long non-coding RNA, described by our group, located on chromosome 8q24.21, up stream to MYC oncogene. We showed by qRT-PCR, that CCAT1 is significantly up-regulated in colon cancer including: adenomatous polyps, primary tumor, normal colonic mucosa adjacent to primary tumor, lymph node metastasis and liver as well as peritoneal metastases. Importantly, no expression was detected in normal colonic tissue, making CCAT1 a potential cancer-specific marker, especially for early cancer detection. Peptide Nucleic Acids [PNAs] are DNA mimics that consist of a neutral backbone allowing excellent hybridization to complementary DNA and RNA targets. BisQ is a novel surrogate base that when incorporated into a PNA sequence [forced intercalation (FIT)-PNA], turns on its fluorescence upon hybridization to a complementary RNA/DNA sequence.

Material and methods: Fresh human tissues of colon cancer peritoneal metastasis were obtained from cytoreductive surgery [n=18] and normal samples were taken from bariatric [n=4] surgery. We performed hybridization analysis for both groups, using confocal microscope. CCAT1 FIT-PNA solution was spray directly on top of the tissues. The samples were also stained for H&E to determine their malignancy and CCAT1 expression levels were measured using qRT-PCR.

Results and discussion: The developed FIT-PNA selectively emits a red [613nm] fluorescence signal [BisQ] when it hybridizes to CCAT1 RNA in living cells. We showed very high specific flourescence of the FIT-PNA in fresh human tissues obtained from peritoneal metastasis of colon cancer, by simply spraying a solution of CCAT1 FIT-PNA on the fresh tissue. This signal was observed within minutes. No flourescence observed when the CCAT1_FIT-PNA was sprayed on normal peritoneum samples used as negative controls. CCAT1 expression levels were confiremed by qRT-PCR and histoloigcal diagnosis was validated by H&E staining and second opinion pathology review. These results highlight the potential role of such FIT-PNA probes for flourescence guided surgery [FGS], using CCAT1 and other potential biomarkers.

Conclusion: CCAT1 may serves as a specific colorectal cancer biomarker when targeted in a real-time manner by FIT-PNA. It may play a role in Flourescence–guided surgery, while detecting and thus eliminating occultmetastasis.