V. Stephen1, J. Carter1, J. Hallion1, M. R. Eichenberger1, E. M. Hattab2, S. Galandiuk1 1University Of Louisville,Department Of Surgery,Louisville, KY, USA 2University Of Louisville,Department Of Pathology,Louisville, KY, USA
Introduction:
Colorectal cancer (CRC) is the second leading cause of cancer-related death in the U.S. MicroRNAs (miRNAs) are small, non-coding RNA molecules that downregulate protein expression and have a role in carcinogenesis by dysregulating oncogenes and tumor suppressor genes. MiRNAs from the miR-200 family (miR-200a, -200b, -200c, -141, -429) are associated with many cancers, playing a role in blocking epithelial to mesenchymal transition (EMT). RASSF2, another target of the miR-200 family, is a negative regulator for the known proto-oncogene K-Ras. We have previously observed that miR-200 is upregulated and RASSF2 is downregulated in CRC cell lines and that modulating miR-200 expression can alter cell proliferation in CRC cell lines. We expanded upon this work by examining miR-200 and RASSF2 expression in fresh frozen CRC tissue and histologically normal adjacent epithelial tissue obtained from CRC patient resection specimens.
Methods:
CRC and normal adjacent tissue samples were obtained from 5 patients diagnosed with stage III CRC from our institution's biorepository. Specimens were cut onto slides and stained. Cells of interest (cancer or normal epithelium) were captured using the ArcturusXT Laser Capture Microdissection System, using H&E slides as reference (Fig. 1A). RNA was then extracted and expression of miR-200 and RASSF2 mRNA was measured via qRT-PCR. Protein was extracted and measured via Western Blot.
Results:
We observed a downregulation of miR-200a, miR-200b, miR-141, and miR-429 (p=0.047, 0.119, 0.007, and 0.027 respectively), and no change in miR-200c (p=0.589) in CRC tissue samples compared to normal adjacent epithelium (Fig. 1B). We also observed a 2.7-fold downregulation of RASSF2 mRNA (p=0.021) (Fig. 1C) and a 6-fold downregulation of RASSF2 protein in CRC tissue relative to normal adjacent epithelium (p=0.092) (Fig. 1D).
Conclusion:
Although our data concerning RASSF2 were consistent with those obtained from CRC cell lines, that for miR-200 was not. RASSF2 is a negative regulator of K-Ras, an oncogenic signaling protein. It is advantageous for cancer cells to downregulate RASSF2 expression in any environment. MiR-200, on the other hand, has two dichotomous roles. Upregulation may be able to block RASSF2, but downregulation helps induce EMT. In cell culture, there is no advantage in promoting EMT and metastasis, but this advantage exists in vivo, particularly in later or more aggressive cancers. Further investigations considering earlier stages of CRC may shed light on this discrepancy. Although we were able to alter RASSF2 expression and cell proliferation by modulating miR-200 expression in cell culture, these data from human tissue identify limitations of the miR-200 family as a therapeutic target in CRC.
