J. Carter1, J. Burton1, N. Galbraith1, M. R. Eichenberger1, S. Galandiuk1 1University Of Louisville,Department Of Surgery,Louisville, KY, USA
Introduction:
We have previously identified increased expression of all members of the miR-200 family in colorectal cancer (CRC) as compared to normal colon epithelium cell lines. The members of the miR-200 family are highly enriched in epithelial tissues and they have been linked to several cancers including CRC. Using our screening data, pathway analysis software identified all members of the miR-200 family to have a validated regulatory role upstream of Ras proteins in the MAPK canonical pathway. Further analysis of the miR-200 family confirmed validated target interaction with Ras associated domain-containing protein 2 (RASSF2). RASSF2 is one of six proteins in the RASSF family that is encoded by the RASSF2 gene. RASSF2 is a negative regulator of Ras and binds directly to K-Ras within the Ras effector domain in a GTP-dependent process. RASSF2 has previously been shown to promote apoptosis, cause cell cycle arrest and is regarded as a novel K-Ras-specific effector and potential tumor suppressor. Therefore, we hypothesize that miR-200 family and RASSF2 expression will differ between CRC cell lines and a normal colon epithelial cell line.
Methods:
Four CRC cell lines (SW116, SW480, HT29, T84) and one normal colon epithelial cell line (CCD841) were acquired for use (ATCC®, Manassas, VA). Total RNA was converted to cDNA. Specific TaqMan® miRNA primers and probes for the miR-200 family and RASSF2 mRNA were used to bind to complementary sequences on target cDNA during qRT-PCR (Life Technologies, Carlsbad, CA). All reactions were completed in duplicate. Statistical analysis was performed using a two sample t-test comparing each CRC cell line to the normal colon epithelial cell line. Western blots were performed on cell lysates to identify RASSF2 protein relative concentrations.
Results:
Data are shown in Figure 1. We observed up-regulation of all members of the miR-200 family in CRC cell lines as compared to the normal colon epithelial cell line (p<0.001). Conversely, RASSF2 mRNA was found to be down-regulated in CRC cell lines compared to the normal colon epithelial cell line (p<0.001). RASSF2 protein was absent in 3 of the 4 CRC cell lines and down-regulated in the remaining CRC cell line (Dukes' B, SW480) compared to the normal colon epithelial cell line.
Conclusions:
As hypothesized, miR-200 expression was increased and RASSF2 mRNA and protein was decreased in CRC cell lines, suggesting a potential association between the miR-200 family and RASSF2. Further study into the gain and loss of function of the miR-200 family should be investigated to determine whether the miR-200 directly targets RASSF2 and influences CRC tumorigenesis.
