J. Burton1, J. Carter1, K. Ramos2, B. G. Oxford3, M. R. Eichenberger1, S. Galandiuk1 1University Of Louisville,Department Of Surgery,Louisville, KY, USA 2University Of Arizona,College Of Medicine,Tuscon, AZ, USA 3University Of Louisville,School Of Medicine,Louisville, KY, USA
Introduction:
Chromosomal instability (CIN) is the major molecular pathway associated with development of sporadic colorectal cancer (CRC). With increasing CIN, mutations in the KRAS gene contribute to the progression from normal mucosa to invasive CRC. K-Ras protein is a product of the KRAS gene and acts within the mitogen-activated protein kinase (MAPK) pathway, a major regulator of cell proliferation.The miR-200 family has been linked to several cancers, including CRC, and is known to target a negative regulator of K-Ras, RASSF2. We have previously identified increased expression of all members of the miR-200 family and decreased expression of RASSF2 in CRC cell lines as compared to a normal colon epithelial cell line. Therefore, we hypothesize that the miR-200 family regulates cell proliferation through targeting of this negative regulator and subsequent K-Ras activity in the MAPK pathway.
Methods:
A K-Ras wild type CRC cell line (HT29) and normal colon epithelial cell line (CCD841) were acquired (ATCC®, Manassas, VA). Cells were grown in appropriate culture medium until confluent. Once grown, cells were harvested and plated into separate wells on a 12-well plate at a concentration of 100,000 cells/mL culture media and then allowed a 24-hour period to adhere. At 24 hours, cells were serum starved for 2 hours and transfected with miR-200 family mimics or antagomirs, and their respective negative controls, using the Lipofectamine®RNAiMAX Transfection Reagent protocol (ThermoFisher Scientific, Waltham, MA). Transfection was stopped after 24 hours and cell number was measured each day for 5 days using an automated cell counter (TC20™ Bio-Rad, Hercules, CA).
Results:
Confirmation of successful transfection was performed. CCD841 cells had significantly increased proliferation when transfected with miRNA mimics of all members of the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) both individually and combined, as compared to the negative control at day 5 (p<0.05). Conversely, we observed a significant decrease in cell proliferation at days 4 and 5 when HT29 cells were transfected with miR-200 antagomirs for the miR-200 family members both individually and combined, compared to the negative control (p<0.05). A representative growth curve is shown in Figure 1.
Conclusions:
These findings suggest that the gain and loss of function of the miR-200 family affects cell proliferation. Overexpression of the miR-200 family increases cell proliferation, an important process in tumor growth, whereas silencing of the miR-200 family decreases cell proliferation, reducing neoplastic progression. miR-200 should be investigated further as a potential therapeutic target in the treatment of colorectal cancer.
