H. L. Roberts1, M. McClain1, J. Rice1, J. Carter1, J. Burton1, S. Galandiuk1 1University Of Louisville School Of Medicine,Department Of Surgery, Division Of Colorectal Surgery,Louisville, KY, USA
Introduction:
Sporadic colon cancer (CC) is commonly caused by chromosomal instability, while hereditary non-polyposis colorectal cancer (HNPCC) is characterized by microsatellite instability (MSI). HNPCC is associated with different clinical manifestations and improved survival but respond poorly to 5-FU based chemotherapy compared to sporadic CC patients.
HCT116 is a widely used experimental Dukes’ D CC cell line not commonly known to be derived from an HNPCC patient. A molecular characteristic of HNPCC is inherited mutations in DNA mismatch repair (MMR) leading to MSI. We have demonstrated different microRNA (miR) expression patterns between HCT116 and other sporadic CC cell lines with respect to miR-99a. This miR is an established inhibitor of mammalian Target of Rapamycin (mTOR). Increases in mTOR protien have been shown to increase cell growth, proliferation, migration, invasion and decrease apoptosis. Thus miR-99a is a potential pathway for CC treatment and metasistis. We hypothesize the influence of miR-99a on the mTOR pathway, with respect to cell proliferation and motility, is dissimilar between MMR proficient (MMR+) and deficient (MMR-) CC cell lines.
Methods:
Dukes C and Dukes D MMR- (HCT15, HCT116), MMR+ (HT29, T84) and normal colon epithelium (CCD841) cell lines (ATCC®) were transfected with miR-99a mimic (99M) and a negative control (M). Transfection efficiency was verified via qPCR. mRNA and protein substrates of both mTOR complexes were analyzed by qPCR and western blot, respectively. Functional assays were performed measuring cell migration and invasion.
Results:
All cell lines were successfully transfected and showing significant upregulation of miR-99a (p<0.001). mRNA levels of proteins of interest were unchanged for all cell lines measured irrespective of transfection group. After transfection with 99M, total mTOR protein was decreased as compared to M- for all cell lines. Migration decreased after transfection with 99M for all cell lines as compared to M- except for the HCT116 cell line (Figure 1). Invasion assays showed no difference in either transfection group.
Conclusion:
HCT116 showed increased migration following transfection with 99M as compared to M- whereas all other cell lines exhibited decreased migration regardless of MMR status and CC stage of the cell line. We intend to investigate further to identify different pathways involved in HNPCC that may permit development of more effective adjuvant therapy for MMR deficient cancer patients with advanced disease.
