Investigating the role of the protein methyltransferases EHMT2 and PRMT5 on the epithelial-to-mesenchymal transition of colorectal cancer

Abstract

The epithelial-to-mesenchymal transition (EMT) is a process by which cancer cells lose their epithelial characteristics and adopt a mesenchymal morphology. EMT is associated with chemoresistance. Dysregulation in protein methylation is associated with EMT in a number of cancer subtypes. This study investigated the influence of 5-fluorouracil (5-FU) resistance on EMT and protein methylation in colorectal cancer (CRC). Four 5-FU resistant CRC cell lines were established from parental cells through dose-escalation exposure of 5-FU. The resistant cells showed several morphological changes throughout the course of treatment. Resistance to 5-FU was confirmed through a cell viability assay. Wound healing and invasion assays demonstrated that the resistant cells experienced a decrease in migration and invasion except for one resistant cell line which displayed higher invasion. Using RT-PCR, the transcript levels of five protein methyltransferases (PMTs) were found to be dysregulated in the resistant cells. Western blotting showed that the protein expressions of EHMT2, PRMT5 and SETD7/9 decreased and that global protein methylation became dysregulated as the cells gained resistance. Western blotting demonstrated that the protein levels of E-cadherin, vimentin and Snail decreased in the resistant cell lines. This study showed that chemoresistance was not associated with EMT. Protein methylation and dysregulation of PMT expression may in part be responsible for the development of 5-FU resistance in the CRC cells. The second part of the project focused on overexpressing EHMT2 and PRMT5 in CRC cells to study their effects on EMT. Attempts were made to utilize molecular cloning techniques to generate EHMT2 and PRMT5 pcDNA3.1 plasmids. EHMT2 pLX304 and pLENTI3.4/V5-DEST plasmids were also purchased and were transfected directly into CRC cells. Through sub-cloning procedures, FLAG-tagged PRMT5 pcDNA3.1 plasmids were generated and transfected into SW480 cells. Western blotting did not reveal the overexpression of PRMT5. The overexpression of the PMTs was also investigated using small-activating RNAs, however the overexpression of the two PMTs was also not detected. It is still unclear whether EHMT2 or PRMT5 induce EMT in CRC.