PO-195 Investigation of the roles of CD90 protein in cell migration, invasion, and colony-forming ability of cancer cells

Introduction CD90 (THY1) is a GPI-anchored glycoprotein expressed mainly in neurons, T cells, endothelial cells, fibroblasts, and some cancer cells involving in cell-cell and cell-matrix interactions. It is also considered as a marker for cancer stem cells (CSCs) possessing important roles in oncogenesis, multiple drug resistance, and metastasis. It suggests that CD90 leads to different functions in different tumours. Material and methods In this study, we used CRISPR-Cas9 method in order to knockout CD90 protein expression in three different cancer cell lines, A549, HGC27, and PANC1 cells. Following validation of the successful knockout using flow cytometer, the effect of CD90 on the biological functions of the cell lines was determined. To this end, Boyden chamber, scratch wound healing and Matrigel invasion assays were used to examine the cell migration and invasion of parental and CD90-knockout cells. Furthermore, soft agar assay was performed in order to observe differences in cell tumorigenicity by means of the ability of CD90 + cells to form colonies with respect CD90 − cells. Finally, a microarray-based mRNA expression profiling of parental and CD90 − cells was performed in order to identify genes with altered expression following knocking out CD90. Results and discussions The migration assay in Boyden chamber method revealed that there were significantly more migrated CD90 +cells compared with CD90− cells. The result obtained from scratch wound healing assay was also consistent with the Boyden chamber assay, as the created scratch was filled with cd90 +cells in a shorter time compared with CD90- cells. Similarly, the invasion assay demonstrated the same results as the migration assay. Consequently, it was demonstrated that CD90 + cells exhibited increased migration and invasion capabilities compared with CD90 − cells. The results from the colony formation assay suggest that CD90 +cells have a significantly higher potential to form colonies compared to CD90- cells. mRNA microarray results also revealed that, CD90- cells differentially expressed six genes. All of the genes were downregulated following CD90 knockout. One of these genes which showed more obvious change was TGF-β2 which the studies are ongoing in this regard. Conclusion The primary outcome of the present study was that CD90 promotes cell migration, viability and colony-forming abilities in three cell lines, A549, HGC27, and PANC1 cells. Furthermore, microarray analysis identified six candidate genes for further examination and functional analysis.