Poster Session - Abstract # 10

Role of Rab27A in Cell Growth of Colorectal Cancer Cells

Harsha Hapugaswatta, Dan A. Dixon

Department of Molecular Biosciences, The university of Kansas, Lawrence, KS, USA

Rab proteins are small GTPase molecules that belong to Ras protein super family. Rab27 subfamily consists two isoforms, Rab27A and Rab27B, that are shown to play major roles in multivesicular body (MVB) trafficking in cells.  Rab27B regulates trafficking of early endosomes whereas Rab27A regulates trafficking of late endosomes.  Our lab has recently discovered that silencing of Rab27B in CRC cells HCT116 and SW480 resulted in dysregulation of cellular recycling system- autophagy leading to suppression of tumor growth.  Rab27A is an isoform of Rab27B with seventy-one percent amino acid content similarity.  Therefore, we intend to investigate the role of Rab27A in CRC cells.  A CRISPR/Cas9 method was used to delete the Rab27A gene from HCT116 cells to create a Rab27A knockout cell line.  The absence of Rab27A was confirmed by qRT-PCR, western blot and immunofluorescence assay.  The elimination of Rab27A by CRISPR/Cas9 did not show the similar autophagy defects as observed in the Rab27B knockout cells.  However, we observed a significant reduction in cell growth in Rab27A knockout cells (KO) when compared to HCT116 wild-type cells.  MTT assay showed a significant (p<0.01) four-fold decrease in cell viability in Rab27A knockout cells.  We confirmed this result using trypan blue cell count observing two-fold decrease in cell growth.  We performed Rab27A knockdown(KD) in HCT116 cells to confirm the cell growth defect is not a result of CRISPR/Cas9 method.  Similar to our knockout cells, Rab27A knocked down cells also showed significant (p<0.05) two-fold decrease in cell viability.  Therefore, future studies will be focused on investigating the role of Rab27A in cell viability and cell growth in CRC cells.  We will also examine the effect of eliminating Rab27A on extracellular vesicle secretion in CRC cells.  Future studies will focus on understanding the mechanism of how Rab27A contributes to cell growth in CRC as well as analyzing the effect of Rab27A knockout on tumor growth in vivo in a xenograft study.