GDPD5, a target of miR-195-5p, is associated with metastasis and chemoresistance in colorectal cancer

Abstract

Purpose: Successful treatment of colorectal cancer (CRC) is greatly impeded by metastasis and chemoresistance, particularly to 5-fluoruracil (5-Fu), one of the staples of clinical intervention in advanced CRC. The purpose of this study is to determine whether the gene glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5) contributes to CRC cell metastasis and 5-Fu resistance. Ultimately, we evaluated the ability of microRNA 195-5p (miR-195-5p) to reduce GDPD5 expression and thus to enhance CRC cell susceptibility to chemotherapy.

Methods: We obtained human primary CRC and adjacent normal tissues from 15 patients who underwent CRC resection. Quantitative real-time polymerase chain reactions (qRT-PCR), western blot, and immunohistochemistry (IHC) were used to determine GDPD5 expression levels in the human CRC tissues, four CRC cell lines, and two 5-Fu resistant CRC cell lines. To measure the impact of GDPD5 on CRC cell chemoresistance, we silenced GDPD5 using GDPD5 siRNA and measured cell survival and apoptosis using MTT assays and TUNEL staining, respectively. Transwell Matrigel invasion assays were performed to explore whether GDPD5 affects the invasion capabilities of 5-Fu resistant CRC cells. Finally, to assess the effects of miR-195-5p on GDPD5 levels and CRC cell chemoresistance, we generated luciferase reporter plasmids with either the wild-type 3'UTR miR-195-5p potential binding sites or mutant binding sites of GDPD5.

Results: We first determined that GDPD5 mRNA and protein are overexpressed in human primary CRC tissues as compared to adjacent normal tissues. GDPD5 overexpression was also evident in four CRC cell lines (Caco-2, HCT8, HCT116 and SW480), as well as in the two 5-Fu resistant CRC cell lines that we generated (HCT116/5-Fu and SW480/5-Fu). Using MTT assays and TUNEL staining of HCT116/5-Fu and SW480/5-Fu cells, we found that GDPD5 silencing sensitizes 5-Fu resistant CRC cells to 5-Fu. Furthermore, GDPD5 silencing in 5-Fu resistant CRC cells reduced the epithelial-to-mesenchymal transition (EMT) and cell invasion, both of which are crucial for CRC metastasis. We then used the bioinformatics algorithm TargetScan to identify the miRNA miR-195-5p, which targets two regions of GDPD5 3'UTR. By generating luciferase reporter plasmids with the 3'UTR miR-195-5p binding sites, we ultimately determined that miR-195-5p increases chemosensitivity and cell apoptosis in 5-Fu resistant CRC cells.

Conclusion: This study identifies the gene GDPD5 as an effector of chemoresistance and metastasis in CRC. Furthermore, our results demonstrate that miR-195-5p is a potent suppressor of GDPD5 and that, as such, it significantly increases chemosensitivity and apoptosis in chemoresistant CRC cells. This study thus not only identifies potential prognostic biomarkers of CRC, but it also opens the possibility for incorporating miR-195-5p into current therapeutic regimens to overcome barriers to successful CRC treatment.

Keywords: Chemoresistance; Colorectal cancer; GDPD5; Metastasis; miR-195-5p.