Elucidating the role of a Rho GTPase-activating protein, DLC1, in melanoma development

Abstract

Aim: (1).To examine the effects of DLC1 overexpression on melanoma markers expression. (2).To investigate the role of DLC1 in melanoma growth; (3).To investigate the role of DLC1 in melanoma metastasis.

Methods: Transduction of A375 melanoma cell line with lentivirus carrying the wildtype DLC1 or the Rho-GAP mutant version of DLC1-K714E. The A375 cell transduced with lentivirus without carrying any gene as negative control. After 72 hours post-transduction, I examined the effects of overexpression of DLC1 or its Rho-GAP mutant on expression levels of markers characteristic of melanoma and epithelial-mesenchymal transition by real-time qPCR and Western blot. Colony forming assay test was conducted to assess the viability of A375 cells in different treatment groups. Wound healing test was conducted to detect the migration capacity of the A375 cells after over-expressing wildtype DLC1 or mutant DLC1-K714E.

Results: Real-time qPCR results showed that higher E-Cadherin expression was detected in the mutant DLC1-K714E than in the control. But there’s no similar phenomenon in the wildtype DLC1 group. In addition, level of SNAIL2 was increased in wildtype DLC1 overexpression, than the control group, whereas there’s no obvious change in the mutant DLC1-K714E group. Western blot did not show any change in both groups. Results of Colony forming assay showed that A375 cells infected by lentivirus carrying DLC1-K714E exhibited resulted in an increase in cell death compared with the control and wildtype DLC1. Wound healing assay did not observe any obvious change between the treatments.

Conclusions: (1). Overexpression of Rho-GAP point mutant of DLC1 resulted in apoptosis of melanoma cell; (2). DLC1 does not seem to play a role in melanoma metastasis; (3) Elevation of DLC1 and E-cadherin expressions in melanoma cells occurred at the mRNA levels.