Figure 6

miR-205-5p targets TNFAIP8 in skin cancer cells and increase sensitivity to vemurafenib. (A) HaCaT cells, and skin cancer SK-MEL-2, A375 and A2058 cell lines were treated with increasing concentrations of vemurafenib for 48 h and relative cell survival/proliferation was measured by MTT assay. The experiments were independently repeated three times. **P < 0.01, ***P < 0.001 compared to vehicle-treated cells. (B) HaCaT cells and skin cancer SK-MEL-2, A375 and A2058 cell lines were transfected with NC mimic or miR-205-5p mimic for 24 h and treated with vemurafenib or vehicle for an additional 48 h and relative cell survival were measured by MTT assay as described in the “Materials and methods” section. *P < 0.05, **P < 0.01, ***P < 0.001 compared to NC mimic vehicle-treated cells or NC mimic and vemurafenib-treated cells. ns-no significance. (C) Melanoma A375 cells were transfected with NC mimic or miR-205-5p mimic for 18 h and then cells were treated with vemurafenib for an additional 24 h. Fifty micrograms of lysates were immunoblotted with anti-cleaved-PARP and anti-β-tubulin antibodies. Western blots were developed using ECL chemiluminescence detection reagents, the blots were scanned using Azure C-500 Biosystem, scans were converted into grayscale and presented. (D) Melanoma A375 cells were transfected with EV of TNFAIP8-Myc plasmid for 18 h first and then cells were treated with vemurafenib or paclitaxel for 24 h as indicated and fifty micrograms lysates were immunoblotted with anti-Myc, anti-cleaved-PARP, and anti-β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents and the blots were scanned using an Odyssey CLx imager. The immunoblot scans were converted into grayscale and presented. (E) Fifty micrograms of lysates from A375 and A2058 cells or A375R and A2058R (vemurafenib resistant) cells were immunoblotted with anti-TNFAIP8, anti-LC3β I/II, and anti-β-actin antibodies. Western blots were developed using ECL chemiluminescence detection reagents, the blots were exposed on X-Ray films, scans were converted into grayscale and presented. (F) Relative cell proliferation/cell survival rate of A375 and A2058 cells, or A375R and A2058R (vemurafenib resistant) cells were measured by using WST-1 reagent (Cayman Chemical) (upper panel) or MTT assay reagent (lower panel) and plotted. **P < 0.01, ***P < 0.001 compared to A375 and A2058 parent cells. Results are representative of three independent experiments. Vem. Vemurafenib.