A Novel Marine Drug, SZ-685C, Induces Apoptosis of MMQ Pituitary Tumor Cells by Downregulating miR-200c

Abstract

Our results suggest that SZ-685C induces MMQ cell apoptosis in a miR-200c-dependent manner. Therefore, SZ-685C might be a useful alternative treatment for pituitary adenoma.

Expression of somatostatin receptor subtype 2 in growth hormone-secreting pituitary adenoma and the regulation of miR-185

“…We chose the rat pituitary adenoma GH3 cell line as a tool. The GH3 cell line (Xie-he Bank, Beijing, China) was cultured in RPMI1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 2.5 % fetal bovine serum (Gibco), 15 % horse serum, 100 mg ml −1 streptomycin, and 100 units ml −1 penicillin (Invitrogen) at 37 °C in a humidified atmosphere of 5 % CO 2 , as described previously [25].…”

“…GH3 cells were transfected in 6-well plates (6 × 104 cells/ ml) with 50 nM of an miR-185 mimic or inhibitor (Ribobio, Guangzhou, China) using Lipofectamine 2000 (Invitrogen) and Opti-MEM medium (Invitrogen) without serum, according to the manufacturer's protocol as described previously [25]. Cells were transfected simultaneously with the appropriate negative control (NC) (Ribobio, Guangzhou, China).…”

“…RNA isolation and reverse transcription from GH3 cells were performed as described previously [25]. Polymerase chain reaction was performed using the real-time PCR System (Bio-rad iQ5, Bio-Rad).…”

“…Western blotting was performed as previously described [25]. Cellular proteins were extracted after transfection with miR-185 mimics or inhibitors, and 30 µg of total protein was subjected to SDS-PAGE immunoblotting analysis.…”

“…MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays were used to assess cell viability as previously described [25]. GH3 cells were seeded in 96-well plates at a density of 4 × 10 5 /well 24 h after transfection with miR-185 mimics and negative controls and cultured in serum-free RPMI 1640 medium for 24 h. A volume of 10 μl of MTT (5 mg ml −1 in PBS, Sigma) was added to each well and incubated at 37 °C in a humidified atmosphere of 5 % CO 2 for 4 h. Acidified isopropyl alcohol (100 µl) was added overnight at 37 °C to dissolve the formazan generated in living GH3 cells.…”