Containing the predicted miR-200a binding site, the putative sequences of
Containing the predicted miR-200a binding site, the putative sequences of the binding site then cloned into a pmirGlO Dual-luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA) to form the reporter vector pmiRGLO-ATB-wild-type (ATB-WT). To mutate the putative binding site of miR200a in ATB gene, the sequence of putative binding site was replaced as indicated and was named as pmiRGLOATB-mutated-type (ATB-MUT). pmirGLO-ATB-WT or pmirGLO-ATB-MUT was cotransfected with miR-200a mimics or miR-200a NC into glioma cells by using Lipofectamie 2000 (Invitrogen, USA). After 48 h transfection for luciferase assay using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Similarly the 3′-UTR of TGF-2 containing the putative miR-200a binding site, the putative sequences of the binding site were cloned into a pmirGlo Dual-luciferase miRNA Target Expression Vector to form the reporter vector TGF-2-wild-type (TGF-2-WT) (GenePharma). To mutate the putative binding site of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28549975 miR-200a in the 3-UTR-containing vector, the sequence of putative binding site was replaced as indicated and was named as TGF-2-mutated-type (TGF-2-MUT). The transfection procedure and measurement of Luciferase activities were handled similarly as described above. All assays were independently performed in triplicate.RNA immunoprecipitationCells were cultured in 6-well plates. When 95 confluency was reached, cell layers were wounded using a 10 L tip to produce a gap, gently washed, and cultured with serum-free medium for 24 h. The wounded gapsEZ-Magna RIP RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA) was used in RNA immunoprecipitation (RIP). RIP was implementedMa et al. Journal of Experimental Clinical Cancer Research (2016) 35:Page 4 ofto pull down endogenous miR-200a associated with ATB in glioma cell lines, and was performed following the manufacturer’s protocol. U251 and A172 cells were lysed by RIP lysis buffer, 100 L of cell lysate was incubated with RIP immunoprecipitation buffer containing magnetic beads conjugated with human anti-Argonaute2 (Ago2) antibody (Millipore) and normal mouse IgG (Millipore) was indicated as negative control. Samples were incubated with Proteinase K buffer and then target RNA was extracted. Purified RNA was subjected to RTQPCR analysis for further study.Western blot analysisTumor xenograft formation assay in nude miceTotal proteins were extracted from the cells using RIPA buffer with PMSF (Beyotime Institute of Biotechnology) on ice, subjected to SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were incubated in 5 nonfat milk dissolved in Tris-buffered saline (TBS) containing 0.1 Tween-20 for 1.5 h at room temperature and then incubated with primary antibodies as follows: TGF-2 (1:1000, Abcam, EUGENE, USA), -actin (1:1000, Santa Cruz Biotechnology). After incubation with secondary antibodies (Goat anti-rabbit or Goat anti-mouse, 1:5000 respectively; ZSGB-BIO, Beijing, China), immune complexes were visualized by SuperSignal?West Femto Trial Kit (Thermo Fisher, USA) and blot bands were scanned using Find-do ?6 Tanon (Tanon, Shanghai, China).Four-week-old female BALB/C athymic nude mice were purchased from the ONO-4059 mechanism of action National Laboratory Animal Center (Beijing, China). Experiments with nude mice were conducted strictly in accordance with a protocol approved by the Administrative Panel on Laboratory.