br Western blot analysis br Cells were
2.4. Western blot analysis
Cells were lysed using radioimmunoprecipitation assay lysis buﬀer (Beyotime, Shanghai, China), and the protein concentration was de-termined using a BCA Protein Assay Kit (Solarbio, Beijing, China). Equal amounts of protein samples were separated by SDS-poly-acrylamide gel electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Piscataway, NJ, USA). The membranes were blocked with 5% skim milk and then incubated with primary Salvinorin A against Bax (1:1000; Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (1:1000; Abcam, Cambridge, UK) and GAPDH (1:1000; Cell Signaling Technology) at 4 °C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 3 h. Immunoreactive bands were
Fig. 2. Knockdown of circPVT1 inhibits NSCLC cell proliferation and induces NSCLC cell apoptosis. (A) RT-qPCR analysis of circPVT1 expression levels in H1299 and A549 cells after transfection. (B) The proliferation of H1299 and A549 cells after transfection was detected by MTT assay. (C) The apoptosis of H1299 and A549 cells after transfection was detected by flow cytometric analysis. (D) Western blot analysis of Bcl-2 and Bax protein expression levels in H1299 and A549 cells after transfection. *P < 0.05 versus sh-NC-transfected cells.
Fig. 3. Knockdown of circPVT1 inhibits NSCLC tumor growth. (A) Tumor volume was calculated every five days, and tumor growth curves were plotted. (B) Four weeks after cell inoculation, the tumors were removed and weighed. (C) RT-qPCR analysis of circPVT1 expression levels in the tumor tissues. (D) Western blot analysis of Bcl-2 and Bax protein expression levels in the tumor tissues. *P < 0.05 versus sh-NC group.
detected by an enhanced chemiluminescence kit (Amersham, Little Chalfont, UK), and in order to eliminate the variations, GAPDH was considered as an inner loading control.
Cell proliferation was determined through (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Cells were seeded at 5 × 103 cells per well in 96-well plates after transfection. At indicated
time points, 20 μl MTT reagent (5 mg/ml; Sigma-Aldrich) was added to each well and the cells were incubated for additional 4 h at 37 °C. Then the medium was removed, and 150 μl of dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added to each well. 10 min later, the purple crystals were dissolved. The absorbance was measured at 490 nm using a mi-croplate reader (MultiskanEX, Lab systems, Helsinki, Finland).
Fig. 4. circPVT1 acts as a ceRNA of miR-497 in NSCLC cells. (A) The putative binding sites of miR-497 on circPVT1 are predicted. (B) Dual-luciferase reporter assay showed the luciferase activity of the combination between miR-497 and circPVT1. (C) RT-qPCR analysis of miR-497 expression levels in H1299 and A549 cells after transfection. *P < 0.05 versus mimics control or sh-NC-transfected cells. (D) RT-qPCR analysis of miR-497 expression levels in NSCLC tissues and matched adjacent normal tissues. (E) The correlation between miR-497 and circPVT1 expression in NSCLC tissues.
2.6. Cell apoptosis analysis
Cell apoptosis was measured using an Annexin-V-FITC Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA). After transfection, the cells were collected, washed three times with PBS, resuspended in 500 μl 1 × binding buﬀer, and double-stained with 5 μl Annexin V-FITC and 5 μl PI at room temperature for 15 min. Then the cells were sub-jected to FACScan flow cytometer (BD Biosciences) equipped with CellQuest software (BD Biosciences).
2.7. Dual-luciferase reporter assay
The sequence fragments of circPVT1 (circPVT1-WT) and Bcl-2 (Bcl-2-WT) 3′-UTR containing the putative target sites for miR-497 were amplified by PCR and cloned into the luciferase reporter psiCHECK2 (Promega, Madison, WI, USA). The binding sites were mutated using a Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA). HEK293 T cells were seeded into 24-well plates and co-trans-fected with luciferase reporter vectors and miR-497 mimics or mimics control using the Lipofectamine 3000 Transfection Reagent. After 48 h, the cells were harvested and lysed. Renilla and firefly luciferase activ-ities were measured using the Dual-Luciferase Reporter Assay system (Promega).