Cell culture and tissue samples
Bladder cancer T24 cells were purchased from American Type Culture Collection (ATCC) and cultured in RPMI 1640 medium (GIBCO) supplemented with 10% fetal bovine serum (GIBCO, Australia) and 1% penicillin streptomycin (GIBCO) in an incubator with humidified 5% CO2 at 37 oC.
Bladder cancer and adjacent non-tumor tissue samples were collected from thirty patients undergoing surgical resection at Shaoxing People's Hospital. Pathological examination was performed to confirm cancer diagnosis. After receiving approval from the ethics committee of Shaoxing People’s Hospital (Zhejiang, China), written informed consent for the present study was obtained from these patients.
Cell transfection
T24 cells were cultured in 6 well plate until reached to 80%, and then they were used for cell transfection. Mature miR-340 mimics were purchased from (Genepharma company, Shanghai). The transfection reagent lipofectamine 2000 and miR-340 mimics (20 μmol/L) were dissolved in the MEMI respectively for 5 minutes. Subsequently, they were mixed and incubated for 25 minutes. Next, the formed complex was incubated with T24 cells for 6 h. After that, the complex was changed to normal medium and cultured for another 48 h. Finally, these cells had been prepared for the subsequent experiments.
Cell proliferation and apoptosis
Thiazolyl blue tetrazolium bromide (MTT) solution (5 mg/mL, 10 μL) was added into the 96-well plate containing cells. After incubated for 4 hours, MTT was removed and DMSO 100 μL was added. Five parallel experiments were carried out to calculate the average absorbance (OD value). The relative percentage of cell viability was calculated as follows: (mean OD value in the experimental group - mean OD value in control group) /mean OD value in control group × 100%.
Cells were collected, washed for two times with PBS. Annexin V-APC (5 μL) and 7-AAD (10 μL) were added into cell medium and incubated for 10 min. Subsequently, flow cytometry was used to analyze cell apoptosis.
RNA extraction and real-time quantitative PCR (RT-qPCR)
Total RNA including miRNA from 60 frozen bladder tissues (30 for tumor tissues and 30 for paired adjacent normal tissues) and T24 cells, was extracted using a miRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Then, we use All-in-One™ miRNA First-Strand cDNA Synthesis and All-in-One™ First-Strand cDNA Synthesis kits (GeneCopoeia, Inc.) to synthesize the miRNA and mRNA into cDNA. Next, RT-qPCR were performed using All-in-One™ miRNA kit and All-in-One™ qPCR Mix (GeneCopoeia, Inc.), respectively, on a CFX96™ Real-Time System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The PCR cycling procedure was first hold 95℃ for 10 min, then 40 cycles at 95℃ for 10 sec, 60℃ for 20 sec, and 72℃ for another 15 sec. The GAPDH and U6 were used as internal control of mRNA and miRNA expression, respectively, and calculated according to 2−ΔΔCt method. All experiments were repeated three times. Primer sequence is as follows: miR-340 forward, 5′-GCGGTTATAAAGCAATGAGA-3′ and reverse primer, 5′-GTGCGTGTCGTGGAGTCG-3′; U6 forward, 5'-CTCGCTTCGGCAGCACA-3' and reverse, 5'-AACGCTTCACGAATTTGCGT-3'; Glut-1 forward, 5′-AACTCTTCAGCCAGGGTCCAC-3′ and reverse, 5′-CACAGTGAAGATGATGAAGAC-3′, PCNA forward, 5’-GCGTG AACCTCACCAGTATGT-3’ and reverse, 5’-TCTTCGGCCCTTAGTGTAATGAT-3’ ; Bax forward, 5′-TGCTTCAGGGTTTCATCCAGG-3′ and reverse, 5′-TGGCAAAGTAGAAAAGGGCGA-3′; GAPDH forward, 5′-TGCACCACCAACTGCTTAGC-3′ and reverse primer, 5′-GGCATGGACTGTGGTCATGAG-3′.
Western blots
Cells were lysed and protein was harvested with RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) with protease inhibitor cocktail (Beyotime Institute of Biotechnology), and quantified by bicinchoninic acid (BCA) analysis (Beyotime). The equal quantities protein lysates were prepared and separated using SDS-PAGE on 10% gels, and transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore). These membranes were blocked with 10% skimmed milk diluted with TBS supplemented with Tween-20 at room temperature for 1 h and incubated with primary antibodies for 2 h. After washing, the membranes were incubated with the secondary horseradish peroxidase-conjugated antibody for 1h. The proteins were visualized using Pierce™ ECL Western Blotting Substrate (Thermo Fisher). The housekeeping gene GAPDH was served as a loading control. Primary anti-bodies against Glut-1, PCNA, PI3K, AKT, Bax and GAPDH (Abcam), p-PI3K and p-AKT (R&D systems), were used according to manufacturer recommendations.
Dual-luciferase reporter assay
The 3’ untranslated regions (UTR) of Glut-1 mRNA (GenBank accession number, NM_006516) was amplified (wild type). The PCR products of Glut-1 3’UTR were cloned the fragment into the SacI and XhoI sites in the pmirGLO vector (Thermo Scientific; Waltham, MA, USA). The corresponding mutant construct was generated by mutating the seed region of miR-340 by site-specific mutagenesis using the following primers: forward primer, 5’-agacaagcaacagcttattttatttt-3’; reverse primer,5’-tcagtaataaaaaaataaaataagctg-3’. T24 cells were co-transfected with miR-NC mimics or miR-340 mimics (20 nmol/L) using lipofectamine 2000 for 48 h. Relative luciferase activity was performed according to manufacturer’s instructions (Promega).