From March 2016 to May 2018, after receiving the written informed consent, Nanjing Drum Tower Hospital affiliated to Nanjing University provided BCa tumors and normal bladder tissues. Tumor tissue and adjacent normal tissue from 84 patients were collected according to institutional protocols. After analysis, it was found that miR-582-5p expressionwas lower in BC tissues compared with normal tissues (Fig. 1A). Moreover, of the 84 patients, 39 had no recurrence at 3 years, and 45 had relapsed, and miR-582-3p was lower in the relapsed tissues than in the non-relapsed tissues (Fig. 1B). The Ethics Committee of Nanjing Drum Tower Hospital approved to carry out this study and obtained the written and informed consent of all patients. All experiments were conducted in accordance with approved guidelines.
Cell lines and culture
American Type Culture Collection (ATCC, Manassas, VA, USA) provided human BC cell lines, T24 and 5637. Cells were cultivated within RPMI1640 medium contained 10% FBS and 1% P/S (Gibico, NY, USA), and incubated within the humid incubator under 37℃ and 5% CO2 conditions.
General Biosystems (Anhui, China) synthesized miR-582-5p mimics and NC mimics. Sequences for CD81 were generated by PCR and inserted into pCS2-CMV vector (GenePharma, Shanghai, China) for CD81 overexpression. According to the manufacturer's instructions, these segments were transfected into T24 and 5637 cells cultured to 80% confluence with Lipofectamine™ 3000 Transfection Reagent (Invitrogen, California, USA).
miRNA was extracted from BC tissues and cell lines by using mirVana microRNA Isolation kits (Invitrogen, California, USA). The Taqman microRNA assay kit (Invitrogen) was performed to detected miR-582-5p levels. U6 RNA was used as an endogenous control. The crease change was calculated by 2−ΔΔCt method. The whole process was repeated three times. The primerswere shown in Table 1.
The total T24 and 5637cell lytic was prepared with RIPA cleavage buffer (Beyotime, Nanjing, China). 10% SDS-PAGE gel separated protein and then was transferred to the PVDF membrane. TBST and 5% skimmed milk powder were used to seal the PVDF membrane. Then, the PVDF membrane was incubated overnight with specific antibodies (Abcam, Cambridge, UK), including anti-CD44, anti-KLF4, anti-ALDH1A1, anti-SOX2, anti-HMGA2 and anti-CD81 at 4°C overnight. β-actin was used as the endogenous control. The second day, after incubation with secondary antibody, the bands were detected by GEL imaging system (Bio-Rad), and the quantification of proteins was analyzed by the software Image J.
Bioinformatics and dual-luciferase reporter gene assay
Firstly, the potential binding sites of miR-582-5p downstream was predicted by using starbase bioinformatics software. Clone the wild (WT) or mutant (MUT) sequence of CD81 3'-UTR into a pGL3-M vector (Promega, WI, USA) to build CD81-3'-UTR-WT or CD81-3'-UTR-MUTvectors. Lipofectamine™ 3000 Transfection Reagent (Invitrogen, California, USA) was used to co-transfect these vectors and miR-582-3p mimics or NC mimics into T24 and 5637 cells. Following 48 h, luciferase activity was assessed.
Sphere formation assay
T24 and 5637 cells were digested by trypsin and washed 3 times in PBS. 1⊆104 cells were inoculated in 6-well cell culture plates containing DMEM/F12 medium (Gibico, NY, USA). After 9–12 days of culture, the spheres were photographed and counted with diameters greater than 50 µm.
ALDEFLUOER kit (Stem Cell Technologies, Vancouver, Canada) was performed to identify cells with high ALDH enzyme activity. In detail, 1 mL single cell suspension was added into 5 µL of activated ALDEFLUOER reagent for 40 min at 37℃. Moreover, Diethylaminobenzaldehyde (DEAB) served as a negative control. Finally, FACS Calibur system was used for flow cytometry, Cellquest graphics software was used for data acquisition and analysis.
The tumor tissues were dewaxed and rehydrated, and then sealed with 0.3% hydrogen peroxide. After antigen repair, the sections were treated with sealing solution. Subsequently, the slices were incubated with the first antibody against CD81 overnight at 4 ℃, and then the second antibody coupled with peroxidase (1:100, Bioss, Beijing, China) was placed at 25℃ for 1 h. Finally, the sections were developed with DAB and observed under microscope.
The mean ± standard deviation (SD) represents data from three independent experiments. Student t-test for two groups and Tukey's multiple comparison test for multi-group comparison. When P < 0.05, the difference is statistically significant.