Tumor suppressive functions of hsa‑miR‑34a on cell cycle, migration and protective autophagy in bladder cancer

Abstract

Bladder cancer (BC) cells exhibit a high basal level of autophagy activity, which contributes to the development of a protective mechanism for cellular survival against current treatments. Hsa‑microRNA‑34a (miR‑34a) presents anti‑tumor function in several types of cancer. However, the functional mechanism of miR‑34a in regulating tumor aggressiveness and protective autophagy of BC remains largely unknown. First, transfected BC cells with miR‑34a mimic exhibited LC3‑II and p62 accumulation through immunofluorescence staining. It was demonstrated that syntaxin 17 (STX17), which is required for autophagosome‑lysosome fusion, was downregulated upon miR‑34a mimic treatment. Mechanistically, miR‑34a reduced the expression of STX17 proteins that directly bind on STX17 3'‑untranslated regions and thus suppressed STX17 mRNA translation to eventually inhibit protective autophagy in BC. Cell viability and colony formation assays revealed that overexpression of miR‑34a in BC cells enhances the chemosensitivity of cisplatin, doxorubicin, epirubicin and mitomycin C. Furthermore, miR‑34a inhibited cell proliferation and triggered G0/G1 cell cycle arrest by inhibiting cyclin D1 and cyclin E2 protein expression. Moreover, miR‑34a suppressed cell motility through the downregulation of epithelial‑mesenchymal transition. In summary, miR‑34a inhibits cell proliferation, motility and autophagy activity in BC, which can benefit BC treatment.

Keywords: autophagy; bladder cancer; hsa‑microRNA‑34a; motility; syntaxin 17.

Figure 1

MiR-34a interrupts autophagic flux in BC cells. (A and B) Human BC cells (5637 and T24) were transfected with negative control (Con.) or miR-34a mimic (100 nM) for 24 h, then protein levels of LC3-I/II and p62 were examined using western blot assay. Protein expression values were quantified using UN-SCAN-IT gel 6.1 software. (C and D) Detection of LC3-II and p62 puncta used immunofluorescence analysis (Red arrow: puncta). Nuclei were counterstained with 40,6-diamidino-2-phenylindole. (E and F) The acidic vesicular organelle presented in BC cells were stained with acridine orange (Red arrow: acidic vesicular organelle). (G and H) The BC cells were incubated with LysoTracker Blue to detect the lysosome in cytoplasm, wherein blue signals indicated the lysosomes. (I) Fluorescence intensity was quantified by ImageJ software. All data are expressed as the means ± SD in triplicate samples. ^*P<0.05, ^**P<0.01, ^***P<0.001 and ^****P<0.0001 relative to the control group. miR, microRNA; BC, bladder cancer; WT, wild-type.

Figure 2

STX17 is involved in the mechanism of miR-34a-mediated autophagy activity. (A) Positions of the putative miR-34a binding sites in 3′UTR of SNAP29, RAB7L1, STX17 and EPG5. (B and C) RT-qPCR assay revealed SNAP29, RAB7L1, STX17 and EPG5 mRNA expression after miR-34a mimic treatment. (D and E) The STX17 protein expression was detected using a western blot assay. (F) Transfection of bladder cancer cells with STX17-3′-UTR plasmid (1 µg/µl) and miR-34a mimic (100 nM) for 24 h, then relative luciferase/Renilla activities were measured. (G-I) The basal levels of STX17 protein and mRNA expression in control stable and miR-34a stable cells were detected using western blotting and RT-qPCR assay, respectively. All data are expressed as the mean ± SD in triplicate samples. ^*P<0.05, ^**P<0.01, ^***P<0.001 and ^****P<0.0001 relative to the control group. STX17, syntaxin 17; miR, microRNA; UTR, untranslated region; RT-qPCR, reverse transcription-quantitative PCR; WT, wild-type; Con, control; ns, not significant.

Figure 3

STX17 overexpression rescues miR-34a-inhibited autophagy activity. (A) BC cells were transfected with vector or STX17 plasmid (1 µg/µl) for 24 h. The overexpressing efficiency of STX17 was verified using western blot assays in 5637 and T24 cells. (B and C) Co-transfection of BC cells with miR-34a mimic and STX17 plasmid for 24 h, the LC3-I/II and p62 protein expression were then analyzed through western blotting. All data are expressed as the mean ± SD in triplicate samples. ^*P<0.05, ^**P<0.01 and ^****P<0.0001 relative to the control group. STX17, syntaxin 17; miR, microRNA; BC, bladder cancer.

Figure 4

MiR-34a confers chemosensitivity in BC cells. (A-D) The miR-34a stable cells were transfected with or without STX17 plasmid (1 µg/µl) for 24 h followed by exposing to various concentrations of chemotherapeutic drugs, including cisplatin (0-50 µM), doxorubicin (0-200 µg/ml), epirubicin (0-200 µM) and mitomycin C (0-200 nM). After 24 h of treatment, resazurin-based cell viability was measured. (E-H) The miR-34a stable cells were treated with chemotherapeutic drugs, including cisplatin (1.5 µM), doxorubicin (0.1 µg/ml), epirubicin (0.1 µM) and mitomycin C (12.5 nM) for 7 days. Cell survival was detected by colony formation assay. All data are expressed as the mean ± SDs in triplicate samples. ^*P<0.05, ^**P<0.01, and ^****P<0.0001 relative to the control group. miR, microRNA; BC, bladder cancer; STX17, syntaxin 17.

Figure 5

MiR-34a promotes G0/G1 cell cycle arrest in BC cells. (A) The miR-34a stable cells were stained with DAPI for nucleus (blue) and Alexa Fluor 546 for Ki-67 (red). Ki-67 fluorescence intensity was quantified by ImageJ software. (B) Colony formation assay was used to measure cell survival in miR-34a stable cells. (C) Effects of miR-34a on cell cycle by flow cytometry in miR-34a stable cells. (D-F) The protein and mRNA levels of cyclin D1 and cyclin E2 were detected by western blot assay and reverse transcription-quantitative PCR assay in miR-34a stable cells, respectively. All data are expressed as the means ± SD in triplicate samples. ^*P<0.05, ^**P<0.01, ^***P<0.001 and ^****P<0.0001 relative to the control group. miR, microRNA; BC, bladder cancer.

Figure 6

MiR-34a inhibits cell motility through EMT suppression. (A) The cell migratory capacity was assessed using a gap closure assay and quantified by counting the migratory cells. (B-I) The levels of EMT-TFs and EMT markers protein and mRNA expression were evaluated using western blot assay and reverse transcription-quantitative PCR assay, respectively. All data are expressed as the mean ± SDs in triplicate samples. ^*P<0.05, ^**P<0.01, ^***P<0.001 and ^****P<0.0001 relative to the control group. miR, microRNA; EMT, epithelial-mesenchymal transition; ns, not significant.

Figure 7

Inhibition of protective autophagy, cell motility, and promotion of cell cycle arrest by hsa-miR-34a treatment in BC. miR, microRNA; BC, bladder cancer.