Fig 1: MiR-362-5p directly targets with QKI. (A) The predicted binding sites within miR-362-5p and wild-type of 3'UTR of QKI (QKI-Wt) and the mutant sequence of 3'UTR of QKI (QKI-Mut). (B) The pmirGLO luciferase vector was inserted with fragments of QKI-Wt or QKI-Mut. Then the constructed pmirGLO luciferase vectors (1 µg) were co-transfected with miR-362-5p mimic (50 pmol) or NC mimic. Luciferase reporter assay was performed after for 48 h transfection. *p < 0.05 vs. NC controls. (C) The relative expression levels of QKI in bladder cancer tissues (N=24) and adjacent tissues (N=24) were analyzed by qRT-PCR. **p < 0.01 vs. adjacent tissues. (D, E) The mRNA and protein expression levels of QKI in bladder cancer cell lines 5637, SW780, UMUC3, and T24 were analyzed by qRT-PCR. (F, G) The 5637 cells were transfected with miR-362-5p mimic (100 pmol) or NC mimic for 48 h. The mRNA and protein levels of QKI were detected by qRT-PCR and western blot. (H, I) The SW780 cells were transfected with miR-362-5p inhibitor (100 pmol) or NC inhibitor for 48 h. The mRNA and protein levels of QKI were detected by qRT-PCR and western blot. The expression was displayed as fold of 5637 or SW780 cells. GAPDH was used as an internal control in western blot. *p < 0.05 vs. corresponding controls.
Fig 2: QKI as a target gene for miR-155 in cardiomyocytes. (a) The bioinformatic analysis showed that miR-155 had a binding site in the 3'-UTR of QKI mRNA of rat, human, and mouse species. (b) Relative luciferase activities of QKI wild-type (QKI-WT) UTRs and mutant UTRs of QKI (QKI-Mutant) were obtained by cotransfection of negative control miRNA or miR-155 mimics and the recombinant plasmid. Relative luciferase activity was calculated as the ratio of Renilla/firefly activities in the cells and normalized to those of the control; n = 8. **P < 0.01 vs. NC. (c) The protein levels of QKI-5 and QKI-6 in NRVMs were tested by western blot; n = 5. *P < 0.05, **P < 0.01 vs. control; #P < 0.05 vs. miR-155; &P < 0.05 vs. AMO-155. (d) QKI mRNA level was detected by qRT-PCR; n = 4. **P < 0.01 vs. control; ##P < 0.01 vs. +NC. (e) The protein expression of QKI-5 and QKI-6 in NRVMs were tested by western blot; n = 4. **P < 0.01 vs. control; #P < 0.05, ##P < 0.01 vs. +NC. (f) The protein expression of QKI-5 and QKI-6 in mice were tested by western blot; n = 3. **P < 0.01 vs. sham group; #P < 0.05, ##P < 0.01 vs. MI group.
Fig 3: MBNL1-AS1 acts as a sponge for miR-362-5p. (A) The predicted binding sites within miR-362-5p and wild-type of MBNL1-AS1 (MBNL1-AS1-Wt) and the mutant sequence of MBNL1-AS1 (MBNL1-AS1-Mut). (B) The pmirGLO luciferase vector was inserted with fragments of MBNL1-AS1-Wt or MBNL1-AS1-Mut. Then the constructed pmirGLO luciferase vectors (1 µg) were co-transfected with miR-362-5p mimic (50 pmol) or NC mimic. Luciferase reporter assay was performed after for 48 h transfection. *p < 0.05 vs. controls. (C) The expression levels of MBNL1-AS1 in bladder cancer tissues (N=24) and adjacent tissues (N=24) were analyzed by qRT-PCR. **p < 0.01 vs. adjacent tissues. (D) The relative expression levels of MBNL1-AS1 in bladder cancer cell lines 5637, SW780, UMUC3, and T24 were analyzed by qRT-PCR. (E) Spearman correlation coefficient analysis was used to evaluate associations between miR-362-5p and MBNL1-AS1 verifed by qRT-PCR (r = -0.5922, p = 0.0023). (F) The 5637 cells were co-transfected MBNL1-AS1 shRNA/NC shRNA (1 µg) and miR-362-5p inhibitor/NC inhibitor (50 pmol) for 48 h. Cell proliferation was detected by stainning BrdU in immunofluorescence assay (bar=50 µm). (G) The cell viability of transfected cells was measured by MTT assay at 48 h. The cell viability was displayed as fold of NC shRNA. (H) The protein levels of QKI, Cyclin D, and p27 was measured by western blot. GAPDH was used as an internal control in western blot. (I) The relative expression levels of miR-362-5p in MBNL1-AS1 shRNA or NC shRNA transfected 5637 cells were analyzed by qRT-PCR. The relative expression was displayed as fold of 5637 cells. **p < 0.01 and *p < 0.05 vs. corresponding controls.
Fig 4: Knockdown of QKI abates the effects of miR-362-5p inhibition on the proliferation of bladder cancer cells. (A, B) The SW780 cells were co-transfected with miR-362-5p inhibitor/NC inhibitor (50 pmol) and QKI siRNA/NC siRNA (50 pmol) for 48 h. Then the cell proliferation and cell viability were determined by staining BrdU (bar=50 µm) and MTT assay. The cell viability was displayed as fold of NC inhibitor. (C) The cell cycle distribution of the transfected cells was measured using flow cytometer after 48 h transfection. The percentage of cells was displayed as fold of NC inhibitor in G1 phase. (D) The protein levels of QKI, Cyclin D, MacroH2A1.1, MacroH2A1.2, p27, c-Fos, PARP-1, and E2F1 in the transfected cells were measured by western blot analysis after 48 h transfection. GAPDH was used as an internal control in western blot. **p < 0.01 and *p < 0.05 vs. corresponding controls.
Fig 5: QKI inhibition abolishes protective effect of AMO-155 on cardiomyocytes. (a, b) QKI was silenced through siRNA transfection, and qRT-PCR and western blot were performed to detect the mRNA and protein level of QKI. siQKI-2 was used to downregulate QKI in the following experiments. (c) The cell viability of NRVMs after treatment with silencing QKI detected by MTT assay; n = 6. (d) Statistical results of TUNEL-positive cells per field. (e) Representative images of TUNEL staining of NRVMs showing the apoptotic cells; n = 4. *P < 0.05. **P < 0.01 vs. control; ##P < 0.01 vs. +NC; &&P < 0.01 vs. +AMO-155. +NC: H2O2+NC; +siQKI: H2O2+siQKI; +AMO-155: H2O2+AMO-155; +AMO-155+siQKI: H2O2+AMO-155+siQKI.
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