Reduction of Bladder Cancer Chemosensitivity Induced by the Effect of HOXA-AS3 as a ceRNA for miR-455-5p That Upregulates Notch1

Abstract

Chemoresistance is one of the main causes of recurrence in bladder cancer patients and leads to poor prognosis. Recently, long non-coding RNAs, like HOXA-AS3, have been reported to regulate chemoresistance in several types of cancer. In this study, we aimed to determine whether HOXA-AS3 can mediate cisplatin resistance in bladder cancer, and its potential mechanism of action. We determined the viability, proliferation, and apoptosis of bladder cancer cells using a CCK-8 assay, EdU staining, and flow cytometry, respectively. We used western blot analysis to assess the expression of markers of epithelial-mesenchymal transition (EMT) and Notch1. We then confirmed expression of these EMT-related markers by immunofluorescence analysis. We found that hypoxia promoted resistance to cisplatin and upregulated the level of HOXA-AS3 in BC cells. Inhibition of HOXA-AS3 enhanced hypoxia-induced cisplatin sensitivity by regulating EMT and Notch1 in BC cells. A dual-luciferase reporter assay confirmed that HOXA-AS3 directly targets miR-455-5p and that Notch1 was a potential target of miRNA-455-5p. We also found that the positive effect of HOXA-AS3 inhibition on cisplatin resistance and tumorigenesis was alleviated when BC cells were transfected with miR-455-5p. Finally, we showed combining HOXA-AS3 small interfering RNA (siRNA) with cisplatin treatment inhibited tumorigenesis in a BALB/c nu/nu mouse model. Our findings indicate that HOXA-AS3 may function as a competing endogenous RNA (ceRNA) of miR-455-5p to regulate Notch1 and play an important role in regulating chemotherapeutic drug sensitivity in BC cells. Therefore, HOXA-AS3 may be a novel therapeutic target for treating bladder cancer.

Keywords: EMT; HOXA-AS3; Notch1; bladder cancer; drug sensitivity.

Figure 1

Hypoxia induces cisplatin resistance and upregulation of HOXA-AS3 in bladder cancer (BC) cells. (A) CCK-8 was used to determine cell viability after treatment with cisplatin under normoxic and hypoxic conditions. *P < 0.05 vs. control. (B) The level of long non-coding RNAs (lncRNAs) after cisplatin treatment under hypoxic or normoxic conditions was examined by qRT-PCR. (C) Gene expression profiling interactive analysis (GEPIA) database analysis of HOXA-AS3 expression in bladder cancer tumor samples and paired normal tissues. (D) Quantitative RT-PCR analysis of HOXA-AS3 levels in bladder cancer tissue and adjacent normal tissue.

Figure 2

Downregulation of HOXA-AS3 enhances cisplatin sensitivity. (A) Quantitative RT-PCR analysis of HOXA-AS3 levels in three bladder cancer cell lines. Cell viability (CCK-8) analysis of bladder cancer cells treated with various concentrations of cisplatin *P < 0.05 vs. UM-UC-3. Correlation curve showing the association of the expression of long non-coding RNA (lncRNA) HOXA-AS3 with sensitivity to cisplatin in bladder cancer (BC) cells. (B–D) Cell viability (CCK-8) analysis of bladder cancer cells transfected with HOXA-AS3 small interfering RNA (siRNA) or a negative control followed by treatment with different concentrations of cisplatin (0, 0.625, 1.25, 2.5, 5, 10 μM). *P < 0.05 vs. negative control. (E) The interference efficiency of HOXA-AS3 siRNA was determined by qRT-PCR. *P < 0.05, **P < 0.01. (F, G) Photomicrographs and bar graphs depicting EdU staining and relative EdU-positive ratio in bladder cancer cells after treatment with cisplatin alone, or cisplatin combined with HOXA-AS3 siRNA. *P < 0.05, **P < 0.01. (H) The number of apoptotic cells in bladder cancer cells transfected with a negative control or HOXA-AS3 siRNA followed by cisplatin treatment, as detected by flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001 vs. negative control plus cisplatin. (I) Knockdown of HOXA-AS3 siRNA enhanced cisplatin sensitivity under hypoxic conditions.

Figure 3

HOXA-AS3 regulates Notch1 expression and reverses hypoxia-induced EMT in bladder cancer (BC) cells. (A, B) Protein levels of EMT markers and Notch1 in bladder cancer cells transfected with a negative control or HOXA-AS3 small interfering RNA (siRNA) under normoxic or hypoxic conditions, by western blot analysis. **P < 0.01, ***P < 0.001. (C, D) Immunofluorescence analysis of E-cadherin and vimentin expression in BC cells transfected with a negative control or HOXA-AS3 siRNA under normoxic or hypoxic conditions.

Figure 4

HOXA-AS3 may function as a competing endogenous RNA of miR-455-5p. (A) The predicted binding site between HOXA-AS3 and miR-455-5p and the mutation in the predicted seed region. (B) Relative luciferase activity of bladder cancer (BC) cells co-transfected with WT-HOXA-AS3 or Mut-HOXA-AS3 and NC, inhibitor NC or miR-455-5p mimics, and inhibitor. *P < 0.05 vs. NC; ^#P < 0.05 vs. inhibitor NC. (C, D) Expression of miR-455-5p in BC cells transfected with HOXA-AS3 small interfering RNA (siRNA) or HOXA-AS3 detected by qRT-PCR. **P < 0.01. (E, F) qRT-PCR results showing the level of miR-455-5p and HOXA-AS3 under hypoxic or control conditions. *P < 0.05, **P < 0.01. (G) Expression of HOXA-AS3 in BC cells transfected with the miR-455-5p mimic or inhibitor detected by qRT-PCR. *P < 0.05 vs. negative control (NC); ^#P < 0.05 vs. inhibitor NC. (H) Expression of Notch1 in BC cells transfected with the miR-455-5p mimic or inhibitor, as detected by qRT-PCR. *P < 0.05 vs. negative control (NC); ^#P < 0.05, ^##P < 0.01 vs. inhibitor NC. (I, J) Relative luciferase activity of BC cells co-transfected with WT-Notch1 or Mut-Notch1 and NC, inhibitor NC or miR-455-5p mimics, and inhibitor. *P < 0.05 vs. negative control (NC); ^#P < 0.05 vs. inhibitor NC.

Figure 5

miR-455-5p mediates the regulatory effect of HOXA-AS3 on cisplatin sensitivity. (A–C) Viability of BC cells (CCK-8 assay) treated with different concentrations of cisplatin for 48 h (0, 0.625, 1.25, 2.5, 5, 10 μM) following transfection with HOXA-AS3 small interfering RNA (siRNA), miR-455-5p inhibitor, or HOXA-AX3 plus miR-455-5p inhibitor. *P < 0.05 vs. negative control. (D–E) Cell proliferation of BC cells (EdU staining) after treatment with cisplatin, or combined with HOXA-AS3 siRNA, or miR-455-5p inhibitor, or HOXA-AS3 siRNA plus miR-455-5p inhibitor. *P < 0.05, **P < 0.01, ***P < 0.001. (F, G) Apoptotic rate of BC cells in different treatment groups (Negative siRNA plus cisplatin, HOXA-AS3 siRNA plus cisplatin, HOXA-AS3 siRNA plus miR-455-5p plus cisplatin) detected by flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

HOXA-AS3 small interfering RNA (siRNA) reversed miR-455-5p-induced EMT. (A) Western blot analysis of the expression of EMT-related proteins. *P < 0.05, **P < 0.01, ***P < 0.001. (B) Immunofluorescence analysis of E-cadherin and vimentin expression in bladder cancer cells transfected with a negative control, HOXA-AS3 siRNA, miR-455-5p inhibitor, or HOXA-AS3 siRNA combined with miR-455-5p inhibitor, followed by cisplatin treatment. (C) Quantitative RT-PCR analysis of Notch1 expression following treatment with HOXA-AS3 siRNA alone, HOXA-AS3 siRNA plus miR-455-5p inhibitor, or the negative control. *P < 0.05, **P < 0.01. (D) Western blot analysis of the expression of Notch1 following treatment with HOXA-AS3 siRNA alone, HOXA-AS3 siRNA plus miR-455-5p inhibitor, or the negative control. *P < 0.05, **P < 0.01.

Figure 7

Suppression of HOXA-AS3 enhances the cisplatin sensitivity of bladder cancer cells in vivo. (A) Volume of tumor xenografts. (B) Body weights of mice measured on day 0–12. (C) Tumor inhibition rate. (D) Tumors isolated from nude mice. (E) Kaplan–Meier curves of mice in the saline groups. (F–H) Notch1 and Ki-67 staining in the different treatment groups (left) and rate of positive staining (right). *P < 0.05, **P < 0.01. (G) TUNEL assay of the level of apoptosis in the different treatment groups. **P < 0.01, ***P < 0.001. (I–L) HOXA-AS3, Notch1, and miR-455-5p expression levels in tumor tissues using qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001. (K) Western blot analysis of HOXA-AS3 and Notch1 expression in tumor tissues. **P < 0.01.