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

Dajin Chen,Hao Yang,Wei Chen,Ying Cai,Ying Wu,Jianghua Chen,Shangzhi Xie,Lan Lan,Yu Cui

doi:10.3389/fonc.2020.572672

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.

Highlights

Bladder cancer (BC) is ranked as the ninth most common cancer worldwide, and men are more than three times more likely to develop the disease than women (1, 2)
We found that HOXA-AS3 expression was significantly increased under hypoxic conditions in BC cells (Figure 1B)
Using a cell counting kit-8 (CCK-8) cell viability assay, we found all three BC cell lines that were transfected with HOXA-AS3 small interfering RNA (siRNA) had increased cisplatin sensitivity compared with the negative controls (Figures 2B–E), while the HOXA-AS3 plasmid had reduced cisplatin sensitivity (Supplemental Figures 2A–D)

Summary

Introduction

Bladder cancer (BC) is ranked as the ninth most common cancer worldwide, and men are more than three times more likely to develop the disease than women (1, 2). Cisplatin is extensively used in the clinic as a first-line treatment for many cancers, including liver, ovarian, non-small cell lung cancer (NSCLC), and BC (3–5); its efficacy is limited due to severe side effects and the development of drug resistance (6). Recent studies have shown abnormal expression of lncRNA in many types of tumors, playing important roles in cancer progression, including cell proliferation, differentiation, invasion, and metastasis (9, 10). Several studies have shown lncRNAs may interact with miRNAs and mutually modulate their expression (11, 12). They may function as competing endogenous RNAs (ceRNAs) with miRNAs and subsequently regulate gene expression by post-transcriptional silencing of the target RNAs (13, 14). Understanding the functions of lncRNAs in cancer has become an area of extensive research

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