Dataset Information

Long non-coding RNA NORAD/miR-224-3p/MTDH axis contributes to CDDP resistance of esophageal squamous cell carcinoma by promoting nuclear accumulation of β-catenin.

ABSTRACT:

Background

Cis-diamminedichloro-platinum (CDDP)-based chemotherapy regimens are the most predominant treatment strategies for patients with esophageal squamous cell carcinoma (ESCC). Dysregulated long non-coding RNAs (lncRNAs) contribute to CDDP resistance, which results in treatment failure in ESCC patients. However, the majority of lncRNAs involved in CDDP resistance in ESCC remain to be elucidated.

Methods

The public Gene Expression Omnibus (GEO) dataset GSE45670 was analysed to reveal potential lncRNAs involved in CDDP resistance of ESCC. Candidate upregulated lncRNAs were detected in ESCC specimens by qRT-PCR to identify crucial lncRNAs. Non-coding RNA activated by DNA damage (NORAD) was selected for further study. Kaplan-Meier analysis and a COX proportional regression model were performed to analyse the potential of NORAD for predicting prognosis of ESCC patients. The role of NORAD in CDDP resistance were determined by conducting gain and loss-of-function experiments in vitro. Fluorescence in situ hybridization (FISH) was performed to determine the subcellular location of NORAD in ESCC cells. A public GEO dataset and bioinformatic algorithms were used to predict the microRNAs (miRNAs) that might be latently sponged by NORAD. qRT-PCR was conducted to verify the expression of candidate miRNAs. Luciferase reporter and Argonaute-2 (Ago2)-RNA immunoprecipitation (RIP) assays were conducted to evaluate the interaction between NORAD and candidate miRNAs. A miRNA rescue experiment was performed to authenticate the NORAD regulatory axis and its effects on CDDP resistance in ESCC cells. Western blotting was conducted to confirm the precise downstream signalling pathway of NORAD. A xenograft mouse model was established to reveal the effect of NORAD on CDDP resistance in vivo.

Results

The expression of NORAD was higher in CDDP-resistant ESCC tissues and cells than in CDDP-sensitive tissues and cells. NORAD expression was negatively correlated with the postoperative prognosis of ESCC patients who underwent CDDP-based chemotherapy. NORAD knockdown partially arrested CDDP resistance of ESCC cells. FISH showed that NORAD was located in the cytoplasm in ESCC cells. Furthermore, overlapping results from bioinformatic algorithms analyses and qRT-PCR showed that NORAD could sponge miR-224-3p in ESCC cells. Ago2-RIP demonstrated that NORAD and miR-224-3p occupied the same Ago2 to form an RNA-induced silencing complex (RISC) and subsequently regulated the expression of metadherin (MTDH) in ESCC cells. The NORAD/miR-224-3p/MTDH axis promoted CDDP resistance and progression in ESCC cells by promoting nuclear accumulation of β-catenin in vitro and in vivo.

Conclusions

NORAD upregulates MTDH to promote CDDP resistance and progression in ESCC by sponging miR-224-3p. Our results highlight the potential of NORAD as a therapeutic target in ESCC patients receiving CDDP-based chemotherapy.

SUBMITTER: Jia Y

PROVIDER: S-EPMC8662861 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundProstate cancer (PC) is common male cancer with high mortality worldwide. Emerging evidence demonstrated that long noncoding RNAs (lncRNAs) play critical roles in various type of cancers including PC by serving as competing endogenous RNAs (ceRNAs) to modulate microRNAs (miRNAs). LncRNA activated by DNA damage (NORAD) was found to be upregulated in PC cells, while the detailed function and regulatory mechanism of NORAD in PC progression remains largely unclear.MethodsExpression of NORAD in PC tissues and cell lines were detected by real-time quantitative PCR (qRT-PCR). NORAD was respectively overexpressed and knocked down by transfection with pcDNA-NORAD and NORAD siRNA into PC-3 and LNCap cells. Cell proliferation, invasion and apoptosis were determined by using CCK-8, Transwell and Flow cytometry assays, respectively. The target correlations between miR-30-5p and NORAD or RAB11A were confirmed by using dual luciferase reporter assay. Moreover, expression levels of RAB11A, the epithelial-mesenchymal transition (EMT) marker proteins and the Wnt pathway related proteins were measured by Western blotting. Tumor xenograft assay was used to study the effect of NORAD on tumor growth in vivo.ResultsNORAD was upregulated in PC tissues and cells. Overexpression of NORAD promoted cell proliferation, invasion, EMT, and inhibited cell apoptosis; while knockdown of NORAD had the opposite effect. NORAD was found to be functioned as a ceRNA to bind and downregulated miR-30a-5p that was downregulated in PC tumor tissues. Rescue experiments revealed that miR-30a-5p could weaken the NORAD-mediated promoting effects on cell proliferation, invasion and EMT. Furthermore, RAB11A that belongs to a member of RAS oncogene family was verified as a target of miR-30a-5p, and reintroduction of RAB11A attenuated the effects of miR-30a-5p overexpression on cell proliferation, invasion, EMT and apoptosis of PC cells. More importantly, silencing RAB11A partially reversed the promoting effects of NORAD overexpression on cell proliferation, invasion and EMT of PC cells via the WNT/β-catenin pathway. Lastly, tumorigenicity assay in vivo demonstrated that NORAD increased tumor volume and weight via miR-30a-5p /RAB11A pathway.ConclusionOur results indicated a significant role of NORAD in mechanisms associated with PC progression. NORAD promoted cell proliferation, invasion and EMT via the miR-30a-5p/RAB11A/WNT/β-catenin pathway, thus inducing PC tumor growth.