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LncRNA ZNF503-AS1 acts as a tumor suppressor in bladder cancer by up-regulating Ca2+ concentration via transcription factor GATA6

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Abstract

Purpose

Ca2+ homeostasis plays a pivotal role in regulating proliferation and apoptosis during cancer development. This study intended to examine the potential tumor-suppressing role of ZNF503 antisense RNA 1 (ZNF503-AS1) in bladder cancer, which may be implicated in the regulation of Ca2+ homeostasis.

Methods

Differentially expressed long non-coding RNAs (lncRNAs) related to bladder cancer were identified using microarray analysis, followed by the verification of transcription factors to which they bind. The relationship between ZNF503-AS1, GATA6 and SLC8A1 was assessed using dual luciferase reporter, RIP and ChIP assays. The expression levels of ZNF503-AS1, GATA6 and SLC8A1 were modulated to examine their effects on the tumorigenic potential, intracellular Ca2+ concentration and Ca2+-ATPase activity in bladder cancer cells. The in vivo tumorigenic ability was validated in nude mice.

Results

Microarray-based expression profile analysis of the GEO GSE61615 dataset revealed that the expression of ZNF503-AS1 was decreased in bladder cancer. Subsequently, we found that ZNF503-AS1 can bind to the transcription factor GATA6 to up-regulate the expression of SLC8A1. ZNF503-AS1 and SLC8A1 were found to be down-regulated in both primary bladder cancer tissues and cells. Exogenous overexpression of ZNF503-AS1 or SLC8A1 attenuated bladder cancer cell proliferation, invasion and migration, but promoted their apoptosis, accompanied by decreased Ca2+-ATPase activities and increased intracellular Ca2+ concentrations. Additional in vivo experiments validated the inhibitory effect of ZNF503-AS1 overexpression on the tumorigenic capacity of bladder cancer cells in nude mice.

Conclusion

ZNF503-AS1 can recruit transcription factor GATA6 to up-regulate SLC8A1 expression, thereby increasing the intracellular Ca2+ concentration and repressing the proliferation, invasion and migration, and enhancing the apoptosis of bladder cancer cells.

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Acknowledgments

We would like show sincere appreciation to the reviewers for critical comments on this article.

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Authors and Affiliations

  1. Department of Urology, The Second Xiangya Hospital, Central South University, No.139 Renmin Road, Changsha, 410011, Hunan Province, People’s Republic of China

    Haiqing He, Shuiqing Wu, Kai Ai, Ran Xu, Zhaohui Zhong, Yinhuai Wang, Lei Zhang, Xiaokun Zhao & Xuan Zhu

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  1. Haiqing He
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Correspondence to Xiaokun Zhao or Xuan Zhu.

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Ethical statement

The study was conducted under approval of the Ethics Committee of The Second Xiangya Hospital, Central South University. All participating patients signed informed consent documentation. Nude mice were used for in vivo studies and were cared for in accordance with the principles of the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health with efforts made to ensure minimal suffering of the animals used in the study.

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Fig. S1

Overexpressed ZNF503-AS1 impedes proliferation, migration, invasion and resistance to apoptosis of bladder cancer J82 cells. A: qRT-PCR and Western blot analysis for the expression of ZNF503-AS1 in J82 cells after sh-ZNF503-AS1 or oe-ZNF503-AS1 transfection. B: EdU assay for the effect of ZNF503-AS1 overexpression on the proliferation of J82 cells (× 200, scale bar = 50 μm), C: quantitative flow cytometric detection for the effect of ZNF503-AS1 overexpression on the apoptosis of J82 cells; D: Transwell assay for the effect of ZNF503-AS1 overexpression on migration and invasion ability of J82 cells (× 200, scale bar = 50 μm); E: qRT-PCR and Western blot analysis for the effect of ZNF503-AS1 overexpression on the expression of MMP-2, MMP-9, Ki67 and Bax in J82 cells. Measurement data were summarized as mean ± standard deviation. Data were analyzed by unpaired t test between two groups from three independent experiments. * p < 0.05 vs. the oe-NC group. (EPS 3481 kb)

Fig. S2

ZNF503-AS1 recruits GATA6 to up-regulate SLC8A1 expression in bladder cancer J82 cells. A: qRT-PCR and Western blot analysis for the SLC8A1 expression in J82 cells overexpressing or silencing SLC8A1. B: qRT-PCR and Western blot analysis for the SLC8A1 expression in J82 cells overexpressing or silencing ZNF503-AS1. * p < 0.05 vs. the sh-NC group, # p < 0.05 vs. the oe-NC group; C: RIP assay to identify the binding of ZNF503-AS1 to GATA6. * p < 0.05 vs. the IgG; C: qRT-PCR and Western blot analysis for determining the SLC8A1 expression in J82 cells, * p < 0.05 vs. the oe-NC + sh-NC group, # p < 0.05 vs. the oe-ZNF503-AS1 + sh-NC group. Measurement data were summarized as mean ± standard deviation. Data were analyzed by unpaired t test between two groups and compared by one-way ANOVA with Tukey post hoc test among multiple groups from three independent experiments. (EPS 1553 kb)

Fig. S3

SLC8A1 inhibits the proliferation and migration of bladder cancer J82 cells by promoting Ca2+ influx. A: EdU assay to detect the effect of SLC8A1 overexpression on J82 cell proliferation; B: quantitative flow cytometric detection of SLC8A1 overexpression on J82 cell apoptosis; C: Transwell assay to detect the effect of SLC8A1 overexpression on J82 cell migration ability; D: Transwell assay for the effect of SLC8A1 overexpression on the invasive ability of J82 cells. Measurement data were summarized as mean ± standard deviation and analyzed by unpaired t test between two groups from three independent experiments. * p < 0.05 vs. the oe-NC group. (EPS 598 kb)

Fig. S4

ZNF503-AS1 promotes Ca2+ and inhibits bladder cancer J82 cell proliferation and migration by regulating the SLC8A1 expression. A, qRT-PCR and Western blot analysis for the expression of SLC8A1 in J82 cells. B: EdU assay to detect the proliferation of J82 cells; C: flow cytometric detection of J82 cell apoptosis; D: Transwell assay to detect J82 cell migration ability (× 400, scale bar = 25 μm); E: Transwell assay to detect J82 cell invasive ability. Measurement data were summarized as mean ± standard deviation. Comparisons among multiple groups were performed using one-way ANOVA or repeated measures ANOVA with Tukey’s post hoc test. The experiment was repeated three times. * p < 0.05 vs. the oe-NC + sh-NC group, # p < 0.05 vs. the oe-ZNF503-AS1 + sh-NC group. (EPS 1032 kb)

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He, H., Wu, S., Ai, K. et al. LncRNA ZNF503-AS1 acts as a tumor suppressor in bladder cancer by up-regulating Ca2+ concentration via transcription factor GATA6. Cell Oncol. 44, 219–233 (2021). https://doi.org/10.1007/s13402-020-00563-z

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