Abstract
BackgroundBladder carcinoma is one of the most common urological cancers. ITPR3, as a ubiquitous endoplasmic reticulum calcium channel protein, was reported to be involved in the development and progression of various types of cancer. However, the potential roles and molecular mechanism of ITPR3 in bladder cancer are still unclear. Herein, we elucidated a novel role of ITPR3 in regulating the proliferation, metastasis, and stemness of bladder cancer cells.MethodsThe expression of ITPR3 in bladder cancer was analyzed using public databases and bladder cancer tissue microarrays. To demonstrate the role of ITPR3 in regulating the NF-ĸB/CD44 pathway and the progression of bladder cancer, a series of molecular biology and biochemistry methods was performed on clinical tissues, along with in vivo and in vitro experiments. The methods used included western blot assay, quantitative RT-PCR assay, immunofluorescence assay, immunohistochemistry (IHC) assays, wound healing assay, Transwell assay, colony formation assay, tumorsphere formation assay, cell flow cytometry analysis, EdU assay, MTT assay, cell transfection, bisulfite sequencing PCR (BSP), a xenograft tumor model and a tail vein cancer metastasis model.ResultsHigher ITPR3 expression was found in bladder cancer tissues and bladder cancer cells compared with the corresponding normal peritumor tissues and SV-HUC-1 cells, which was attributed to demethylation in the ITPR3 promoter region. ITPR3 promoted the proliferation of bladder cancer by accelerating cell cycle transformation and promoted local invasion and distant metastasis by inducing epithelial-to-mesenchymal transition (EMT). Meanwhile, ITPR3 maintained the cancer stemness phenotype by regulating CD44 expression. NF-κB, which is upstream of CD44, also played a critical role in this process.ConclusionsOur study clarifies that ITPR3 serves as an oncogene in bladder cancer cells and represents a novel candidate for bladder cancer diagnosis and treatment.
Highlights
Bladder carcinoma is one of the most common urological cancers
The results showed that Inositol 1 (ITPR3) was significantly upregulated in Bladder cancer (BCa) tissues compared with corresponding normal peritumor tissues and positively correlated with clinicopathological stages (Ta-T4) and tumor invasion degree (NMIBC and Muscle invasive bladder cancer (MIBC)) (Fig. 1a, b)
From the analysis of GEO datasets (GSE3167, GDS183), the GEPIA website and the The Cancer Genome Atlas (TCGA) dataset, we found that ITPR3 was overexpressed in bladder cancer and positively correlated with clinical stages, which was consistent with the result from immunohistochemistry (IHC)
Summary
ITPR3, as a ubiquitous endoplasmic reticulum calcium channel protein, was reported to be involved in the development and progression of various types of cancer. The potential roles and molecular mechanism of ITPR3 in bladder cancer are still unclear. We elucidated a novel role of ITPR3 in regulating the proliferation, metastasis, and stemness of bladder cancer cells. Most bladder cancers are transitional cell carcinomas, with more than 70% of new patients having non-muscle-invasive BCa (stage Tis, Ta, T1) and less than 30% having muscle-invasive BCa (stage T2, T3, T4). As a calcium channel regulatory protein between the ER and mitochondria, the biological effects and expression of ITPR3 in human cancer development and progression are still unclear, especially in BCa
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