ITPR3 Facilitates Tumor Growth, Metastasis and Stemness by Inducing the NF-ĸB/CD44 Pathway in Urinary Bladder Carcinoma
Background: Bladder 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.
Methods: The 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.
Results: Higher 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.
Conclusions: Our study clarifies that ITPR3 serves as an oncogene in bladder cancer cells and represents a novel candidate for bladder cancer diagnosis and treatment.
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Supplementary Figure 1: Bioinformatics analysis of ITPR3 expression in bladder cancer and correlation between ITPR3 and overall survival in BCa patients. (A) ITPR3 mRNA expression in bladder cancer tissue and normal adjacent tissue from the GEPIA website based on TCGA database. (B, C) ITPR3 mRNA expression in bladder cancer tissues and adjacent tissues from the TCGA dataset and GEO (GSE3167, GDS183) dataset. (D) ITPR3 is significantly upregulated in bladder cancer (left), as is ITPR3 mRNA expression in multiple kinds of cancers (right), which was analyzed from the Oncomine database. (E) ITPR3 was overexpressed in bladder cancer compared with normal tissues in Sanchez-Carbayo’s research from the Oncomine dataset. (F) Kaplan-Meier analysis of overall survival (OS) and disease-free survival (DFS) in BCa patients with high (n =201) and low (n =201) ITPR3 mRNA expression from TCGA database.
Supplementary Figure 2: The expression of ITPRs in BCa and the relationship between ITPR3 and ITPR1 or ITPR2. (A) Analysis of ITPR1, ITPR2, and ITPR3 mRNA expression in bladder cancer and normal tissues from the TCGA dataset. (B) ITPR1, ITPR2, and ITPR3 mRNA expression levels in SV-HUC-1, 5637, and 253J cells were detected by a qRT-PCR assay. (C) ITPR1, ITPR2, and ITPR3 protein levels were examined by a western blot assay in 5637 cells transfected with shITPR3 or shCon.
Supplementary Figure 3: Significant overexpression of ITPR3 in bladder cancer may be due to demethylation of its promoter. (A) The methylation status of ITPR3 in SV-HUC-1, 5637 and 253J cells was analyzed by bisulfite sequencing PCR. The 43 CpG sites in 10 clones were represented. The solid circle represents the methylated cytosine, and the hollow circle represents the unmethylated cytosine. (B) Analysis of the linear correlation between the mRNA level and methylation level of ITPR3 based on TCGA data. (C) Predicted CpG islands in the promoter region of ITPR3. Numbers indicate the positions in bp relative to the transcription start site. The blue regions represent the CpG islands, and the red vertical bars are the CpG loci in these input sequences. The promoter methylation level (D) and mRNA expression level (E) of ITPR3 in bladder cancer and normal tissue were examined through the UALCAN website based on the TCGA dataset. (F) ITPR3 protein levels were detected by a western blot assay in SV-HUC-1 cells after treatment with different doses of 5-Aza (0 µM, 2.5 µM and 5 µM) for 72 hours. β-actin was used as an internal loading control.
Supplementary Figure 4: GSEA analysis of ITPR3 in the TCGA dataset and significantly changed cell signaling pathways in 50 hallmark gene sets by ITPR3 expression. (A) Heat map of the top 100 genes upregulated or repressed in the ITPR3 high‐expression and ITPR3 low‐expression BCa patient groups. (B) The significantly changed cell signaling pathways in 50 hallmark gene sets from GSEA analysis of ITPR3 expression in the BCa TCGA dataset.
Supplementary Figure 5: ITPR3 was enriched in the CSC. (A) The morphological differences of 5637 and CSCs derived from 5637 cells were captured by contrast microscopy. (B) 253J was also observed in the same way. (C) The expression of ITPR3 and cancer stem cell markers such as CD44, SOX2, and OCT4 was detected by western blot assay in 5637 and CSCs derived from 5637 cells. (D) The expression of ITPR3 and cancer stem cell markers such as CD44, SOX2, and OCT4 was detected by a western blot assay in 253J and CSCs derived from 253J cells.
Posted 29 Dec, 2020
Received 08 Jan, 2021
On 08 Jan, 2021
Received 30 Dec, 2020
Received 27 Dec, 2020
On 21 Dec, 2020
On 21 Dec, 2020
On 20 Dec, 2020
Invitations sent on 20 Dec, 2020
On 20 Dec, 2020
On 20 Dec, 2020
On 20 Dec, 2020
On 19 Dec, 2020
ITPR3 Facilitates Tumor Growth, Metastasis and Stemness by Inducing the NF-ĸB/CD44 Pathway in Urinary Bladder Carcinoma
Posted 29 Dec, 2020
Received 08 Jan, 2021
On 08 Jan, 2021
Received 30 Dec, 2020
Received 27 Dec, 2020
On 21 Dec, 2020
On 21 Dec, 2020
On 20 Dec, 2020
Invitations sent on 20 Dec, 2020
On 20 Dec, 2020
On 20 Dec, 2020
On 20 Dec, 2020
On 19 Dec, 2020
Background: Bladder 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.
Methods: The 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.
Results: Higher 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.
Conclusions: Our study clarifies that ITPR3 serves as an oncogene in bladder cancer cells and represents a novel candidate for bladder cancer diagnosis and treatment.
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