Guofeng Qian

Decreased TRPM7 inhibits activities and induces apoptosis of bladder cancer cells via ERK1/2 pathway.

Transient receptor potential melastatin 7 (TRPM7) functions as a Mg2+/Ca2+-permeable channel fused with a kinase domain and regulates various physical processes and diseases. However, its effects on pathogenesis of human bladder cancer (BCa) has not been clarified yet. Our microarray analysis has suggested that calcium signaling pathway is connected with bladder cancer via MAPK pathway. Therefore, we aim to investigate the mechanism of TRPM7 in BCa tumorigenesis by using BCa tissues compared with normal bladder epithelium tissues, as well as using distinct BCa cell lines (EJ, 5637 and T24). We observed increased TRPM7 expression and dysregulation of proteins involved in Epithelial-Mesenchymal Transition (EMT) in BCa tissues. Moreover, knockdown of TRPM7 in BCa cells reversed the EMT status, accompanied by increase of reactive oxygen species (ROS). Furthermore, TRPM7 deficiency could inhibit BCa cell proliferation, migration and invasion, as well as induce p-ERK1/2 and suppress PI3K/AKT at the protein level. Downregulation of TRPM7 promoted cell cycle arrest at G0/G1 phase and apoptosis in vitro, which could be recovered by pre-treatment with U0126 to deactivate ERK1/2, suggesting a close correlation between TRPM7 and the MAPK signaling pathway. Furthermore, a NOD/SCID mouse model transplanted using the BCa cells was established, revealing delayed tumor growth by reduced protein activity and mRNA transcription of TRPM7 in vivo. Our results suggested TRPM7 might be essential for BCa tumorigenesis by interfering BCa cell proliferation, motility and apoptosis.

Capsaicin Suppresses Cell Proliferation, Induces Cell Cycle Arrest and ROS Production in Bladder Cancer Cells through FOXO3a-Mediated Pathways

Capsaicin (CAP), a highly selective agonist for transient receptor potential vanilloid type 1 (TRPV1), has been widely reported to exhibit anti-oxidant, anti-inflammation and anticancer activities. Currently, several therapeutic approaches for bladder cancer (BCa) are available, but accompanied by unfavorable outcomes. Previous studies reported a potential clinical effect of CAP to prevent BCa tumorigenesis. However, its underlying molecular mechanism still remains unknown. Our transcriptome analysis suggested a close link among calcium signaling pathway, cell cycle regulation, ROS metabolism and FOXO signaling pathway in BCa. In this study, several experiments were performed to investigate the effects of CAP on BCa cells (5637 and T24) and NOD/SCID mice. Our results showed that CAP could suppress BCa tumorigenesis by inhibiting its proliferation both in vitro and in vivo. Moreover, CAP induced cell cycle arrest at G0/G1 phase and ROS production. Importantly, our studies revealed a strong increase of FOXO3a after treatment with CAP. Furthermore, we observed no significant alteration of apoptosis by CAP, whereas Catalase and SOD2 were considerably upregulated, which could clear ROS and protect against cell death. Thus, our results suggested that CAP could inhibit viability and tumorigenesis of BCa possibly via FOXO3a-mediated pathways.

Downregulation of TRPM7 suppressed migration and invasion by regulating epithelial–mesenchymal transition in prostate cancer cells

Metastasis is a leading cause of death in patients with prostate cancer (PCa). Transient receptor potential channel 7 (TRPM7) functions as a Mg2+/Ca2+-permeable channel as well as a protein kinase that regulate various cellular processes including cell adhesion, migration and survival. However, the function of TRPM7 in metastasis of PCa remains largely unknown. Microarray analysis suggested that calcium signaling pathway was significantly altered in metastatic PCa tissues, compared with benign prostatic hyperplasia tissues. Bioinformatics analysis using microarray data and database for annotation, visualization and integrated discovery database revealed altered genes involved in calcium signaling pathway were significantly upregulated in TRPM7 deficiency PCa cells, which was also confirmed by experimental verification. Therefore, we aim to investigate the role of TRPM7 in human PCa cell migration and invasion as well as the underlying mechanisms. We observed that TRPM7 was upregulated in PCa cells and tissues compared with prostate hyperplasia cells and tissues. Further investigations suggested that TRPM7 deficiency suppressed migration and invasion of distinct PCa cell lines while overexpression of TRPM7 increased migration of PCa cells. In addition, knockdown of TRPM7 in PCa cells reversed the epithelial–mesenchymal transition (EMT) status, accompanied by downregulation of MMPs and upregulation of E-cadherin. Taken together, our study indicated that downregulation of TRPM7 could inhibit migration and invasion via reversing EMT status in PCa cells.

Co-expression network analysis identified FCER1G in association with progression and prognosis in human clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most common solid lesion within kidney, and its prognostic is influenced by the progression covering a complex network of gene interactions. In current study, the microarray data GSE66272 containing ccRCC and adjacent normal tissues was analyzed to identify 4042 differentially expressed genes, on which weighted gene co-expression network analysis was performed. Then 12 co-expressed gene modules were identified. The highest association was found between blue module and pathological stage (r = -0.77) by Pearsons correlation analysis. Functional enrichment analysis revealed that biological processes of blue module focused on inflammatory response, immune response, chemotaxis (all p < 1e-10). In the significant module, a total of 38 network hub genes were identified, FCER1G exhibited the highest correlation (r = 0.95) with ccRCC progression. In addition, FCER1G was hub node in the protein-protein interaction network of the genes in blue module as well. Thus, FCER1G was subsequently selected for validation. In the test set GSE53757 and RNA-sequencing data, FCER1G expression was also positively correlated with four stages of ccRCC progression (p < 0.001). Receiver operating characteristic (ROC) curve indicated that FCER1G could distinguish localized (pathological stage I, II) from non-localized (pathological stage III, IV) ccRCC (AUC=0.74, p < 0.001). Besides, FCER1G could be a prognostic gene in clinical practice as well, revealed by survival analysis based on RNA-sequencing data (p < 0.05). In conclusion, using weighted gene co-expression analysis, FCER1G was identified and validated in association with ccRCC progression and prognosis, which might improve the prognosis by influencing immune-related pathways.

Knockdown of SIRT1 Suppresses Bladder Cancer Cell Proliferation and Migration and Induces Cell Cycle Arrest and Antioxidant Response through FOXO3a-Mediated Pathways

Bladder cancer (BCa) is one of the most common tumors, but its underlying mechanism has not been fully clarified. Our transcriptome analysis suggested a close link of Sirtuins, Peroxisome Proliferator-Activated Receptor (PPAR), cell cycle regulation, reactive oxygen species (ROS) metabolism, and Forkhead Box Class O (FOXO) signaling pathway in BCa. SIRT1 is a key member of Sirtuins, playing important roles in aging and energy metabolism, which has been reported to be involved in various metabolic diseases and tumors. We observed that SIRT1 was upregulated in BCa tissues at both mRNA and protein levels. By establishing a SIRT1-knockdown BCa cell model, our results suggested that proliferation and viability were suppressed. Moreover, migration rate was inhibited as well, possibly via reduction of epithelial-mesenchymal transition (EMT). In addition, cell cycle arrest was significantly induced, consisting with strongly decreased proteins involved (CDK2/4/6). Furthermore, ROS production was slightly reduced, accompanied by increasing of antioxidant enzymes and total/acetylated FOXO3a. Consistently with our Path-net analysis, we observed no significant alteration of apoptosis in the SIRT1-knockdown BCa cells. Taken together, our results suggested that SIRT1 deficiency in BCa cells could suppress cell viability by activating antioxidant response and inducing cell cycle arrest possibly via FOXO3a-related pathways.

Identification of key genes and pathways in human clear cell renal cell carcinoma (ccRCC) by co-expression analysis

Human clear cell renal cell carcinoma (ccRCC) is the most common solid lesion within kidney, and its prognostic is influenced by the progression covering a complex network of gene interactions. In our study, we screened differential expressed genes, and constructed protein-protein interaction (PPI) network and a weighted gene co-expression network to identify key genes and pathways associated with the progression of ccRCC (n = 56). Functional and pathway enrichment analysis demonstrated that upregulated differentially expressed genes (DEGs) were significantly enriched in response to wounding, positive regulation of immune system process, leukocyte activation, immune response and cell activation. Downregulated DEGs were significantly enriched in oxidation reduction, monovalent inorganic cation transport, ion transport, excretion and anion transport. In the PPI network, top 10 hub genes were identified (TOP2A, MYC, ALB, CDK1, VEGFA, MMP9, PTPRC, CASR, EGFR and PTGS2). In co-expression network, 6 ccRCC-related modules were identified. They were associated with immune response, metabolic process, cell cycle regulation, angiogenesis and ion transport. In conclusion, our study illustrated the hub genes and pathways involved in the progress of ccRCC, and further molecular biological experiments are needed to confirm the function of the candidate biomarkers in human ccRCC.

Silencing of HJURP induces dysregulation of cell cycle and ROS metabolism in bladder cancer cells via PPARγ-SIRT1 feedback loop

Holliday Junction Recognition Protein (HJURP) is a centromeric histone chaperone involving in de novo histone H3 variant CenH3 (CENP-A) recruitment. Our transcriptome and in vivo study revealed that HJURP is significantly upregulated in bladder cancer (BCa) tissues at both mRNA and protein levels. Knockdown of HJURP inhibited proliferation and viability of BCa cell lines revealed by CCK-8, colony formation and Ki-67-staining assays, and induced apoptosis and reactive oxygen species (ROS) production, as well as triggered cell cycle arrest at G0/G1 phase possibly via loss of CENP-A. Interestingly, in the HJURP-reduced BCa cells the levels of PPARγ and acetylated-p53 were increased, while the ratio of phosphorylated/total SIRT1 protein was decreased. Moreover, after treatment of the BCa cells using PPARγ antagonist (GW9662) and SIRT1 agonist (resveratrol, RSV) respectively, thee phenotypes of cell cycle arrest, increased ROS production and inhibited proliferation rate were all rescued. Taken together, our results suggested that HJURP might regulate proliferation and apoptosis via the PPARγ-SIRT1 negative feedback loop in BCa cells.

Overexpression of COL3A1 confers a poor prognosis in human bladder cancer identified by co-expression analysis

Human bladder cancer (BCa) is one of the worldwide cancers in men and women populations, with the etiology and mechanism unknown. In our study, we constructed a weighted gene co-expression network to identify gene modules associated with the progression of BCa (n = 93). In the significant module (R2 = 0.48), a total of 103 network hub genes were identified, and 4 of them were hub nodes in the protein-protein interaction network as well. In validation, COL3A1 showed a higher correlation with the disease progression than any other hub genes in hub module in the test set (p < 0.001). Functional and pathway enrichment analysis demonstrated that COL3A1 is overrepresented in pathway of focal adhesion, which associated with tumor progression and might cause metastasis. Gene set enrichment analysis (GSEA) also demonstrated that the gene set of “MAPK signaling pathway” and focal adhesion related pathways were enriched in BCa samples with COL3A1 highly expressed (FDR < 0.05). Considering the clinicopathological parameters, highly-expressed COL3A1 was closely correlated with local recurrence and BCa stage. Survival analysis revealed that BCa patients with higher expression of COL3A1 had a significantly shorter overall survival time and disease free survival time.In conclusion, based on the co-expression analysis, COL3A1 was identified in the association with progression and prognosis of BCa, which might refer a poor prognosisprobably by regulating MAPK signaling pathway.

A novel correlation between ATP5A1 gene expression and progression of human clear cell renal cell carcinoma identified by co‑expression analysis

Clear cell renal cell carcinoma (ccRCC) is the most common solid lesion within kidneys, and its prognostic is influenced by the progression covering a complex network of gene interactions. In our study, a weighted gene co-expression network was constructed to identify gene modules associated with the progression of ccRCC (n=35). In the significant module (R2 = −0.53), a total of 13 network hub genes were identified, and 2 of them were hub nodes in the protein-protein interaction network as well. In validation, ATP5A1 showed a higher correlation with the disease progression than any other hub gene in the hub module (P=0.001219). In the test set (n=202), ATP5A1 was also highly expressed in normal kidney than ccRCC tissues of each grade (P<0.001). Functional and pathway enrichment analysis demonstrated that ATP5A1 is overrepresented in pathway of oxidative phosphorylation, which associated with tumorigenesis and tumor progression. Gene set enrichment analysis (GSEA) also demonstrated that the gene set of ‘oxidative phosphorylation’ and metabolic pathways were enriched in ccRCC samples with ATP5A1 highly expressed (P<0.05). In conclusion, based on the co-expression analysis, ATP5A1 was validated to be associated with progression of ccRCC, probably by regulating tumor-related phosphorylation.

Co-expression network analysis identified six hub genes in association with progression and prognosis in human clear cell renal cell carcinoma (ccRCC)

Human clear cell renal cell carcinoma (ccRCC) is one of the most common types of malignant adult kidney tumors. We constructed a weighted gene co-expression network to identify gene modules associated with clinical features of ccRCC (n = 97). Six hub genes (CCNB2, CDC20, CEP55, KIF20A, TOP2A and UBE2C) were identified in both co-expression and protein-protein interaction (PPI) networks, which were highly correlated with pathologic stage. The significance of expression of the hub genes in ccRCC was ranked top 4 among all cancers and correlated with poor prognosis. Functional analysis revealed that the hub genes were significantly enriched in cell cycle regulation and cell division. Gene set enrichment analysis suggested that the samples with highly expressed hub gene were correlated with cell cycle and p53 signaling pathway. Taken together, six hub genes were identified to be associated with progression and prognosis of ccRCC, and they might lead to poor prognosis by regulating p53 signaling pathway.