Orai1 and STIM1 are critical for cell migration and proliferation of clear cell renal cell carcinoma

The intracellular Ca²⁺ regulation has been implicated in tumorigenesis and tumor progression. Notably, store-operated Ca²⁺ entry (SOCE) is a major Ca²⁺ entry mechanism in non-excitable cells, being involved in cell proliferation and migration in several types of cancer. However, the expression and biological role of SOCE have not been investigated in clear cell renal cell carcinoma (ccRCC). Here, we demonstrate that Orai1 and STIM1, not Orai3, are crucial components of SOCE in the progression of ccRCC. The expression levels of Orai1 in tumor tissues were significantly higher than those in the adjacent normal parenchymal tissues. In addition, native SOCE was blunted by inhibiting SOCE or by silencing Orai1 and STIM1. Pharmacological blockade or knockdown of Orai1 or STIM1 also significantly inhibited RCC cell migration and proliferative capability. Taken together, Orai1 is highly expressed in ccRCC tissues illuminating that Orai1-mediated SOCE may play an important role in ccRCC development. Indeed, Orai1 and STIM1 constitute a native SOCE pathway in ccRCC by promoting cell proliferation and migration.     

Serum exosomal miR-210 as a potential biomarker for clear cell renal cell carcinoma

Exosomal microRNAs (miRNAs) are suggested to reflect molecular changes occurring in their cells of origin and are potential indicators in the early detection of cancers. This study aimed to determine whether certain exosomal miRNAs from tumor tissue can be used as noninvasive biomarkers for clear cell renal cell carcinoma (ccRCC). Based on ccRCC miRNA expression profiles and the literature, we selected six miRNAs (miR-210, miR-224, miR-452, miR-155, miR-21, and miR-34a) and analyzed their expression in tissues, sera, and serum exosomes through quantitative real-time polymerase chain reaction in hypoxia-induced (with CoCl₂) renal cell lines. miR-210, miR-224, miR-452, miR-155, and miR-21 were upregulated in tumor tissues compared with normal tissues. Serum miR-210 and miR-155 levels were higher in patients with ccRCC than in healthy controls (HCs). Furthermore, only exosomal miR-210 was significantly upregulated in patients with ccRCC than in HCs. Moreover, receiver operating characteristic (ROC) curve analysis revealed an area under the ROC curve of 0.8779 (95% confidence interval, 0.7987-0.9571) and a sensitivity and specificity of 82.5% and 80.0%, respectively. Moreover, exosomal miR-210 was upregulated at an advanced stage, and Fuhrman grade and metastasis decreased significantly one month after surgery. Acute hypoxia exposure activates miR-210 and release of exosomes with upregulated miR-210 in both normal and tumor RCC cell lines and interferes with vacuole membrane protein 1 mRNA expression, especially in the metastatic ccRCC cell line. In conclusion, Serum exosomal miR-210 originating from tumor tissue has potential as a novel noninvasive biomarker for the detection and prognosis of ccRCC.     

SIRT3过表达通过调控细胞代谢转换抑制肾肿瘤细胞的增殖

目的:研究SIRT3对肾透明细胞癌(clear cell renal cell carcinoma, ccRCC)769-P细胞增殖和抗氧化能力的影响,并进一步探究其作用机制。方法:在769-P细胞的基础上构建SIRT3过表达稳转细胞系;利用CCK-8试剂检测769-P SIRT3过表达细胞的增殖速度;利用CellROX~Deep Red染料并结合流式细胞分析检测SIRT3过表达对769-P细胞中ROS水平的影响;利用定量蛋白质组学和代谢组学的方法,探究SIRT3对769-P细胞的作用机制。结果:CCK-8实验结果表明,769-P SIRT3过表达细胞的生长速度与对照细胞相比下降了约48%;定量蛋白质组学分析显示,769-P SIRT3过表达细胞中ALDOA、ALDOA、ENO2、PKM、LDHA、LDHB表达量下调约0.4至0.7倍,SDHB和CS上调约1.3倍;代谢组学分析显示,PEP、pyruvic acid、lactate、carnitine水平下降约0.4至0.7倍,isocitric acid和acetyl-CoA水平升高分别约1.3和2.8倍;分析还显示SIRT3过表达上调SOD2、TXN、GPX4和GLRX5的表达量约1.3至2倍,降低ROS水平约40%,增强细胞对过氧化氢的耐受力。结论:SIRT3过表达引起769-P细胞的代谢转换,从而抑制其增殖;且上调769-P细胞中抗氧化酶的表达,降低ROS水平,增强细胞的抗氧化能力。     

High-level expression of Notch1 increased the risk of metastasis in T1 stage clear cell renal cell carcinoma

Background

Although metastasis of clear cell renal cell carcinoma (ccRCC) is basically observed in late stage tumors, T1 stage metastasis of ccRCC can also be found with no definite molecular cause resulting inappropriate selection of surgery method and poor prognosis. Notch signaling is a conserved, widely expressed signal pathway that mediates various cellular processes in normal development and tumorigenesis. This study aims to explore the potential role and mechanism of Notch signaling in the metastasis of T1 stage ccRCC.

Methodology/Principal Findings

The expression of Notch1 and Jagged1 were analyzed in tumor tissues and matched normal adjacent tissues obtained from 51 ccRCC patients. Compared to non-tumor tissues, Notch1 and Jagged1 expression was significantly elevated both in mRNA and protein levels in tumors. Tissue samples of localized and metastatic tumors were divided into three groups based on their tumor stages and the relative mRNA expression of Notch1 and Jagged1 were analyzed. Compared to localized tumors, Notch1 expression was significantly elevated in metastatic tumors in T1 stage while Jagged1 expression was not statistically different between localized and metastatic tumors of all stages. The average size of metastatic tumors was significantly larger than localized tumors in T1 stage ccRCC and the elevated expression of Notch1 was significantly positive correlated with the tumor diameter. The functional significance of Notch signaling was studied by transfection of 786-O, Caki-1 and HKC cell lines with full-length expression plasmids of Notch1 and Jagged1. Compared to the corresponding controls, all cell lines demonstrated significant promotion in cell proliferation and migration while cell cycle remained unaffected.

Conclusions/Significance

High-level expression of Notch signaling increased the risk of metastasis in T1 stage ccRCC by stimulating the proliferation and migration of tumor cells, which may be helpful for the selection of suitable operation method and prognosis of ccRCC.     

MicroRNA-187, down-regulated in clear cell renal cell carcinoma and associated with lower survival,inhibits cell growth and migration though targeting B7-H3

Aberrantly expressed microRNAs (miRNAs) are frequently associated with the aggressive malignant behavior of human cancers, including clear cell renal cell carcinoma (ccRCC). Based on the preliminary deep sequencing data, we hypothesized that miR-187 may play an important role in ccRCC development. In this study, we found that miR-187 was down-regulated in both tumor tissue and plasma of ccRCC patients. Lower miR-187 expression levels were associated with higher tumor grade and stage. All patients with high miR-187 expression survived 5 years, while with low miR-187 expression, only 42% survived. Suppressed in vitro proliferation, inhibited in vivo tumor growth, and decreased motility were observed in cells treated with the miR-187 expression vector. Further studies showed that B7 homolog 3 (B7-H3) is a direct target of miR-187. Over-expression of miR-187 decreased B7-H3 mRNA level and repressed B7-H3-3′-UTR reporter activity. Knockdown of B7-H3 using siRNA resulted in similar phenotype changes as that observed for overexpression of miR-187. Our data suggest that miR-187 is emerging as a novel player in the disease state of ccRCC. miR-187 plays a tumor suppressor role in ccRCC.     

RASSF6 promotes p21Cip1/Waf1-dependent cell cycle arrest and apoptosis through activation of the JNK/SAPK pathway in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is a highly aggressive and common pathological subtype of renal cancer. This cancer is characterized by biallelic inactivation of the von Hippel–Lindau (VHL) tumor suppressor gene, which leads to the accumulation of hypoxia-inducible factors (HIFs). Although therapies targeted at HIFs can significantly improve survival, nearly all patients with advanced ccRCC eventually succumb to the disease. Thus, additional oncogenic events are thought to be involved in the development of ccRCC tumors. In this study, we investigated the role of RASSF6 in ccRCC. Downregulation of RASSF6 was commonly observed in primary tumors relative to matched adjacent normal tissues. Moreover, functional studies established that ectopic re-expression of RASSF6 in ccRCC cells inhibited cell proliferation, clonogenicity, and tumor growth in mice, whereas silencing of RASSF6 dramatically enhanced cell proliferation in vitro and in vivo. Mechanistic investigation suggested that RASSF6 triggers p21^Cip1/Waf1 accumulation to induce G₁ cell cycle arrest and promote apoptosis upon exposure to pro-apoptotic agents, and both of these mechanisms appear to be mediated by activated JNK signaling. Together, these findings suggest that RASSF6 may play a tumor suppressor role in the progression of ccRCC.     

HAO2 inhibits malignancy of clear cell renal cell carcinoma by promoting lipid catabolic process

Hydroxy acid oxidase 2 (HAO2) has been reported to inhibit tumor progression through metabolic pathway. The current study was designed to evaluate the prognostic significance and probable mechanism of HAO2 in patients with clear cell renal cell carcinoma (ccRCC). The study screened The Cancer Genome Atlas Kidney Clear Cell Carcinoma (TCGA-KIRC) database for patients with ccRCC having complete clinical information and HAO2 expression. Low HAO2 was associated with shorter overall survival (OS) and shorter disease-free survival (DFS). Gene set enrichment analysis (GSEA) showed HAO2 was associated with neutral lipid catabolic process, metabolic process, lipid oxidation, epithelial–mesenchymal transition (EMT), and Kirsten rat sarcoma viral oncogene signaling (KRAS). Western blot analysis and immunohistochemistry analysis checked HAO2 expression in ccRCC cancer tissues, normal tissues, and renal cancer cell lines. HAO2 was downregulated in ccRCC cancer tissues and ccRCC cell lines when compared with their control group. Overexpression of HAO2 by plasmid promoted lipid catabolic process, eliminated lipid accumulation, inhibited KRAS expression, controlled the proliferation, migration, and invasion activity of ccRCC tumor cells. Our results indicated that HAO2 inhibits malignancy ccRCC by promoting lipid catabolic process, HAO2 could be an effective molecular marker and treatment for ccRCC.     

KIF20B promotes the progression of clear cell renal cell carcinoma by stimulating cell proliferation

Renal cell carcinoma (RCC) is a common urinary system cancer with high morbidity and mortality rate. Clear cell renal cell carcinoma (ccRCC) is a highly aggressive and common type of RCC. More and effective therapeutic targets are badly needed for the treatment of ccRCC. Kinesin family protein (KIF)20B, also named M-phase phosphoprotein 1, was reported as a microtubule-associated, plus-end-directed kinesin. KIF20B was involved in multiple cellular processes such as cytokinesis. Multiple studies indicated the oncogenic role for KIF20B in several types of tumors, including breast cancer and bladder cancer. However, the possible role of KIF20B in the progression of renal carcinoma is still unknown. Herein, our study demonstrated that KIF20B was relatively highly expressed in ccRCC tissues. In addition, KIF20B was inversely related to the clinical features including tumor size and T stage. We further found that inhibition of the KIF20B expression by a specific short hairpin RNA obviously reduces proliferation of ccRCC cells both in vitro and in vivo. Our study reveals the involvement of KIF20B in ccRCC progression. Generally, KIF20B is a promising novel therapeutic for the treatment of clear cell RCC.     

G Protein γ subunit 7 loss contributes to progression of clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is a common urinary neoplasm, looking for useful candidates to establish scientific foundation for the therapy of ccRCC is urgent. We downloaded genomic profiles of GSE781, GSE6244, GSE53757, and GSE66271 from the Gene Expression Omnibus (GEO) database. GEO2R was used to analyze the derivative genes, while hub genes were screened by protein-protein interactions and cytoscape. Further, overall survival, gene methylation, gene mutation, and gene expression were all analyzed using bioinformatics tools. Colony formation and cell-cycle assay were used to detect the biological function of GNG7 in vitro. We found that GNG7 was downregulated in ccRCC tissues and negatively associated with overall survival in ccRCC patients. We also found that promoter methylation and frequent gene mutation were responsible for GNG7 gene suppression. GNG7 low expression was related to upregulation of enhancer of zeste homolog 2 and downregulation of disabled homolog 2-interacting protein. Further, Gene Set Enrichment Analysis results showed that mTOR1, E2F, G2M, and MYC pathways were all significantly altered in response to GNG7 low expression. In vitro, A498 and 786-O cells in which GNG7 expression was silenced, exhibited a lower G1 phase when compared to the negative control cells. Taken together, our findings suggest that GNG7 is a tumor suppressor gene in ccRCC progression and represents a novel candidate for ccRCC treatment.     

Study of FABP's interactome and detecting new molecular targets in clear cell renal cell carcinoma

Fatty acids (FAs) play a crucial role in the development of clear cell renal cell carcinoma (ccRCC), FAs function requires the participation of fatty-acid-binding protein (FABP). Current studies have shown that different members of the FABP's family play different roles in the tumorigenesis of ccRCC. Therefore, the systematic analysis of FABPs will be of great significance. However, the diverse expression patterns and prognostic values of nine FABPs have yet to be elucidated. In this study, through multiple analysis and verification of multiple databases, such as ONCOMINE, The Human Protein Atlas, UALCAN, Gene Expression Profiling Interactive Analysis, and cBioPortal, we found that the expression of FABP1 was significantly downregulated and the expression of FABP5/6/7 was significantly upregulated in ccRCC compared with renal tissues, and the patients with high messenger RNA (mRNA) levels of the FABP5/6/7 or low mRNA levels of FABP1 were predicted to have a lower overall survival or disease-free survival. Further analysis by the protein–protein interaction (PPI), Gene Ontology pathway, and Kyoto Encyclopedia of Genes and Genomes pathway showed that FABPs were mainly involved in the peroxisome proliferator-activated receptor (PPAR) pathway. In coexpression analysis, we found that FABP1/5/6/7 was coexpressed with transforming growth factor-β1 (TGF-β1), PPARA, and LPL. This study implied that FABP1/5/6/7 could act as an important tumor biomarker of ccRCC; the role of FABPs may be related to PPAR or TGF-β pathway.     

葡萄糖6-磷酸脱氢酶调控细胞周期蛋白E1和细胞周期蛋白依赖性激酶2促进细胞增殖及其在肾透明细胞癌中的预后价值

肾透明细胞癌(clear cell renal cell carcinoma,ccRCC)是一种转移率高、预后差的细胞代谢性疾病,对其有效诊疗及预后分子标志物的研究十分重要。葡萄糖6-磷酸脱氢酶(glucose 6-phosphatedehydrogenase, G6PD)在ccRCC中高表达,并提示患者不良预后,其促进ccRCC细胞增殖的分子机制有待进一步揭示。本研究发现,降低G6PD可抑制细胞周期G₁/S期转化并显著抑制ccRCC细胞增殖。G6PD可在细胞水平调控G₁/S期转化及增殖相关因子Cyclin D1,CDK4,CDK6,Cyclin E1和CDK2基因表达。TCGA数据库分析结果表明,ccRCC 中Cyclin D1,Cyclin E1 和 CDK2的mRNA 水平显著升高,而CDK4表达无明显差异,CDK6表达却显著降低。相关性分析结果显示,G6PD与Cyclin D1呈显著负相关(P<0.0001),G6PD与CDK4,CDK6之间无显著相关性(P>0.05),G6PD与Cyclin E1(P<0.0001)以及CDK2(P<0.05)显著正相关。进一步免疫组化检测结果表明,Cyclin E1和 CDK2在ccRCC肿瘤组织中表达显著升高。生存预后分析结果显示,Cyclin D1高表达提示ccRCC患者整体预后更为良好,CDK4和CDK6表达水平在ccRCC患者总生存率预测中无意义;而Cyclin E1和CDK2高表达均可提示ccRCC患者预后不良。进一步细胞水平检测发现,Cyclin E1、CDK2表达降低可显著逆转G6PD促进ccRCC细胞增殖的能力。综上,与增殖相关因子Cyclin D1,CDK4和CDK6相比,G6PD有可能通过促进Cyclin E1和CDK2表达升高而发挥促进 ccRCC肿瘤细胞增殖的作用,并且这3者的异常高表达有望成为ccRCC患者不良预后的独立生存预测因素。     

Increased Expression of Pregnancy Up-Regulated Non-Ubiquitous Calmodulin Kinase Is Associated with Poor Prognosis in Clear Cell Renal Cell Carcinoma

Purpose

The aims of this study were to evaluate the clinical significance and potential prognostic value of pregnancy up-regulated non-ubiquitous calmodulin kinase (PNCK) in clear cell renal cell carcinoma (ccRCC) patients.

Materials and Methods

The expression of PNCK mRNA was determined in 24 paired samples of ccRCCs and adjacent normal tissues using real-time RT-PCR. The expression of PNCK was determined in 248 samples of ccRCCs and 92 paired samples of adjacent normal tissues by immunohistochemical analysis. Statistical analysis was performed to define the relationship between PNCK expression and the clinical features of ccRCC.

Results

The mRNA level of PNCK was significantly higher in tumorous tissues than in the adjacent non-tumorous tissues (p<0.001). An immunohistochemical analysis of 92 paired tissue specimens showed that PNCK expression was higher in tumorous tissues than in the adjacent non-tumorous tissues (p<0.001). Moreover, there was a significant correlation between the PNCK expression and various clinicopathological parameters such as Fuhrman grade (p = 0.011), tumor size (p<0.001), T stage (p<0.001) and N stage (p = 0.015). Patients with higher PNCK expression had shorter overall survival time than those with lower PNCK expression (p<0.001). Multivariate analysis indicated that PNCK expression was an independent predictor for poor survival of ccRCC patients.

Conclusions

To our knowledge, this is the first study that determines the relationship between PNCK and prognosis in ccRCC. We found that increased PNCK expression is associated with poor prognosis in ccRCC. PNCK may represent a novel prognostic marker for ccRCC.     

Identification of differentially expressed genes in clear cell renal cell carcinoma by analysis of full-length enriched cDNA library

Summary Renal cell carcinoma (RCC) is the most common malignancy in adult kidney, and accounts for 3% of malignancies worldwide with increasing incidence. Clear cell RCC (ccRCC) is the major type in RCC. Resection by surgery is the main treatment because the response of ccRCC to traditional therapies is very poor. To identify the tumor-associated genes for better understanding the molecular mechanism of ccRCC, the full-length enriched cDNA libraries of ccRCC and normal kidney tissues were constructed by the oligo-capping method. Nucleotide sequences of the cDNA libraries of ccRCC and normal kidney tissues were sequenced. From the sequence analysis of 19,425 and 12,400 clones of ccRCC and normal kidney tissues, 4356 and 3055 genes were identified, respectively. By comparing the gene-expression patterns of ccRCC and normal tissues, the up- or down-regulated genes were identified. Among these identified genes, the differential expression of annexin A2 and argininosuccinate synthetase genes were further confirmed by quantitative real-time PCR and Western blot analysis.     

Knockdown of ubiquitin-like modifier-activating enzyme 2 promotes apoptosis of clear cell renal cell carcinoma cells

Small ubiquitin-related modifier (SUMO) proteins are involved in the development of tumors. Ubiquitin-like modifier-activating enzyme 2 (UBA2) is an important member of the SUMO modification system; however, its role in clear cell renal cell carcinoma (ccRCC) is unclear. Therefore, we investigated the expression and function of UBA2 in ccRCC. Both mRNA and protein expression levels of UBA2 were found to be higher in ccRCC than in normal renal tissues and significantly related to the tumor size, Fuhrman grade, and tumor stage. UBA2 knockdown inhibited ccRCC cell growth, promoted apoptosis in vitro and in vivo, and decreased the abundance of a p53 mutant, c-Myc, and key enzymes of the SUMO modification system. Meanwhile, overexpression of UBA2 had the opposite effects. Overexpression of the p53 mutant or c-Myc alleviated the effects of UBA2 knockdown on ccRCC cell proliferation and apoptosis. In conclusion, targeting UBA2 may have a therapeutic potential against ccRCC.Subject terms: Oncogenes, Oncogenesis     

基质金属蛋白酶和组织金属蛋白酶抑制剂在肾细胞癌组织中的表达

目的:基质金属蛋白酶及组织金属蛋白酶抑制剂在肾细胞癌转移中占有重要的作用,研究肾细胞癌组织中MMP-2、MMP-9、TIMP-1和TIMP-2的表达情况,为肾癌转移的治疗提供理论依据。方法:选取36例肾细胞癌肾组织标本,从相同的肾细胞癌组织及癌旁肾组织获得对照样本,均进行根治性肾切除手术切除。肿瘤分期按TNM分期标准。为了统计评估,肿瘤1期和2期为低级,3期以上为高级。RT-PCR检测肿瘤和正常组织中的MMP-2、MMP-9、TIMP-1和TIMP-2的表达。结果:不同样本MMPs和TIMPs表达水平各不相同。肾细胞癌组织中MMP-2、MMP-9、TIMP-1、TIMP-2在肾细胞癌中的表达明显高于正常肾组织(P0.05)。在肾细胞癌的肿瘤分期方面,MMP-2与MMP-9和肿瘤的分期显著相关,随着肿瘤分期的增加,MMP-2与MMP-9的表达明显升高(P0.05),而TIMP-1与TIMP-2与肿瘤的分期无关。结论:肾细胞癌组织中TIMP-2、MMP-2,MMP-9,TIMP-1的mRNA表达显著高于正常肾组织,抑制MMPS的表达将成为治疗肾细胞癌转移的新的方向。     

肾透明细胞癌预后相关miRNAs的生物信息学分析

目的寻找可作为肾透明细胞癌（ccRCC）生物标志物的miRNA,以及ccRCC与正常组织间miRNA差异表达情况。 方法利用TCGA数据库下载ccRCC中miRNA表达数据,分析肿瘤与正常组织间差异表达miRNA。使用Kaplan-Meier曲线对患者进行生存分析,筛选出表达情况与临床预后相关的miRNA。通过生物信息学对miRNA的靶基因进行预测,然后运用FunRich软件和ClueGO对靶基因进行GO和KEGG富集分析。 结果通过TCGA数据库分析发现,ccRCC较正常组织差异表达miRNA共54个,其中上调33个,下调21个。通过生存分析发现hsa-miR-21和hsa-miR-155与患者预后相关,P≤0.05。进一步通过Perl软件在Targetscan、miRDB、miRTarBase、miRPath这四个数据库中预测miRNA靶基因并将结果取交集,共发现129个靶基因。GO和KEGG分析结果表明,这些靶基因主要与转录因子活性、信号转导以及FoxO、TNF等信号通路密切相关。 结论通过生物信息学分析发现了ccRCC与正常组织的差异表达miRNA;其中hsa-miR-21和hsa-miR-155与患者总体生存率相关,并通过调控靶基因参与相关的信号通路进而影响ccRCC的发生发展进程,提示hsa-miR-21和hsa-miR-155可能是ccRCC潜在的生物标志物。