Identification of Potential Serum Proteomic Biomarkers for Clear Cell Renal Cell Carcinoma

Objective

To investigate discriminating protein patterns and serum biomarkers between clear cell renal cell carcinoma (ccRCC) patients and healthy controls, as well as between paired pre- and post-operative ccRCC patients.

Methods

We used magnetic bead-based separation followed by matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) to identify patients with ccRCC. A total of 162 serum samples were analyzed in this study, among which there were 58 serum samples from ccRCC patients, 40 from additional paired pre- and post-operative ccRCC patients (n = 20), and 64 from healthy volunteers as healthy controls. ClinProTools software identified several distinct markers between ccRCC patients and healthy controls, as well as between pre- and post-operative patients.

Results

Patients with ccRCC could be identified with a mean sensitivity of 88.38% and a mean specificity of 91.67%. Of 67 m/z peaks that differed among the ccRCC, healthy controls, pre- and post-operative ccRCC patients, 24 were significantly different (P<0.05). Three candidate peaks, which were upregulated in ccRCC group and showed a tendency to return to healthy control values after surgery, were identified as peptide regions of RNA-binding protein 6 (RBP6), tubulin beta chain (TUBB), and zinc finger protein 3 (ZFP3) with the m/z values of 1466.98, 1618.22, and 5905.23, respectively.

Conclusion

MB-MALDI-TOF-MS method could generate serum peptidome profiles of ccRCC, and provide a new approach to identify potential biomarkers for diagnosis as well as prognosis of this malignancy.     

缺血后处理对大鼠肾脏miRNAs表达谱的影响

目的：观察缺血后处理对Wistar大鼠肾组织miRNAs表达的影响。方法：将20 只Wistar 大鼠随机分为缺血再灌注组（I/R 组）和缺血后处理组（IPO 组）,每组10 只。I/R 组行剖腹手术,分离双肾动脉后切除右肾,夹闭左肾动脉阻断血供,45 min 后恢复; IPoC组行剖腹手术,分离左肾动脉后将其夹闭,在阻断血液供应45 min 后恢复血供时采取10 s再灌、10 s停灌处理,反复5个周 期。在所有实验完成6 小时后,处死大鼠切除左肾,分别提取两组肾组织中的总RNA和miRNA,利用miRNA微阵列对其进行杂 交检测,通过芯片扫描和数据聚类分析,获取两组大鼠肾脏组织miRNAs 表达谱,并进行qRT-PCR 验证,筛选差异表达的 miRNAs。结果：通过qRT-PCR 验证共筛选出7 种表达差异较大的miRNAs,其中下调的三种,分别是miR-27a,miR-665, miR-let7f,上调的四种,分别为miR-532-3p,miR-205,miR-122,miR-291b。结论：差异表达miRNAs 可能参与缺血后处理减弱缺血 再灌注损伤的作用机制中。     

LncRNAs Expression Signatures of Renal Clear Cell Carcinoma Revealed by Microarray

Background

Long noncoding RNAs (lncRNAs) are an important class of pervasive genes involved in a variety of biological functions. They are aberrantly expressed in many types of cancers. In this study, we described lncRNAs profiles in 6 pairs of human renal clear cell carcinoma (RCCC) and the corresponding adjacent nontumorous tissues (NT) by microarray.

Methodology/Principal Findings

With abundant and varied probes accounting 33,045 LncRNAs in our microarray, the number of lncRNAs that expressed at a certain level could be detected is 17157. From the data we found there were thousands of lncRNAs that differentially expressed (≥2 fold-change) in RCCC tissues compared with NT and 916 lncRNAs differentially expressed in five or more of six RCCC samples. Compared with NT, many lncRNAs were significantly up-regulated or down-regulated in RCCC. Our data showed that down-regulated lncRNAs were more common than up-regulated ones. ENST00000456816, {"type":"entrez-nucleotide","attrs":{"text":"X91348","term_id":"1418768","term_text":"X91348"}}X91348, {"type":"entrez-nucleotide","attrs":{"text":"BC029135","term_id":"23271405","term_text":"BC029135"}}BC029135, {"type":"entrez-nucleotide","attrs":{"text":"NR_024418","term_id":"212286160","term_text":"NR_024418"}}NR_024418 were evaluated by qPCR in sixty-three pairs of RCCC and NT samples. The four lncRNAs were aberrantly expressed in RCCC compared with matched histologically normal renal tissues.

Conclusions/Significance

Our study is the first one to determine genome-wide lncRNAs expression patterns in RCCC by microarray. The results displayed that clusters of lncRNAs were aberrantly expressed in RCCC compared with NT samples, which revealed that lncRNAs differentially expressed in tumor tissues and normal tissues may exert a partial or key role in tumor development. Taken together, this study may provide potential targets for future treatment of RCCC and novel insights into cancer biology.     

High Throughput Kinomic Profiling of Human Clear Cell Renal Cell Carcinoma Identifies Kinase Activity Dependent Molecular Subtypes

Despite the widespread use of kinase-targeted agents in clear cell renal cell carcinoma (CC-RCC), comprehensive kinase activity evaluation (kinomic profiling) of these tumors is lacking. Thus, kinomic profiling of CC-RCC may assist in devising a classification system associated with clinical outcomes, and help identify potential therapeutic targets. Fresh frozen CC-RCC tumor lysates from 41 clinically annotated patients who had localized disease at diagnosis were kinomically profiled using the PamStation®12 high-content phospho-peptide substrate microarray system (PamGene International). Twelve of these patients also had matched normal kidneys available that were also profiled. Unsupervised hierarchical clustering and supervised comparisons based on tumor vs. normal kidney and clinical outcome (tumor recurrence) were performed and coupled with advanced network modeling and upstream kinase prediction methods. Unsupervised clustering analysis of localized CC-RCC tumors identified 3 major kinomic groups associated with inflammation (A), translation initiation (B), and immune response and cell adhesions (C) processes. Potential driver kinases implicated include PFTAIRE (PFTK1), PKG1, and SRC, which were identified in groups A, B, and C, respectively. Of the 9 patients who had tumor recurrence, only one was found in Group B. Supervised analysis showed decreased kinase activity of CDK1 and RSK1-4 substrates in those which progressed compared to others. Twelve tumors with matching normal renal tissue implicated increased PIM’s and MAPKAPK’s in tumors compared to adjacent normal renal tissue. As such, comprehensive kinase profiling of CC-RCC tumors could provide a functional classification strategy for patients with localized disease and identify potential therapeutic targets.     

肾透明细胞癌相关微小RNA的研究进展

微小核糖核酸(miRNAs)是一类长约22个核苷酸的非编码单链小核糖核酸分子,miRNA通过与靶mRNA 3'端非翻译序列完全或部分互补结合,导致靶mRNA降解或转录后翻译抑制,从而调控靶基因的表达.最新研究显示人类血清/血浆中miRNA表达稳定,并在肿瘤患者血清中发现多种miRNA,其中的一些已经被证实与肾癌发生及发展相关,以往miRNA与肾癌的研究方.向多集中于肾癌组织,尽管发现很多有差异的miRNA,但不同研究者之间的结果常难以相互验证,而最近研究证实血清miRNA具有组织相关性和器官特异性,并对某些肿瘤具有高敏感性和特异性,因此其有望成为新的肿瘤标志物.肾癌是国内泌尿系统的第二常见恶性肿瘤,而且其近年来发病率和死亡率有逐年增高的趋势.由于肾透明细胞癌是肾癌的主要亚型,因此本文就血清miRNA在肾透明细胞癌的表达及其作用的研究进展作一综述.     

Identification of Novel miRNAs and miRNA Expression Profiling in Wheat Hybrid Necrosis

MicroRNAs (miRNAs) play essential roles in a vast array of biological processes, including growth and development, defense against viral infection, and responses to environmental changes in plant. Wheat hybrid necrosis is an interesting genetic phenomenon observed frequency and it is lethal or semi lethal, resulting in gradual death or loss of productivity. However, the molecular basis and mechanisms associated with hybrid necrosis in wheat are still not well understood. Here, we report the population and expression profiles of miRNAs in wheat hybrid necrosis. We identified a total of 57 conserved miRNA families as well as 182 putative novel miRNAs. Expression profiling revealed that expression of 49 known miRNAs and 165 novel miRNAs was changed in hybrid necrosis. And the expression levels of some miRNAs and their predicated targets have been confirmed by qRT-PCR. These results indicate that these miRNAs, especially miR159, miR166, miR167 and miR5072 could be involved in the extensive regulation of gene expression in response to hybrid necrosis.     

COX-2 与mPGES-1 在肾透明细胞癌中的表达及临床意义

目的:探讨环氧合酶-2(COX-2)和膜结合型前列腺素E2合成酶1(mPGES-1)在肾透明细胞癌组织中的表达及临床意义。方法:采用免疫组化SP法分别检测49例肾透明细胞癌组织标本和21例正常肾组织标本中COX-2和mPGES-1的表达。结果:COX-2在正常肾组织中的阳性表达率为4.8%,在肾透明细胞癌组织中的阳性表达率为53.1%(P<0.05);mPGES-1在正常肾组织中的阳性表达率为4.8%,在肾透明细胞癌组织中的阳性表达率为40.8%(P<0.05);COX-2和mPGES-1的高表达均与肾透明细胞癌的病理分级和临床分期无相关性(P>0.05);COX-2和mPGES-1在肾透明细胞癌中的表达呈正相关(P<0.05),r=0.5。结论:COX-2和mPGES-1在肾透明细胞癌发生及发展过程中共同发挥重要作用;COX-2和mPGES-1可能成为肾透明细胞癌新的治疗靶点。     

肾透明细胞癌MAPK9的表达与索拉非尼疗效的关系

目的:探讨肾透明细胞癌组织中MAPK9表达及其与肾透明细胞癌主要临床病理特征之间的关系,分析MAPK9对索拉非尼靶向治疗疗效的关系。方法:回顾分析2006年5月-2013年10月经病理确诊的32例肾透明细胞癌转移患者的MAPK9的表达情况。所有患者术后均服用索拉菲尼并随访;同时选取10例距肿瘤3 cm癌旁正常肾组织作为对照。对比分析肾透明细胞癌患者与对照组MAPK9的阳性表达率,分析MAPK9阳性表达与疗效的相关性,并分析MAPK9阳性表达与生存率之间的关系。结果:肾透明细胞癌组MAPK9阳性表达21例(65.6%),较正常对照组2例(20.0%)有显著性差异(P0.01)。TNMⅠ~Ⅱ期MAPK9阳性表达12例(85.7%),较TNMⅢ~Ⅳ期10例(55.6%)有显著性差异(P0.05);核分级1~2级MAPK9阳性表达18例(81.8%),较3~4级4例(40.0%)有显著性差异(P0.05)。MAPK9阳性表达患者与阴性表达患者疗效有显著性差异(P0.05)。MAPK9阳性患者PFS为(646±103)d,较阴性患者PFS为(502±89)d,有显著性差异(P0.05)。MAPK9阳性患者OS为(866±75)d,较阴性患者OS为(657±62)d,有显著性差异(P0.01)。结论:MAPK9在肾透明细胞癌中高表达,与肿瘤的临床TNM分期、病理分级相关。MAPK9阳性表达患者其靶向治疗疗效越好,提示MAPK9可能是一个预测索拉菲尼靶向治疗肾透明细胞癌疗效的潜在因子。     

Identification of Metastamirs as Metastasis-associated MicroRNAs in Clear Cell Renal Cell Carcinomas

MicroRNAs (miRNAs) play a pivotal role in cancerogenesis and cancer progression, but their specific role in the metastasis of clear cell renal cell carcinomas (ccRCC) is still limited. Based on microRNA microarray analyses from normal and cancerous samples of ccRCC specimens and from bone metastases of ccRCC patients, we identified a set of 57 differentially expressed microRNAs between these three sample groups of ccRCC. A selected panel of 33 miRNAs was subsequently validated by RT-qPCR on total 57 samples. Then, 30 of the 33 examined miRNAs were confirmed to be deregulated. A stepwise down-regulation of miRNA expression from normal, over primary tumor to metastatic tissue samples, was found to be typical. A total of 23 miRNAs (miR-10b/-19a/-19b/-20a/-29a/-29b/-29c/-100/-101/-126/-127/-130/-141/-143/-145/-148a/-192/-194/-200c/-210/-215/-370/-514) were down-regulated in metastatic tissue samples compared with normal tissue. This down-regulated expression in metastatic tissue in comparison with primary tumor tissue was also present in 21 miRNAs. In cell culture experiments with 5-aza-2''-deoxycytidine and trichostatin A, epigenetic modifications were shown as one reason of this down-regulation. The altered miRNA profiles, comprising newly identified metastasis-associated miRNAs, termed metastamir and the predicted miRNA-target interactions together with the significant correlations of miRNAs that were either lost or newly appeared in the studied sample groups, afford a solid basis for further functional analyses of individual miRNAs in RCC metastatic progression.     

Gas6 and the Receptor Tyrosine Kinase Axl in Clear Cell Renal Cell Carcinoma

Background

The molecular biology of renal cell carcinoma (RCC) is complex and not fully understood. We have recently found that the expression of the receptor tyrosine kinase Axl in the RCC tumors independently correlates with survival of the patients.

Principal Findings

Here, we have investigated the role of Axl and its ligand Gas6, the vitamin-K dependent protein product of the growth arrest-specific gene 6, in clear cell RCC (ccRCC) derived cells. The Axl protein was highly expressed in ccRCC cells deficient in functional von Hippel-Lindau (VHL) protein, a tumor suppressor gene often inactivated in ccRCC. VHL reconstituted cells expressed decreased levels of Axl protein, but not Axl mRNA, suggesting VHL to regulate Axl expression. Gas6-mediated activation of Axl in ccRCC cells resulted in Axl phosphorylation, receptor down-regulation, decreased cell-viability and migratory capacity. No effects of the Gas6/Axl system could be detected on invasion. Moreover, in ccRCC tumor tissues, Axl was phosphorylated and Gas6 γ-carboxylated, suggesting these molecules to be active in vivo.

Significance

These results provide novel information regarding the complex function of the Gas6/Axl system in ccRCC.     

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.     

异常激活的葡萄糖-6-磷酸脱氢酶通过上调周期蛋白D1表达促进肾透明细胞癌增殖

葡萄糖6-磷酸脱氢酶（glucose 6-phosphate dehydrogenase,G6PD）为磷酸戊糖途径的调节酶。研究表明,G6PD与多种恶性肿瘤的发生密切相关。然而,G6PD在肾透明细胞癌（clear cell renal cell carcinoma, ccRCC）中的功能及其作用机制却鲜有报道。本研究通过TCGA数据分析发现,G6PD在肾透明细胞癌TNM Ⅲ/Ⅳ期mRNA表达水平显著升高,与患者的性别、原发肿瘤直径、淋巴结转移、远端转移、病灶一侧的偏重性、病理分级以及TNM临床分期密切相关。并且,G6PD异常激活有可能成为评价肾透明细胞癌患者不良预后的分子。细胞系检测结果提示,与对照293T细胞及恶性程度较低的786-O细胞相比,恶性程度较高的Caki-1细胞中的G6PD表达及活性明显增加。基因稳定转染结合CCK8分析结果显示,G6PD过表达或异常激活可显著提高293T及786-O细胞的增殖能力,并且促进786-O细胞中周期蛋白D1基因表达上调。综上,本研究通过TCGA数据库分析和稳定细胞系检测及CCK8分析,结果显示,G6PD在肾透明细胞癌中异常激活,并可上调细胞周期蛋白D1表达,进而促进肿瘤细胞增殖。该研究为进一步揭示肾透明细胞癌分子发病机制以及开发有效的靶向治疗方案提供了借鉴。     

Role of IL13RA2 in Sunitinib Resistance in Clear Cell Renal Cell Carcinoma

Vascular endothelial growth factor (VEGF) and mammalian target of rapamycin are well-known therapeutic targets for renal cell carcinoma (RCC). Sunitinib is an agent that targets VEGF receptors and is considered to be a standard treatment for metastatic or unresectable clear cell RCC (ccRCC). However, ccRCC eventually develops resistance to sunitinib in most cases, and the mechanisms underlying this resistance are not fully elucidated. In the present study, we established unique primary xenograft models, KURC1 (Kyoto University Renal Cancer 1) and KURC2, from freshly isolated ccRCC specimens. The KURC1 xenograft initially responded to sunitinib treatment, however finally acquired resistance. KURC2 retained sensitivity to sunitinib for over 6 months. Comparing gene expression profiles between the two xenograft models with different sensitivity to sunitinib, we identified interleukin 13 receptor alpha 2 (IL13RA2) as a candidate molecule associated with the acquired sunitinib-resistance in ccRCC. And patients with high IL13RA2 expression in immunohistochemistry in primary ccRCC tumor tends to have sunitinib-resistant metastatic site. Next, we showed that sunitinib-sensitive 786-O cells acquired resistance in vivo when IL13RA2 was overexpressed. Conversely, shRNA-mediated knockdown of IL13RA2 successfully overcame the sunitinib-resistance in Caki-1 cells. Histopathological analyses revealed that IL13RA2 repressed sunitinib-induced apoptosis without increasing tumor vasculature in vivo. To our knowledge, this is a novel mechanism of developing resistance to sunitinib in a certain population of ccRCC, and these results indicate that IL13RA2 could be one of potential target to overcome sunitinib resistance.