Mounting evidence has demonstrated the vital importance of tumor-associated macrophages (TAMs) and exosomes in the formation of the premetastatic niche. However, the molecular mechanisms by which tumor-derived exosomal miRNAs interact with TAMs underlying premetastatic niche formation and colorectal cancer liver metastasis (CRLM) remain largely unknown. Transmission electron microscopy and differential ultracentrifugation were used to verify the existence of exosomes. In vivo and in vitro assays were used to identify roles of exosomal miR-934. RNA pull-down assay, dual-luciferase reporter assay, etc. were applied to clarify the mechanism of exosomal miR-934 regulated the crosstalk between CRC cells and M2 macrophages. In the present study, we first demonstrated the aberrant overexpression of miR-934 in colorectal cancer (CRC), especially in CRLM, and its correlation with the poor prognosis of CRC patients. Then, we verified that CRC cell-derived exosomal miR-934 induced M2 macrophage polarization by downregulating PTEN expression and activating the PI3K/AKT signaling pathway. Moreover, we revealed that hnRNPA2B1 mediated miR-934 packaging into exosomes of CRC cells and then transferred exosomal miR-934 into macrophages. Interestingly, polarized M2 macrophages could induce premetastatic niche formation and promote CRLM by secreting CXCL13, which activated a CXCL13/CXCR5/NFκB/p65/miR-934 positive feedback loop in CRC cells. These findings indicate that tumor-derived exosomal miR-934 can promote CRLM by regulating the crosstalk between CRC cells and TAMs. These findings reveal a tumor and TAM interaction in the metastatic microenvironment mediated by tumor-derived exosomes that affects CRLM. The present study also provides a theoretical basis for secondary liver cancer.

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Tumor-derived exosomal miR-934 induces macrophage M2 polarization to promote liver metastasis of colorectal cancer

// Mengge Huang 1, * , Zhenyu Zhong 2, * , Mengxin Lv 3 , Jing Shu 4 , Qiang Tian 5 , Junxia Chen 3 1 College of Clinical Medicine, Southwest Medical University, Luzhou 646000, China 2 The First Clinical College, Chongqing Medical University, Chongqing 400016, China 3 Department of Cell Biology and Genetics, Chongqing Medical University, Chongqing 400016, China 4 Department of Clinical Laboratory, The Affiliated Hospital of Southwest Medical University, Luzhou 646000, China 5 Department of Cell Biology and Genetics, Southwest Medical University, Luzhou 646000, China * These authors have contributed equally to this work Correspondence to: Qiang Tian, email: 122398613@qq.com Junxia Chen, email: chjunxia@126.com Keywords: lncRNA, circRNA, ceRNA, microarray, bladder carcinoma Received: February 10, 2016      Accepted: May 16, 2016      Published: May 30, 2016 ABSTRACT Accumulating evidences indicate that long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) play important roles in tumorigenesis. However, the mechanisms remain largely unknown. To explore lncRNAs and circRNAs expression profiling and their biological functions in bladder cancer, we surveyed the lncRNA/circRNA and mRNA expression profiles of bladder cancer and para-cancer tissues using microarray for four patients. Thousands of significantly changed lncRNAs and mRNAs as well as hundreds of circRNAs were identified. Five dysregulated lncRNAs and four mRNAs were confirmed by quantitative real-time PCR in 30 pairs of samples. GO and KEGG pathway enrichment analyses were executed to determine the principal functions of the significantly deregulated genes. Further more, we constructed correlated expression networks including coding-noncoding co-expression (CNC), competing endogenous RNAs (ceRNA), cis regulation, lncRNAs-transcription factor (TF)-mRNA with bioinformatics methods. Co-expression analysis showed lncRNA APLP2 expression is correlated with apoptosis-related genes, including PTEN and TP53INP1. CeRNA network inferred that lncRNA H19 and circRNA MYLK could bind competitively with miRNA-29a-3p increasing target gene DNMT3B, VEGFA and ITGB1 expressions. Moreover, the nearby genes pattern displayed that overexpressing ADAM2 and C8orf4 are cis-regulated by lncRNA RP11-359E19.2, involving in progression of bladder cancer. In addition, lncRNAs-TF-mRNA diagram indicated that lncRNA BC041488 could trans-regulate CDK1 mRNA expression through SRF transcription factor. Taken together, these results suggested lncRNAs and circRNAs could implicate in the pathogenesis and development of bladder cancer. Our findings provide a novel perspective on lncRNAs and circRNAs and lay the foundation for future research of potential roles of lncRNAs and circRNAs in bladder carcinoma.

Comprehensive analysis of differentially expressed profiles of lncRNAs and circRNAs with associated co-expression and ceRNA networks in bladder carcinoma

It is well-established that the chemokine C-X-C motif ligand 13 (CXCL13) and its receptor, the G-protein coupled receptor (GPCR) CXCR5, play fundamental roles in inflammatory, infectious and immune responses Originally identified as a B-cell chemoattractant, CXCL13 exerts important functions in lymphoid neogenesis, and has been widely implicated in the pathogenesis of a number of autoimmune diseases and inflammatory conditions, as well as in lymphoproliferative disorders Current evidence also indicates that the CXCL13:CXCR5 axis orchestrates cell-cell interactions that regulate lymphocyte infiltration within the tumor microenvironment, thereby determining responsiveness to cytotoxic and immune-targeted therapies In this review, we provide a comprehensive perspective of the involvement of CXCL13 and its receptor in cancer progression Studies in recent years postulated novel roles for this chemokine in controlling the cancer cell phenotype, and suggest important functions in the growth and metastatic dissemination of solid tumors Carcinogens have been found to induce CXCL13 production, and production of this chemokine within the tumor milieu has been shown to impact the proliferation, migration, and invasive properties of cancer cells Thus, the complex networks of cellular interactions involving tumoral CXCL13 and CXCR5 integrate to promote cancer cell autonomous and non-autonomous responses, highlighting the relevance of autocrine and paracrine interactions in dictating the cancer phenotype Dissecting the molecular and signaling events regulated by CXCL13 and how this chemokine dynamically controls the interaction between the cancer cell and the tumor microenvironment is key to identify novel effectors and therapeutic targets for cancer treatment

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CXCL13 and Its Receptor CXCR5 in Cancer: Inflammation, Immune Response, and Beyond

The NRF2/KEAP1 pathway represents one of the most important cell defense mechanisms against exogenous or endogenous stressors. Indeed, by increasing the expression of several cytoprotective genes, the transcription factor NRF2 can shelter cells and tissues from multiple sources of damage including xenobiotic, electrophilic, metabolic, and oxidative stress. Importantly, the aberrant activation or accumulation of NRF2, a common event in many tumors, confers a selective advantage to cancer cells and is associated to malignant progression, therapy resistance, and poor prognosis. Hence, in the last years, NRF2 has emerged as a promising target in cancer treatment and many efforts have been made to identify therapeutic strategies aimed at disrupting its prooncogenic role. By summarizing the results from past and recent studies, in this review, we provide an overview concerning the NRF2/KEAP1 pathway, its biological impact in solid and hematologic malignancies, and the molecular mechanisms causing NRF2 hyperactivation in cancer cells. Finally, we also describe some of the most promising therapeutic approaches that have been successfully employed to counteract NRF2 activity in tumors, with a particular emphasis on the development of natural compounds and the adoption of drug repurposing strategies.

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Potential Applications of NRF2 Inhibitors in Cancer Therapy.

Background Bladder cancer is one of the most common urinary malignancies, and has a high recurrence rate and poor outcomes. In order to identify novel diagnostic and prognostic biomarkers for bladder cancer, we conducted a meta-analysis to analyze the association between long non-coding RNA (lncRNA) expression and survival in bladder cancer. Materials and methods We searched literature from databases using our inclusion and exclusion criteria. STATA 14.0 software was used to analyze the data from collected studies and to construct the forest plots. A different effect size was selected for each meta-analysis. Results After selection, 30 articles were found to be eligible. The present meta-analysis contains data from 13 articles about clinicopathological characteristics, six articles about diagnosis, and 16 articles about prognosis. In the present study, we found that many lncRNAs could function as potential diagnostic and prognostic markers in bladder cancer. Among these findings, UCA1 was expected to be a diagnostic biomarker for bladder cancer, while the aberrant expression of HOTAIR and GAS5 was associated with poor disease-free survival/recurrence-free survival/disease-specific survival. Conclusion Overall, the present study is the first meta-analysis to assess the association between expression of lncRNAs and clinical value in patients with bladder cancer. LncRNAs hold promise as novel diagnostic and prognostic markers in bladder cancer.

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LncRNA as a diagnostic and prognostic biomarker in bladder cancer: a systematic review and meta-analysis

The chemokine ligands and their receptors play critical roles in cancer progression and patients outcomes. We found that CXCL13 was significantly upregulated in ccRCC tissues compared with normal tissues in both The Cancer Genome Atlas (TCGA) cohort and a validated cohort of 90 pairs ccRCC tissues. Statistical analysis showed that high CXCL13 expression related to advanced disease stage and poor prognosis in ccRCC. We also revealed that serum CXCL13 levels in ccRCC patients (n = 50) were significantly higher than in healthy controls (n = 40). Receiver operating characteristic (ROC) curve revealed that tissue and serum CXCL13 expression might be a diagnostic biomarker for ccRCC with an area under curve (AUC) of 0.809 and 0.704, respectively. CXCL13 was significantly associated with its receptor, CXCR5, in ccRCC tissues, and ccRCC patients in high CXCL13 high CXCR5 expression group have a worst prognosis. Functional and mechanistic study revealed that CXCL13 promoted the proliferation and migration of ccRCC cells by binding to CXCR5 and activated PI3K/AKT/mTOR signaling pathway. These results suggested that CXCL13/CXCR5 axis played a significant role in ccRCC and might be a therapeutic target and prognostic biomarker.

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CXCL13/CXCR5 Axis Predicts Poor Prognosis and Promotes Progression Through PI3K/AKT/mTOR Pathway in Clear Cell Renal Cell Carcinoma

// Fengjin Zhao 1,* , Tianxin Lin 1,2,* , Wang He 1 , Jinli Han 1 , Dingjun Zhu 1 , Kaishun Hu 3 , Weicong Li 1 , Zaosong Zheng 1 , Jian Huang 1 and Wenlian Xie 1 1 Department of Urology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China 2 Lin Bai-xin Research Center, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China 3 Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China * These Authors contributed equally to this work Correspondence: Wenlian Xie, email: // Keywords : lincRNA, bladder cancer, proliferation, apoptosis Received : September 29, 2014 Accepted : November 24, 2014 Published : November 25, 2014 Abstract Long intergenic noncoding RNAs (lincRNAs) play important roles in regulating various biological processes in cancer, including proliferation and apoptosis. However, the roles of lincRNAs in bladder cancer remain elusive. In this study, we identified a novel lincRNA, which we termed AATBC. We found that AATBC was overexpressed in bladder cancer patient tissues and positively correlated with tumor grade and pT stage. We also found that inhibition of AATBC resulted in cell proliferation arrest through G1 cell cycle mediated by cyclin D1, CDK4, p18 and phosphorylated Rb. In addition, inhibition of AATBC induced cell apoptosis through the intrinsic apoptosis signaling pathway, as evidenced by the activation of caspase-9 and caspase-3. The investigation for the signaling pathway revealed that the apoptosis following AATBC knockdown was mediated by activation of phosphorylated JNK and suppression of NRF2. Furthermore, JNK inhibitor SP600125 could attenuate the apoptotic effect achieved by AATBC knockdown, confirming the involvement of JNK signaling in the induced apoptosis. Moreover, mouse xenograft model revealed that knockdown of AATBC led to suppress tumorigenesis in vivo . Taken together, our study indicated that AATBC might play a critical role in pro-proliferation and anti-apoptosis in bladder cancer by regulating cell cycle, intrinsic apoptosis signaling, JNK signaling and NRF2. AATBC could be a potential therapeutic target and molecular biomarker for bladder cancer.

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Knockdown of a novel lincRNA AATBC suppresses proliferation and induces apoptosis in bladder cancer

Background/Aims: Long non-coding RNAs (lncRNAs) have emerged as new regulators and biomarkers in several cancers. However, few lncRNAs have been well characterized in clear cell renal cell carcinoma (ccRCC). Methods: We investigated the lncRNA expression profile by microarray analysis in 5 corresponding ccRCC tissues and adjacent normal tissues. Lung cancer–associated transcript 1 (LUCAT1) expression was examined in 90 paired ccRCC tissues by real-time PCR and validated by The Cancer Genome Atlas (TCGA) database. Kaplan-Meier analysis was used to examine the prognostic value of LUCAT1 and CXCL2 in ccRCC patients. Loss and gain of function were performed to explore the effect of LUCAT1 on proliferation and invasion in ccRCC cells. Western blotting was performed to evaluate the underlying mechanisms of LUCAT1 in ccRCC progression. Chemokine stimulation assay was performed to investigate possible mechanisms controlling LUCAT1 expression in ccRCC cells. Enzyme-linked immunosorbent assays were performed to determine serum CXCL2 in ccRCC patients and healthy volunteers. Receiver operating characteristic curve analysis was performed to examine the clinical diagnostic value of serum CXCL2 in ccRCC. Results: We found that LUCAT1 was significantly upregulated in both clinical ccRCC tissues (n = 90) and TCGA ccRCC tissues (n = 448) compared with normal tissues. Statistical analysis revealed that the LUCAT1 expression level positively correlated with tumor T stage (P < 0.01), M stage (P < 0.01), and TNM stage (P < 0.01). Overall survival and disease-free survival time were significantly shorter in the high-LUCAT1-expression group than in the low-LUCAT1-expression group (log-rank P < 0.01). LUCAT1 knockdown inhibited ccRCC cell proliferation and colony formation, induced cell cycle arrest at G1 phase, and inhibited cell migration and invasion. Overexpression of LUCAT1 promoted proliferation, migration, and invasion of ccRCC cells. Mechanistic investigations showed that LUCAT1 induced cell cycle G1 arrest by regulating the expression of cyclin D1, cyclin-dependent kinase 4, and phosphorylated retinoblastoma transcriptional corepressor 1. Moreover, LUCAT1 promoted proliferation and invasion in ccRCC cells partly through inducing the phosphorylation of AKT and suppressing the phosphorylation of GSK-3β. We also revealed that chemokine CXCL2, upregulated in ccRCC, induced LUCAT1 expression and might be a diagnostic and prognostic biomarker in ccRCC. Conclusions: LUCAT1 was upregulated in ccRCC tissues and renal cancer cell lines, and significantly correlated with malignant stage and poor prognosis in ccRCC. LUCAT1 promoted proliferation and invasion in ccRCC cells through the AKT/GSK-3β signaling pathway. We also revealed that LUCAT1 overexpression was induced by chemokine CXCL2. These findings indicate that the CXCL2/LUCAT1/AKT/GSK-3β axis is a potential therapeutic target and molecular biomarker for ccRCC.

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Long Non-Coding RNA LUCAT1 Promotes Proliferation and Invasion in Clear Cell Renal Cell Carcinoma Through AKT/GSK-3β Signaling Pathway.

To study the detailed mechanisms of tumorigenesis and clinical outcomes of centrosomal protein 55 (CEP55) overexpression in renal cell carcinoma. Microarray analysis was performed to explore differentially expressed genes in five pairs of RCC tissues. Data of CEP55 expression and corresponding clinical information for 532 RCC patients of TCGA database were downloaded from cBioPortal. The expression of CEP55 in RCC tissues and cells was determined by real-time quantitative reverse transcription PCR (qRT-PCR), Western blot analysis and immunohistochemistry (IHC). Cells were transfected with siRNAs or lentivirus to regulate the expression of CEP55. The effects of CEP55 on proliferation, migration, invasion and epithelial-to-mesenchymal transition (EMT) of RCC cells were determined by MTS, migration and invasion assay and Western blot analysis. CEP55, one of the most upregulated genes in microarray analysis, was overexpressed in RCC tissues and cells. CEP55 expression was significantly correlated with poor outcome including neoplasm disease stage, histologic grade and TNM status, as well as survival status of patients. In vitro experiments showed that downregulation of CEP55 could dramatically inhibit RCC cell proliferation, migration and invasion, while overexpression of CEP55 could promote these biological behaviors. We further demonstrated that CEP55 knockdown suppressed epithelial–mesenchymal transition (EMT), which was mediated via upregulation of E-cadherin and downregulation of N-cadherin and ZEB1, through PI3K/AKT/mTOR pathway. In contrast, overexpression of CEP55 could promote EMT in RCC cells via the activation of PI3K/AKT/mTOR pathway. Importantly, inhibition of PI3K/AKT/mTOR pathway reduced the effects of CEP55 on the migration, invasion and EMT of RCC cells. Our study showed that CEP55 could promote EMT through PI3K/AKT/mTOR pathway and might be an effective prognostic marker in RCC.

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CEP55 promotes epithelial–mesenchymal transition in renal cell carcinoma through PI3K/AKT/mTOR pathway

Previous years have witnessed the importance of long non-coding RNAs (lncRNAs) in cancer research. The lncRNA Pvt1 oncogene (non-protein coding) (PVT1) was revealed to be upregulated in various cancer types. The aim of the present study was to investigate the function of PVT1 in clear cell renal cell carcinoma (ccRCC). The expression of PVT1 in ccRCC was analyzed using reverse transcription-quantitative polymerase chain reaction, and it was revealed that PVT1 expression was upregulated in ccRCC tissues compared with that in normal adjacent tissues. Next, PVT1 expression from The Cancer Genome Atlas datasets was validated, and it was also revealed that the high expression of PVT1 was associated with advanced disease stage and a poor prognosis. Furthermore, the knockdown of PVT1 induced apoptosis by increasing the expression of poly ADP ribose polymerase and Bcl-2-associated X protein, and promoted cell cycle arrest at the G1 phase by decreasing the expression of cyclin D1. Study of the mechanism involved indicated that PVT1 promoted the progression of ccRCC partly through activation of the epidermal growth factor receptor pathway. Altogether, the results of the present study suggested that PVT1 serves oncogenic functions and may be a biomarker and therapeutic target in ccRCC.

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Knockdown of long non-coding RNA PVT1 induces apoptosis and cell cycle arrest in clear cell renal cell carcinoma through the epidermal growth factor receptor pathway

Zaosong Zheng

Papers

CXCL13/CXCR5 Axis Predicts Poor Prognosis and Promotes Progression Through PI3K/AKT/mTOR Pathway in Clear Cell Renal Cell Carcinoma

Knockdown of a novel lincRNA AATBC suppresses proliferation and induces apoptosis in bladder cancer

Long Non-Coding RNA LUCAT1 Promotes Proliferation and Invasion in Clear Cell Renal Cell Carcinoma Through AKT/GSK-3β Signaling Pathway.

CEP55 promotes epithelial–mesenchymal transition in renal cell carcinoma through PI3K/AKT/mTOR pathway

Knockdown of long non-coding RNA PVT1 induces apoptosis and cell cycle arrest in clear cell renal cell carcinoma through the epidermal growth factor receptor pathway