Weimin Yao

Pseudogene PTENP1 Functions as a Competing Endogenous RNA to Suppress Clear-Cell Renal Cell Carcinoma Progression

PTENP1 is a pseudogene of the PTEN tumor suppression gene (TSG). The functions of PTENP1 in clear-cell renal cell carcinoma (ccRCC) have not yet been studied. We found that PTENP1 is downregulated in ccRCC tissues and cells due to methylation. PTENP1 and PTEN are direct targets of miRNA miR21 and their expression is suppressed by miR21 in ccRCC cell lines. miR21 expression promotes ccRCC cell proliferation, migration, invasion in vitro, and tumor growth and metastasis in vivo. Overexpression of PTENP1 in cells expressing miR21 reduces cell proliferation, invasion, tumor growth, and metastasis, recapitulating the phenotypes induced by PTEN expression. Overexpression of PTENP1 in ccRCC cells sensitizes these cells to cisplatin and gemcitabine treatments in vitro and in vivo. In clinical samples, the expression of PTENP1 and PTEN is correlated, and both expressions are inversely correlated with miR21 expression. Patients with ccRCC with no PTENP1 expression have a lower survival rate. These results suggest that PTENP1 functions as a competing endogenous RNA (ceRNA) in ccRCC to suppress cancer progression. Mol Cancer Ther; 13(12); 3086–97. ©2014 AACR.

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, X91348, BC029135, 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.

MicroRNA-34a functions as an anti-metastatic microRNA and suppresses angiogenesis in bladder cancer by directly targeting CD44

BackgroundMetastasis have considered as an important clinical obstacle in the treatment of human cancer including bladder cancer. Post-transcriptional regulation has emerged as robust effectors of metastasis. MiRNAs are involved in cancer development and progression, acting as tumor suppressors or oncogenes. In this study, we focus on it that microRNA-34a functions as an anti-metastatic microRNA and suppress angiogenesis in bladder cancer by directly targeting CD44.MethodsThe expression of mir-34a was detected by quantitative real-time PCR. Oligonucleotide and lentivirus were used to overexpress miR-34a. Tube formation assay and transwell assay were used to examine the effect on bladder cancer tube formation, migration and invasion in vitro. Animal models were used to examine the effect on metastasis and angiogenesis in vivo. Luciferase assay was carried out to verify the precise target of miR-34a.ResultsWe not only proved that mir-34a was significantly downregulated in bladder cancer tissues and cell lines but also that circulating miR-34a levels are reduced in bladder cancer, and their levels were positively relevance. Gain-of-function experiments investigated that increased mir-34a expression suppressed tube formation and reduced cell migration and invasion. In vivo metastasis, assays also demonstrated that overexpression of mir34a markedly inhibited bladder cancer metastasis. CD31, an endothelial cell–specific marker which stained in T24 tumors to evaluate for blood vessel density, the immunohistochemistry results showed that blood vessel quantification reduced dramatically in the T24 tumors over-expressing mir-34a. Combining with our previous studies and bioinformatics analysis, we expected that CD44 gene was a direct target of mir-34a, siRNA-mediated knockdown of CD44 partially phenocopied mir-34a overexpression suggesting that the pro-apoptotic role of mir-34a may be mediated primarily through CD44 regulation, whereas restoring the expression of CD44 attenuated the function of mir-34a in bladder cancer cells. Additionally, we identified that EMT (epithelial-mesenchymal transition) related proteins could be regulated by mir-34a which indicated that mir-34a could partially reserve EMT.ConclusionOur study defines a major metastasis and angiogenesis suppressive role for mir-34a, a microRNA functions as a tumor suppressor in bladder cancer by directly targeting CD44, which would be helpful as a therapeutic approach to block bladder cancer metastasis.

Analysis of Altered MicroRNA Expression Profiles in Proximal Renal Tubular Cells in Response to Calcium Oxalate Monohydrate Crystal Adhesion: Implications for Kidney Stone Disease

Background Calcium oxalate monohydrate (COM) is the major crystalline component in kidney stones and its adhesion to renal tubular cells leads to tubular injury. However, COM-induced toxic effects in renal tubular cells remain ambiguous. MicroRNAs (miRNAs) play an important role in gene regulation at the posttranscriptional levels. Objective The present study aimed to assess the potential changes in microRNAs of proximal renal tubular cells in response to the adhesion of calcium oxalate monohydrate (COM) crystals. Methodology Lactate dehydrogenase (LDH) activity and DAPI staining were used to measure the toxic effects of HK-2 cells exposed to COM crystals. MicroRNA microarray and mRNA microarray were applied to evaluate the expression of HK-2 cells exposed to COM crystals. Quantitative real-time PCR (qRT-PCR) technology was used to validate the microarray results. Target prediction, Gene Ontology (GO) analysis and pathway analysis were applied to predict the potential roles of microRNAs in biological processes. Principal Findings Our study showed that COM crystals significantly altered the global expression profile of miRNAs in vitro. After 24 h treatment with a dose (1 mmol/L), 25 miRNAs were differentially expressed with a more than 1.5-fold change, of these miRNAs, 16 were up-regulated and 9 were down-regulated. A majority of these differentially expressed miRNAs were associated with cell death, mitochondrion and metabolic process. Target prediction and GO analysis suggested that these differentially expressed miRNAs potentially targeted many genes which were related to apoptosis, regulation of metabolic process, intracellular signaling cascade, insulin signaling pathway and type 2 diabetes. Conclusion Our study provides new insights into the role of miRNAs in the pathogenesis associated with nephrolithiasis.

miR-490-5p suppresses tumour growth in renal cell carcinoma through targeting PIK3CA.

Dysregulated micro‐RNAs have been reported in many human cancers, including renal cell carcinoma. Recent studies indicated that miR‐490 is involved in tumour development and progression. However, the expression profile and function in renal cell carcinoma remains unknown.

lncRNA PVT1 and its splicing variant function as competing endogenous RNA to regulate clear cell renal cell carcinoma progression

Long non-coding RNAs (lncRNAs) exert critical regulatory roles in the development and progression of several cancers. Plasmacytoma variant translocation 1 (PVT1), an lncRNA, was shown to be upregulated in clear cell renal cell carcinoma (ccRCC) in our study, while Kaplan-Meier curve and Cox regression analysis showed that high expression of PVT1 was associated with poor overall survival (OS) and disease free survival (DFS) in ccRCC patients. In vitro experiments revealed that PVT1 promoted renal cancer cell proliferation, migration, and invasion, while in vivo studies confirmed its oncogenic roles in ccRCC. Further bioinformatic analysis and RNA immunoprecipitation revealed that PVT1 could function as an oncogenic transcript partly through sponging miR-200s to regulate BMI1, ZEB1 and ZEB2 expression. Besides, a novel splicing variant of PVT1 lacking exon 4 (PVT1ΔE4) was found to have a higher expression in ccRCC and could also promote cell proliferation and invasion as the full-length transcript did. Besides, SRSF1 decreased the inclusion of exon 4 of full-length transcript and increased the relative expression of PVT1ΔE4 in ccRCC. Mechanistic investigations indicated that PVT1ΔE4 could also upregulate the expression of BMI1, ZEB1 and ZEB2 through interacting with miR-200s. Our study helps reveal new molecular events in ccRCC and provides promising diagnostic and therapeutic targets for this disease.

Fibulin-1 is epigenetically down-regulated and related with bladder cancer recurrence.

BackgroundBladder cancer is one of the most common cancers worldwide. Fibulin-1, a multi-functional extracellular matrix protein, has been demonstrated to be involved in many kinds of cancers, while its function in bladder cancer remains unclear. So here we investigated the expression and function of fibulin-1 in Bladder cancer.MethodsWe used real-time PCR, Western blot analysis and immunohistochemistry to determine the expression of fibulin-1 in Bladder cancer cells and patient tissues respectively. Methylation-specific PCR and quantitative sequencing were used to examine the methylation status of FBLN1 gene promoter. Eukaryotic expression plasmid and lentiviral vector were used to overexpress fibulin-1 in Bladder cancer cells 5637, HT-1376 to investigate its function in vitro and in vivo.ResultsWe identified that fibulin-1 was significantly down-regulated in bladder cancer, and its dysregulation was associated with non-muscle-invasive bladder cancer (NMIBC) grade and recurrence. The promoter region of FBLN1 was generally methylated in bladder cancer cell lines and tissues, further investigation in patient tissues showed that the methylation status was associated with the fibulin-1 expression. Overexpression of fibulin-1 significantly suppressed tumor growth, induced tumor cell apoptosis, decreased cell motility, and inhibited angiogenesis in cultured bladder cancer cells and xenograft tumor in nude mice.ConclusionsAltogether, our results indicated that fibulin-1 expression is associated with NMIBC grade and recurrence, it is epigenetically down-regulated and functions as a tumor suppressor gene and angiogenesis inhibitor in bladder cancer.

Laparoscopic Renal Cryoablation Versus Laparoscopic Partial Nephrectomy for the Treatment of Small Renal Masses: A Systematic Review and Meta-analysis of Comparative Studies

BACKGROUND For small renal masses (SRMs), open partial nephrectomy represents the therapeutic standard of care, and laparoscopic partial nephrectomy (LPN) has provided encouraging outcomes. Laparoscopic renal cryoablation (LRC) could be regarded as an alternative to surgical excision in selected patients, if perioperative complication rates and oncologic results are comparable. However, the short- and long-term outcomes of LRC versus LPN have not been adequately assessed. This study evaluated the safety and efficacy of LRC compared with LPN in the treatment of SRMs. MATERIALS AND METHODS A systematic search of the Medline, Scopus, and CNKI databases and the Cochrane Library was performed up to October 1, 2013. Outcomes of interest assessing the two techniques included demographic and clinical baseline characteristics, surgical and oncological variables, renal function, and complications. RESULTS Nine eligible trials (555 cases and 642 controls) assessing LRC versus LPN were identified, including two prospective and seven retrospective studies. Patients undergoing LRC were significantly older (weighted mean difference [WMD], 6.48 years; 95% confidence interval [CI], 3.12-9.83; P<.001) and had a higher solitary kidney rate (odds ratio [OR]=3.76; 95% CI, 2.05-6.92; P<.001). Although LRC was associated with shorter operative time (WMD, -54.28 minutes; 95% CI, -83.79 to -24.78; P<.001), less blood loss (WMD, -111.75 mL; 95% CI, -147.96 to -75.53; P<.001), lower risk of conversion (OR=0.17; 95% CI, 0.05-0.60; P=.005), and fewer overall complications (OR=0.53; 95% CI, 0.29-0.98; P=.04), especially the rate of intraoperative complications (OR=0.20; 95% CI, 0.07-0.58; P=.003) and major complications (OR=0.45; 95% CI, 0.25-0.81; P=.008), patients having LPN might still benefit from a significantly lower local recurrence rate (OR=13.03; 95% CI, 4.20-40.39; P<.001) and lower distant metastasis rate (OR=9.05; 95% CI, 2.31-35.51; P=.002). CONCLUSIONS Compared with LPN, LRC was associated with reliable perioperative safety, comparable renal function, and fewer complications; however, LRC may still result in a higher risk of tumor progression. Therefore, our meta-analysis suggested that LRC was associated with worse oncological outcomes than LPN but that LRC may be indicated in selected patients with significant comorbidity. Because of the inherent limitations of the included studies, further large sample, prospective, multicenter, and long-term follow-up studies are awaited to corroborate these findings.

Alternative Splicing of EZH2 pre-mRNA by SF3B3 Contributes to the Tumorigenic Potential of Renal Cancer

Purpose: Deregulation or mutation of the EZH2 gene causes various tumors, including clear cell renal cell carcinoma (ccRCC). Although several splice variants of EZH2 have been identified, little is known about how EZH2 splicing is regulated or the contribution of alternative splicing to its protumorigenic functions. Experimental Design: We conducted RT-PCR, Western blot analysis, and IHC techniques to examine EZH2 and its alternative splicing transcript expression in renal cancer tissue and renal cancer cell lines. Proliferation, migration, clonogenicity, and tumorigenicity of renal cancer cells either exhibiting knockdown of EZH2 or its splicing factor SF3B3 were assessed by CCK8, Transwell assay, and murine xenograft experiments. Results: We found that the inclusion of alternative EZH2 exon 14 was significantly increased in ccRCC samples and renal cancer cell lines. In ccRCC lines, enforced expression of EZH2Δ14 inhibited, and EZH2 promoted, cell growth, migration, proliferation, and tumorigenicity in a xenograft model. Mechanistic studies demonstrated that EZH2Δ14 isoform functions as a dominant-negative inhibitor of full-length EZH2. Coexpression of EZH2Δ14 variant with full-length EZH2 not only abrogated DAB2IP and HOXA9 suppression but also inhibited EZH2-driven tumorigenesis. Strikingly, the splicing factor SF3B3 stimulates inclusion of exon14 and has pro-proliferative activity. Importantly, the upregulation of SF3B3 expression observed in clinical ccRCC samples parallels the increased inclusion of EZH2 exon14, and the SF3B3 level is associated with higher tumor stage and poor overall survival. Conclusions: These results suggest SF3B3 as a key regulator of EZH2 pre-mRNA splicing and SF3B3 may represent a novel prognostic factor and potential therapeutic target in ccRCC. Clin Cancer Res; 23(13); 3428–41. ©2016 AACR.

Regulation of glucose metabolism by p62/SQSTM1 through HIF1α

ABSTRACT The signaling adaptor sequestosome 1 (SQSTM1)/p62 is frequently overexpressed in tumors and plays an important role in the regulation of tumorigenesis. Although great progress has been made, biological roles of p62 and relevant molecular mechanisms responsible for its pro-tumor activity remain largely unknown. Here, we show that p62 knockdown reduces cell growth and the expression of glycolytic genes in a manner that depends on HIF1α activity in renal cancer cells. Knockdown of p62 decreases HIF1α levels and transcriptional activity by regulating mTORC1 activity and NF-κB nuclear translocation. Furthermore, p62 interacts directly with the von Hippel-Lindau (VHL) E3 ligase complex to modulate the stability of HIF1α. Mechanistically, p62 binds to the VHL complex and competes with HIF1α. Expression of p62 inhibits the interaction of DCNL1 (also known as DCUN1D1) with CUL2 and attenuates the neddylation of CUL2, and thus downregulates the VHL E3 ligase complex activity. Functionally, HIF1α expression is required for p62-induced glucose uptake, lactate production and soft agar colony growth. Taken together, our findings demonstrate that p62 is a crucial positive regulator of HIF1α, which is a facilitating factor in p62-enhanced tumorigenesis. Highlighted Article: p62 is a crucial positive regulator of HIF1α, which is a facilitating factor in p62-enhanced tumorigenesis.