Objective: To explore the potential pathogenesis of clear cell renal cell carcinoma (ccRCC) based on the HIF-1α/ACLY signaling pathway, as well as to provide new ideas for the treatment of ccRCC. Methods: Seventy-eight ccRCC cases diagnosed at the First Affiliated Hospital of Soochow University, Suzhou, China were collected. The VHL mutation was examined using exon sequencing. The expression of HIF-1α/ACLY in VHL-mutated ccRCC was evaluated using immunohistochemical staining and further validated in VHL-mutated ccRCC cell lines (786-O, A498, UM-RC-2, SNU-333, and Caki-2) using Western blot. The mRNA and protein levels of ACLY were detected using real-time quantitative PCR and Western blot after overexpression or interference with HIF-1α in ccRCC cell lines. HeLa cells were treated with CoCl₂ and hypoxia (1%O₂) to activate HIF-1α and then subject to the detection of the ACLY mRNA and protein levels. The potential molecular mechanism of HIF-1α-induced ACLY activation was explored through JASPAR database combined with chromatin immunoprecipitation assay (ChIP) and luciferase reporter gene assay. The effect of HIF-1α/ACLY regulation axis on lipid accumulation was detected using BODIPY staining and other cell biological techniques. The expression of ACLY was compared between patients with ccRCC and those with benign lesions, and the feasibility of ACLY as a prognostic indicator for ccRCC was explored through survival analysis. Results: Exon sequencing revealed that 55 (70.5%) of the 78 ccRCC patients harbored a VHL inactivation mutation, and HIF-1α expression was associated with ACLY protein levels. The protein levels of ACLY and HIF-1α in ccRCC cell lines carrying VHL mutation were also correlated to various degrees. Overexpression of HIF-1α in A498 cells increased the mRNA and protein levels of ACLY, and knockdown of HIF-1α in Caki-2 cells inhibited the mRNA and protein levels of ACLY (P<0.001 for all). CoCl₂ and hypoxia treatment significantly increased the mRNA and protein levels of ACLY by activating HIF-1α (P<0.001 for all). The quantification of transcriptional activity of luciferase reporter gene and ChIP-qPCR results suggested that HIF-1α could directly bind to ACLY promoter region to transcriptionally activate ACLY expression and increase ACLY protein level (P<0.001 for all). The results of BODIPY staining suggested that the content of free fatty acids in cell lines was associated with the levels of HIF-1α and ACLY. The depletion of HIF-1α could effectively reduce the accumulation of lipid in cells, while the overexpression of ACLY could reverse this process. At the same time, cell function experiments showed that the proliferation rate of ccRCC cells with HIF-1α knockdown was significantly decreased, and overexpression of ACLY could restore proliferation of these tumor cells (P<0.001). Survival analysis further showed that compared with the ccRCC patients with low ACLY expression, the ccRCC patients with high ACLY expression had a poorer prognosis and a shorter median survival (P<0.001). Conclusions: VHL mutation-mediated HIF-1α overexpression in ccRCC promotes lipid synthesis and tumor progression by activating ACLY. Targeting the HIF-1α/ACLY signaling axis may provide a theoretical basis for the clinical diagnosis and treatment of ccRCC.
Humans ; Carcinoma, Renal Cell/pathology* ; Kidney Neoplasms/pathology* ; HeLa Cells ; Von Hippel-Lindau Tumor Suppressor Protein/genetics* ; Mutation ; Signal Transduction ; Luciferases/therapeutic use* ; Hypoxia/genetics* ; RNA, Messenger ; Lipids/therapeutic use* ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic

OBJECTIVE: To explore the mechanism by which ginsenoside 20(S)-Rg3 upregulates the expression of tumor suppressor von Hippel-Lindau (VHL) gene in ovarian cancer cells. METHODS: Ovarian cancer cell line SKOV3 treated with 20(S)-Rg3 were examined for mRNA and protein levels of VHL, DNMT1, DNMT3A and DNMT3B by real-time PCR and Western blotting, respectively. The changes in VHL mRNA expression in SKOV3 cells in response to treatment with 5-Aza-CdR, a DNA methyltransferase inhibitor, were detected using real-time PCR. VHL gene promoter methylation was examined with methylation-specific PCR and VHL expression levels were determined with real-time PCR and Western blotting in non-treated or 20(S)-Rg3-treated SKOV3 cells and in 20(S)-Rg3-treated DNMT3A-overexpressing SKOV3 cells. VHL and DNMT3A protein levels were detected by immunohistochemistry in subcutaneous SKOV3 cell xenografts in nude mice. RESULTS: Treatment of SKOV3 cells with 20(S)-Rg3 significantly upregulated VHL and downregulated DNMT3A expressions at both the mRNA and protein levels ( CONCLUSIONS Ginsenoside 20(S)-Rg3 upregulates VHL expression in ovarian cancer cells by suppressing DNMT3A-mediated DNA methylation.
Animals ; Cell Line, Tumor ; DNA Methylation ; Female ; Gene Expression ; Ginsenosides/pharmacology* ; Humans ; Mice ; Mice, Nude ; Ovarian Neoplasms/genetics* ; Promoter Regions, Genetic ; Von Hippel-Lindau Tumor Suppressor Protein/genetics*

OBJECTIVE: To detect VHL gene mutation in a pedigree affected with von Hippel-Lindau syndrome (VHL). METHODS: Clinical data of the pedigree was reviewed. Patients were subjected to Sanger sequencing to detect mutation of the VHL gene. Structure of pVHL was predicted by 3D modeling using the swiss-model. RESULTS: A novel c.426delT(p.V142fs) [NM_000551] mutation was found in exon 2 of the VHL gene. 3D modeling suggested that the alpha-structure of pVHL is completely absent. CONCLUSION The novel c.426delT(p.V142fs) mutation probably underlies the VHL in this pedigree.
DNA Mutational Analysis ; Exons ; Humans ; Mutation ; Pedigree ; Von Hippel-Lindau Tumor Suppressor Protein ; genetics ; von Hippel-Lindau Disease ; genetics

OBJECTIVE: To analyze the germline variations of genes RET, VHL, SDHD and SDHB in patients with pheochromocytoma and/or paraganglioma and to evaluate variations of these genes in Chinese patients. METHODS: Patients who were treated in Peking University First Hospital from September 2012 to March 2014 and diagnosed with pheochromocytoma and/or paraganglioma by pathologists were included in this study. Twelve patients were included in total, of whom 11 had pheochromocytoma, and 1 had paraganglioma. Deoxyribonucleic acid (DNA) was extracted from the leukocytes of peripheral blood of the patients. The exons 10, 11, 13-16 of the RET gene, and all exons of VHL, SDHB and SDHD genes and their nearby introns (±20 bp) were amplified with polymerase chain reactions, and the products were sent to a biotechnology company for sequencing. The sequencing results were compared with wildtype sequences of these genes to identify variations. One of the patients was diagnosed with multiple endocrine neoplasia type 2A. A family analysis was performed in his kindred, and his family members received genetic tests for the related variations. RESULTS: Three patients were found to have germline gene variations. A c.136C>T (p.R46X) variation of the SDHB gene was found in a patient with malignant pheochromocytoma. A c.1901G>A (C634Y) variation, as well as c.2071G>A (p.G691S) and c.2712C>G (p.S904S) variations of the RET gene were found in a patient with multiple endocrine neoplasia type 2A. After a family analysis, five family members of this patient were found to have the same variations. c.2071G>A (p.G691S) and c.2712C>G (p.S904S) variations of the RET gene were also found in a clinical sporadic patient without evidence of malignancy. A patient with congenital single ventricle malformation and pheochromocytoma was included in this study, and no variation with clinical significance was found in the four genes of this patient. CONCLUSION 25% (3/12) patients with pheochromocytoma or paraganglioma were found to have missense or nonsense germline gene variations in this study, including the c.136C>T (p.R46X) variation of the SDHB gene, the c.1901G>A (C634Y) variation of the RET gene, and c.2071G>A (p.G691S) and c.2712C>G (p.S904S) variations of the RET gene. The former two variations have already been confirmed to be pathogenic. The existence of these variations in Chinese patients with pheochromocytoma and/or paraganglioma was validated in this study, which supports the conclusion that genetic testing is necessary to be generally performed in patients with pheochromocytoma and/or paraganglioma.
Adrenal Gland Neoplasms/genetics* ; Genetic Testing ; Germ-Line Mutation ; Humans ; Paraganglioma/genetics* ; Pheochromocytoma/genetics* ; Proto-Oncogene Proteins c-ret/genetics* ; Succinate Dehydrogenase/genetics* ; Von Hippel-Lindau Tumor Suppressor Protein/genetics*

LncRNA H19 encoded by the H19 imprinting gene plays an important regulatory role in the cell. Recently study has found that in hypoxic cells, the expression of H19 gene changes, and the transcription factors and protein involved in the expression change accordingly. Through the involvement of specific protein 1 (SP1), hypoxia-inducible factor-1α (HIF-1α) binds directly to the H19 promoter and induces the up-regulation of H19 expression under hypoxic conditions. The tumor suppressor protein p53 may also mediate the expression of the H19 gene, in part by interfering with HIF-la activity under hypoxia stress. The miR675-5p encoded by exon 1 of H19 promotes hypoxia response by driving the nuclear accumulation of HIF-1α and reducing the expression of VHL gene, which is a physiological HIF-1α inhibitor. In addition, under the condition of hypoxia, the expression of transporter on cell membrane changes, and the transition of the intracellular glucose metabolism pathway from aerobic oxidation to anaerobic glycolysis is also involved in the involvement of H19. Therefore, H19 may be a key gene that maintains intracellular balance under hypoxic conditions and drives adaptive cell survival under conditions of hypoxia stress.
Cell Hypoxia ; genetics ; Genes, Tumor Suppressor ; physiology ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; RNA, Long Noncoding ; Up-Regulation ; physiology ; Von Hippel-Lindau Tumor Suppressor Protein ; genetics

BACKGROUNDVon Hippel-Lindau (VHL) disease is a hereditary tumor disorder caused by mutations or deletions of the VHL gene. Few studies have documented the clinical phenotype and genetic basis of the occurrence of VHL disease in China. This study armed to present clinical and genetic analyses of VHL within a five-generation VHL family from Northwestern China, and summarize the VHL mutations and clinical characteristics of Chinese families with VHL according to previous studies.

METHODSAn epidemiological investigation of family members was done to collect the general information. A retrospective study of clinical VHL cases was launched to collect the relative clinical data. Genetic linkage and haplotype analysis were used to make sure the linkage of VHL to disease in this family. The VHL gene screening was performed by directly analyzing DNA sequence output. At last, we summarized the VHL gene mutation in China by the literature review.

RESULTSA five-generation North-western Chinese family afflicted with VHL disease was traced in this research. The family consisted of 38 living family members, of whom nine were affected. The individuals afflicted with VHL exhibited multi-organ tumors that included pheochromocytomas (8), central nervous system hemangioblastomas (3), pancreatic endocrine tumors (2), pancreatic cysts (3), renal cysts (4), and paragangliomas (2). A linkage analysis resulted in a high maximal LOD score of 8.26 (theta = 0.0) for the marker D3S1263, which is in the same chromosome region as VHL. Sequence analysis resulted in the identification of a functional C>T transition mutation (c. 499 C>T, p.R167W) located in exon 3 of the 167 th codon of VHL. All affected individuals shared this mutation, whereas the unaffected family members and an additional 100 unrelated healthy individuals did not. To date, 49 mutations have been associated with this disease in Chinese populations. The most frequent VHL mutations in China are p.S65 W, p.N78 S, p.R161Q and p.R167 W.

CONCLUSIONSThe results supported the notion that the genomic sequence that corresponds to the 167 th residue of VHL is a mutational hotspot. Further research is needed to clarify the molecular role of VHL in the development of organ-specific tumors.

Adolescent ; Adult ; Asian Continental Ancestry Group ; China ; Female ; Haplotypes ; genetics ; Humans ; Male ; Middle Aged ; Mutation ; Pedigree ; Retrospective Studies ; Von Hippel-Lindau Tumor Suppressor Protein ; genetics ; Young Adult ; von Hippel-Lindau Disease ; diagnosis ; genetics

BACKGROUNDVascular endothelial growth factor-targeted agents are standard treatments in advanced clear-cell renal cell carcinoma (ccRCC), but biomarkers of activity are lacking. The aim of this study was to investigate the association of Von Hippel-Lindau (VHL) gene status, vascular endothelial growth factor receptor (VEGFR) or stem cell factor receptor (KIT) expression, and their relationships with characteristics and clinical outcome of advanced ccRCC.

METHODSA total of 59 patients who received targeted treatment with sunitinib or pazopanib were evaluated for determination at Cancer Hospital and Institute, Chinese Academy of Medical Sciences between January 2010 and November 2012. Paraffin-embedded tumor samples were collected and status of the VHL gene and expression of VEGFR and KIT were determined by VHL sequence analysis and immunohistochemistry. Clinical-pathological features were collected and efficacy such as response rate and Median progression-free survival (PFS) and overall survival (OS) were calculated and then compared based on expression status. The Chi-square test, the Kaplan-Meier method, and the Lon-rank test were used for statistical analyses.

RESULTSOf 59 patients, objective responses were observed in 28 patients (47.5%). The median PFS was 13.8 months and median OS was 39.9 months. There was an improved PFS in patients with the following clinical features: Male gender, number of metastatic sites 2 or less, VEGFR-2 positive or KIT positive. Eleven patients (18.6%) had evidence of VHL mutation, with an objective response rate of 45.5%, which showed no difference with patients with no VHL mutation (47.9%). VHL mutation status did not correlate with either overall response rate (P = 0.938) or PFS (P = 0.277). The PFS was 17.6 months and 22.2 months in VEGFR-2 positive patients and KIT positive patients, respectively, which was significantly longer than that of VEGFR-2 or KIT negative patients (P = 0.026 and P = 0.043).

CONCLUSIONVHL mutation status could not predict the efficacy of sunitinib or pazopanib. Further investigation of VHL/VEGFR pathway components is needed.

Adult ; Aged ; Antineoplastic Agents ; therapeutic use ; Carcinoma, Renal Cell ; genetics ; mortality ; pathology ; Disease-Free Survival ; Female ; Humans ; Immunohistochemistry ; Indoles ; therapeutic use ; Kidney Neoplasms ; genetics ; mortality ; pathology ; Male ; Middle Aged ; Prognosis ; Pyrimidines ; therapeutic use ; Pyrroles ; therapeutic use ; Sulfonamides ; therapeutic use ; Vascular Endothelial Growth Factor A ; genetics ; Von Hippel-Lindau Tumor Suppressor Protein ; genetics ; Young Adult

OBJECTIVETo construct a lentiviral expression vector for human VHL and its shRNA vector, and study the effect of VHL on proliferation and apoptosis of renal cell carcinoma cell lines.

METHODSLentiviral vectors pZsGreen1-VHL and pLL3.7-shVHL were constructed and transfected into 293T cells with 3 packaging plasmids by Lipofectamine(TM) 2000 reagent. The supernatant was collected to infect A498 and Caki-1 cells, respectively. VHL mRNA and protein levels were detected by RT-PCR and Western blotting, respectively. The effect of VHL on the proliferation, cell cycle and cell apoptosis were analyzed by MTS and flow cytometry.

RESULTSThe recombinant lentiviral vectors were successfully constructed. The proliferation of A498 cells with reconstituted wild-type VHL was significantly inhibited, while the proliferation of Caki-1 cells with VHL knockdown was significantly enhanced as compared with the control cells (P<0.05). VHL induced G0/G1-S cell cycle arrest. The apoptosis rate of A498 cells with reconstituted wild-type VHL was significantly increased while that of Caki-1 cells with VHL knockdown was significantly lowered compared with the control cells (P<0.05).

CONCLUSIONVHL can inhibit the proliferation and induce apoptosis of renal cell carcinoma cells.

Apoptosis ; Carcinoma, Renal Cell ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Genetic Vectors ; Humans ; Lentivirus ; Plasmids ; RNA, Messenger ; RNA, Small Interfering ; Transfection ; Von Hippel-Lindau Tumor Suppressor Protein ; genetics

BACKGROUNDVon Hippel-Lindau disease (VHL), a heritable autosomal dominant disease characterized by neoplasia in multiple organ systems, has rarely been reported in Asia. We genetically investigated a unique Chinese family with VHL disease and performed an analysis of the VHL protein stability.

METHODSGenomic deoxyribonucleic acid (DNA) extracted from peripheral blood was amplified by polymerase chain reaction (PCR) to three exons of the VHL gene in 9 members of the Chinese family with VHL disease. PCR products were directly sequenced. We estimated the effects of VHL gene mutation on the stability of pVHL, which is indicated by the free energy difference between the wild-type and the mutant protein (ΔΔG).

RESULTSThe Chinese family was classified as VHL type 1. Three family members, including two patients and a carrier, had a T to G heterozygotic missense mutation at nucleotide 515 of the VHL gene exon 1. This missense mutation resulted in the transition from leucine to arginine in amino acid 101 of the VHL protein. There was low stability of the VHL protein (the ΔΔG was 12.71 kcal/mol) caused by this missense mutation.

CONCLUSIONSWe first reported a family with this VHL gene mutation in Asia. This missense mutation is predicted to significantly reduce the stability of the VHL protein and contribute to the development of the renal cell carcinoma (RCC) phenotype displayed by this family. The genetic characterization and protein stability analysis of families with VHL disease are important for early diagnosis and prevention of the disease being passed on to their offspring.

Adult ; China ; Female ; Humans ; Male ; Middle Aged ; Mutation, Missense ; Protein Stability ; Von Hippel-Lindau Tumor Suppressor Protein ; chemistry ; genetics ; von Hippel-Lindau Disease ; genetics

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder and its clinical manifestation including haemangioblastomas of the central nervous system, renal cell carcinoma, haeochromocytomas, and pancreatic cyst. The deletion, mutation and promoter methylation of VHL gene can cause VHL disease. Swine is considered as an ideal model for human disease because of its physiological and anatomical similarity to human. We cloned pig VHL gene that is 2 725 bp in length. VHL highly expressed in adrenal gland, liver, pancreas, heart and testis. We designed 5 shRNAs and screened the most effective interference RNA fragment with a knockdown efficiency of 72%. Porcine embryonic fibroblasts stably transfected with pGenesil-shRNA vector were used as donor cells for nuclear transfer and there was no significant difference of embryo development compared with the control group. Moreover, VHL was efficiently knocked-down with efficiency of 71% in porcine cloned blastocyst, these results lay a solid foundation for constructing the VHL knock-down model of pig.
Animals ; Cloning, Molecular ; Disease Models, Animal ; Embryo, Mammalian ; Gene Knockdown Techniques ; Swine ; Von Hippel-Lindau Tumor Suppressor Protein ; genetics ; von Hippel-Lindau Disease ; genetics