Downregulation of CLDN7 due to promoter hypermethylation is associated with human clear cell renal cell carcinoma progression and poor prognosis.

Abstract:

BACKGROUND:Metastasis is the primary cause of death in renal cell carcinoma (RCC). Loss of cell-to-cell adhesion, including tight junctions (TJs) is the initial step in the process of metastasis. Claudin-7 (CLDN7) is a major component of TJs. However, the clinical significance and its regulation of kidney tumorigenesis remain poorly understood. METHODS:A total of 120 fresh clear cell RCC (ccRCC) specimens and 144 primary RCC and adjacent nonmalignant renal paraffin specimens were obtained from Department of Urology, Peking University First Hospital. Expression of CLDN7 in ccRCC tissues and cell lines were determined using bioinformatic data mining, quantitative real-time PCR (qRT-PCR), Western blotting and immunostaining. The clinical significance of CLDN7 expression and promoter DNA methylation status was analyzed in ccRCC patients from Peking University First Hospital and The Cancer Genome Atlas. Additionally, the methylation specific-PCR, bisulfite genomic sequencing and demethylation analysis of CLDN7 were performed. Biological functions of CLDN7 were investigated by examining cell proliferation using MTS assays and EdU incorporation assays, cell migration by in vitro wound healing assays and transwell migration assays, cell invasion by transwell invasion assays, and cell apoptosis by flow cytometry. Mouse model experiments were performed to confirm the effects of CLDN7 on tumor growth and metastasis in vivo. The molecular mechanism of CLDN7 function was investigated using gene-set enrichment analysis (GSEA) and high-throughput cDNA sequencing (RNA-Seq) and confirmed by qRT-PCR, Western blot and immunostaining in vitro and in vivo. RESULTS:Our findings revealed that CLDN7 is frequently downregulated via hypermethylation of its promoter in ccRCC. CLDN7 can help predict aggressive tumor status and poor prognosis in ccRCC patients. Interestingly, hypermethylation of the CLDN7 promoter was related to advanced ccRCC status and poor prognosis. Moreover, overexpression of CLDN7 induced cell apoptosis, suppressed proliferation, migration and invasion abilities of ccRCC cells both in vitro and in vivo. Additionally, GSEA and RNA-Seq results showed that CLDN7 had negative effects in cancer-associated signaling pathways and (epithelial-mesenchymal transition) EMT-related pathways. These results were validated by qRT-PCR, Western blot and immunostaining. CONCLUSIONS:We have demonstrated a previously undescribed role of CLDN7 as a ccRCC suppressor and suggest that loss of CLDN7 potentiates EMT and tumor progression. CLDN7 may serve as a functional tumor suppressor in tumor progression and a potential biomarker and target in patients with ccRCC.

journal_name

J Exp Clin Cancer Res

authors

Li Y,Gong Y,Ning X,Peng D,Liu L,He S,Gong K,Zhang C,Li X,Zhou L

doi

10.1186/s13046-018-0924-y

subject

Has Abstract

pub_date

2018-11-14 00:00:00

pages

276

issue

1

eissn

0392-9078

issn

1756-9966

pii

10.1186/s13046-018-0924-y

journal_volume

37

pub_type

杂志文章
  • PCBP1 depletion promotes tumorigenesis through attenuation of p27Kip1 mRNA stability and translation.

    abstract:BACKGROUND:Poly C Binding Protein 1 (PCBP1) is an RNA-binding protein that binds and regulates translational activity of subsets of cellular mRNAs. Depletion of PCBP1 is implicated in various carcinomas, but the underlying mechanism in tumorigenesis remains elusive. METHODS:We performed a transcriptome-wide screen to ...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:10.1186/s13046-018-0840-1

    authors: Shi H,Li H,Yuan R,Guan W,Zhang X,Zhang S,Zhang W,Tong F,Li L,Song Z,Wang C,Yang S,Wang H

    更新日期:2018-08-07 00:00:00

  • CT cystoscopy in the evaluation of bladder tumors.

    abstract::The aim of the present study was to assess the role of virtual cystoscopy in the identification of bladder tumors. Fifteen patients (11 men and 4 women, median age: 61 years, range: 46-74 years) with a positive finding of bladder tumor at fiber-optic cystoscopy were studied by multislice-CT. Scans were downloaded to a...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:

    authors: Marini M,Stasolla A,D'Aprile MR,Colaiacomo MC,Lo Mele L,Maccioni F,Iori S,Binda B,Izzo L

    更新日期:2003-03-01 00:00:00

  • Identification of sentinel node in breast cancer.

    abstract::Axillary lymphadenectomy is a very important procedure in the staging of breast cancer patients. However, it is associated with a significant morbidity rate. On the other hand, using early diagnosis we can see a high number of cases where the lymph nodes are negatives. With the intention of avoiding unnecessary axilla...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:

    authors: Zerwes FP,Frasson AL,Gutfilen B,Barbosa da Fonseca LM

    更新日期:2002-06-01 00:00:00

  • Prohibitin overexpression predicts poor prognosis and promotes cell proliferation and invasion through ERK pathway activation in gallbladder cancer.

    abstract:BACKGROUND:Prohibitin (PHB), a pleiotropic protein overexpressed in several tumor types, has been implicated in the regulation of cell proliferation, invasive migration and survival. However, PHB expression and its biological function in gallbladder cancer (GBC) remain largely unknown. METHODS:PHB and p-ERK protein ex...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:10.1186/s13046-016-0346-7

    authors: Cao Y,Liang H,Zhang F,Luan Z,Zhao S,Wang XA,Liu S,Bao R,Shu Y,Ma Q,Zhu J,Liu Y

    更新日期:2016-04-16 00:00:00

  • Hemangioblastoma of the filum terminale. Case report and review of the literature.

    abstract::Hemangioblastomas of the filum terminale are particularly rare tumors. The authors present the case of a 62-year-old woman with a 6 month history of low-back-pain, who underwent surgery for the removal of an hemangioblastoma affecting the cauda at L2-L3 level. This highly vascularized tumour is tightly adherent to the...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章,评审

    doi:

    authors: Ciappetta P,Occhiogrosso G,Domenicucci M,D'Andrea G,Bastianello S,Frati A

    更新日期:2007-06-01 00:00:00

  • Cisplatin triggers cancer stem cell enrichment in platinum-resistant cells through NF-κB-TNFα-PIK3CA loop.

    abstract:BACKGROUND:Parallel to complex alteration in molecular and cellular events, enrichment of cancer stem cells (CSC) contributes significantly in deliberation and maintenance of cisplatin resistance. Cisplatin mediated CSC enrichment is well established in various cancers, yet the underlying mechanism is largely unknown. ...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:10.1186/s13046-017-0636-8

    authors: Thakur B,Ray P

    更新日期:2017-11-23 00:00:00

  • ANGPTL1 attenuates colorectal cancer metastasis by up-regulating microRNA-138.

    abstract:BACKGROUND:Angiopoietin-like protein 1 (ANGPTL1) has been reported to suppress migration and invasion in lung and breast cancer, acting as a novel tumor suppressor candidate. Nevertheless, its effects on colorectal cancer (CRC) remain poorly defined. In this study, we aim to demonstrate the biological function of ANGPT...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:10.1186/s13046-017-0548-7

    authors: Chen H,Xiao Q,Hu Y,Chen L,Jiang K,Tang Y,Tan Y,Hu W,Wang Z,He J,Liu Y,Cai Y,Yang Q,Ding K

    更新日期:2017-06-12 00:00:00

  • Radionuclide therapy with bone seeking radionuclides in palliation of painful bone metastases.

    abstract::Radionuclide therapy with bone seeking radiopharmaceuticals is one of the oldest interventions in nuclear medicine and has been now in use for many years as an effective method of palliating painful bone metastases. Clinical indications have slowly but progressively increased starting from end-stage prostate cancer wi...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章,评审

    doi:

    authors: Maini CL,Sciuto R,Romano L,Bergomi S

    更新日期:2003-12-01 00:00:00

  • Lipoxin A4 reverses mesenchymal phenotypes to attenuate invasion and metastasis via the inhibition of autocrine TGF-β1 signaling in pancreatic cancer.

    abstract:BACKGROUND:Pancreatic cancer is a lethal disease in part because of its potential for aggressive invasion and metastasis. Lipoxin A4 (LXA4) is one of the metabolites that is derived from arachidonic acid and that is catalyzed by 15-lipoxygenase (15-LOX), and it has recently been reported to exhibit anti-cancer effects....

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:10.1186/s13046-017-0655-5

    authors: Zong L,Chen K,Jiang Z,Chen X,Sun L,Ma J,Zhou C,Xu Q,Duan W,Han L,Lei J,Li X,Ma Q,Wang Z

    更新日期:2017-12-11 00:00:00

  • Magnetic resonance of the breast: correlation between enhancement patterns and microvessel density in malignant tumors.

    abstract::The continuous growth of a solid tumor is contingent upon the induction and formation of new vascular structures (angiogenesis). Several studies have established a close correlation between the microvessel density (MVD) of a tumor and its growth capacity. Therefore, a non-invasive method to accurately assess tumor MVD...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:

    authors: Carriero A,Ambrossini R,Mattei PA,Angelucci D,Bonomo L

    更新日期:2002-09-01 00:00:00

  • Serum miR-22 as potential non-invasive predictor of poor clinical outcome in newly diagnosed, uniformly treated patients with diffuse large B-cell lymphoma: an explorative pilot study.

    abstract:BACKGROUND:Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous group of tumors, with aggressive clinical course that renders prognostication and choice of treatment strategy difficult. Chemo-immunotherapy with rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP) is the current first-line trea...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:10.1186/s13046-018-0768-5

    authors: Marchesi F,Regazzo G,Palombi F,Terrenato I,Sacconi A,Spagnuolo M,Donzelli S,Marino M,Ercolani C,Di Benedetto A,Blandino G,Ciliberto G,Mengarelli A,Rizzo MG

    更新日期:2018-05-02 00:00:00

  • Mimicking tumor hypoxia and tumor-immune interactions employing three-dimensional in vitro models.

    abstract::The heterogeneous tumor microenvironment (TME) is highly complex and not entirely understood. These complex configurations lead to the generation of oxygen-deprived conditions within the tumor niche, which modulate several intrinsic TME elements to promote immunosuppressive outcomes. Decoding these communications is n...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章,评审

    doi:10.1186/s13046-020-01583-1

    authors: Bhattacharya S,Calar K,de la Puente P

    更新日期:2020-05-01 00:00:00

  • Sustained delivery of siRNA/PEI complex from in situ forming hydrogels potently inhibits the proliferation of gastric cancer.

    abstract:BACKGROUND:Gastric cancer remains a major cause of mortality and morbidity worldwide. In recent years, gene-based therapeutic strategies were confirmed promising in cancer inhibition and attracted great attention. RNA interference (RNAi) is a powerful tool for gene therapy and has been widely employed to aid in treatme...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:10.1186/s13046-016-0334-y

    authors: Peng H,Yang H,Song L,Zhou Z,Sun J,Du Y,Lu K,Li T,Yin A,Xu J,Wei S

    更新日期:2016-03-31 00:00:00

  • Transplatin, a cisplatin trans-isomer, may enhance the anticancer effect of 5-fluorouracil.

    abstract::Transplatin (TDDP), a trans-isomer of cisplatin (CDDP), is well known to have faint cytotoxicity because its geometric structure allows less adduct formation with DNA than does CDDP. However, TDDP might have the potential to enhance the anticancer effect of 5-fluorouracil (5-FU) as well as CDDP. In this study, five ga...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:

    authors: Nakata B,Yamagata S,Kanehara I,Shirasaka T,Hirakawa K

    更新日期:2006-06-01 00:00:00

  • Low-molecular-weight heparins are superior to vitamin K antagonists for the long term treatment of venous thromboembolism in patients with cancer: a cochrane systematic review.

    abstract:BACKGROUND:Cancer and its therapies increase the risk of venous thromboembolism. Compared to patients without cancer, patients with cancer anticoagulated for venous thromboembolism are more likely to develop recurrent thrombotic events and major bleeding. Addressing all important outcomes including harm is of great imp...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章,评审

    doi:10.1186/1756-9966-27-21

    authors: Akl EA,Barba M,Rohilla S,Terrenato I,Sperati F,Muti P,Schünemann HJ

    更新日期:2008-07-18 00:00:00

  • Detection of CD44 variants in fine needle aspiration biopsies of thyroid tumor by RT-PCR.

    abstract::Increased expression of CD44 variants is often observed in differentiated thyroid carcinomas. To establish a method for gene diagnosis of thyroid without any additional invasions to the patients, RNA in leftover cells inside the needle used for fine needle aspiration biopsies (FNABs) was extracted to detect the expres...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:

    authors: Takano T,Sumizaki H,Amino N

    更新日期:1997-09-01 00:00:00

  • Targeting of tubulin polymerization and induction of mitotic blockage by Methyl 2-(5-fluoro-2-hydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylate (MBIC) in human cervical cancer HeLa cell.

    abstract:BACKGROUND:Microtubule Targeting Agents (MTAs) including paclitaxel, colchicine and vinca alkaloids are widely used in the treatment of various cancers. As with most chemotherapeutic agents, adverse effects and drug resistance are commonly associated with the clinical use of these agents. Methyl 2-(5-fluoro-2-hydroxyph...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:10.1186/s13046-016-0332-0

    authors: Hasanpourghadi M,Karthikeyan C,Pandurangan AK,Looi CY,Trivedi P,Kobayashi K,Tanaka K,Wong WF,Mustafa MR

    更新日期:2016-03-31 00:00:00

  • FAK-ERK activation in cell/matrix adhesion induced by the loss of apolipoprotein E stimulates the malignant progression of ovarian cancer.

    abstract:BACKGROUND:Extracellular matrix (ECM) is a mediator of tumor progression. However, whether the alterations of the intraperitoneal ECM prior to tumor establishment affects the malignant progression of ovarian cancer remains elusive. METHODS:Apolipoprotein (ApoE) knock-out mice was used to analyze the intraperitoneal EC...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:10.1186/s13046-018-0696-4

    authors: Lai H,Zhao X,Qin Y,Ding Y,Chen R,Li G,Labrie M,Ding Z,Zhou J,Hu J,Ma D,Fang Y,Gao Q

    更新日期:2018-02-20 00:00:00

  • High performance of targeted next generation sequencing on variance detection in clinical tumor specimens in comparison with current conventional methods.

    abstract:BACKGROUND:Next generation sequencing (NGS) is being increasingly applied for assisting cancer molecular diagnosis. However, it is still needed to validate NGS accuracy on detection of DNA alternations based on a large number of clinical samples, especially for DNA rearrangements and copy number variations (CNVs). This...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:10.1186/s13046-017-0591-4

    authors: Su D,Zhang D,Chen K,Lu J,Wu J,Cao X,Ying L,Jin Q,Ye Y,Xie Z,Xiong L,Mao W,Li F

    更新日期:2017-09-07 00:00:00

  • The Chk1 inhibitor SAR-020106 sensitizes human glioblastoma cells to irradiation, to temozolomide, and to decitabine treatment.

    abstract:BACKGROUND:Glioblastoma is the most common and aggressive brain tumour in adults with a median overall survival of only 14 months after standard therapy with radiation therapy (IR) and temozolomide (TMZ). In a novel multimodal treatment approach we combined the checkpoint kinase 1 (Chk1) inhibitor SAR-020106 (SAR), dis...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:10.1186/s13046-019-1434-2

    authors: Patties I,Kallendrusch S,Böhme L,Kendzia E,Oppermann H,Gaunitz F,Kortmann RD,Glasow A

    更新日期:2019-10-21 00:00:00

  • G-quadruplex ligand RHPS4 radiosensitizes glioblastoma xenograft in vivo through a differential targeting of bulky differentiated- and stem-cancer cells.

    abstract:BACKGROUND:Glioblastoma is the most aggressive and most lethal primary brain tumor in the adulthood. Current standard therapies are not curative and novel therapeutic options are urgently required. Present knowledge suggests that the continued glioblastoma growth and recurrence is determined by glioblastoma stem-like c...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:10.1186/s13046-019-1293-x

    authors: Berardinelli F,Tanori M,Muoio D,Buccarelli M,di Masi A,Leone S,Ricci-Vitiani L,Pallini R,Mancuso M,Antoccia A

    更新日期:2019-07-16 00:00:00

  • The late effects of proton irradiation on cell growth, cell cycle arrest and apoptosis in a human melanoma cell line.

    abstract::The aim of this work is the in vitro study of the late effects of single proton irradiation on HTB63 human melanoma cell growth, cell cycle and cell death. The experimental conditions were focused on analyzing the effects of irradiation on the periphery of tumour that can be, in clinical practice, close to critical or...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:

    authors: Ristic-Fira A,Nikolic D,Petrovic I,Ruzdijic S,Raffaele L,Sabini MG,Cirrone GA,Cuttone G,Farruggia G,Masotti L,Kanazir DT

    更新日期:2001-03-01 00:00:00

  • circGFRA1 and GFRA1 act as ceRNAs in triple negative breast cancer by regulating miR-34a.

    abstract:BACKGROUD:Accumulating evidences indicate that circular RNAs (circRNAs), a class of non-coding RNAs, play important roles in tumorigenesis. However, the function of circRNAs in triple negative breast cancer (TNBC) is largely unknown. METHODS:We performed circRNA microarrays to identify circRNAs that are aberrantly exp...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:10.1186/s13046-017-0614-1

    authors: He R,Liu P,Xie X,Zhou Y,Liao Q,Xiong W,Li X,Li G,Zeng Z,Tang H

    更新日期:2017-10-16 00:00:00

  • Ectopic endometrium in human foetuses is a common event and sustains the theory of müllerianosis in the pathogenesis of endometriosis, a disease that predisposes to cancer.

    abstract:BACKGROUND:Endometriosis is a gynecological disease defined by the histological presence of endometrial glands and stroma outside the uterine cavity. Women with endometriosis have an increased risk of different types of malignancies, especially ovarian cancer and non-Hodgkin's lymphoma. Though there are several theorie...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:10.1186/1756-9966-28-49

    authors: Signorile PG,Baldi F,Bussani R,D'Armiento M,De Falco M,Baldi A

    更新日期:2009-04-09 00:00:00

  • Hypermethylation and loss of retinoic acid receptor responder 1 expression in human choriocarcinoma.

    abstract:BACKGROUND:Human placental development resembles tumorigenesis, due to the invasive and fusogenic potential of trophoblasts. However, these features are tightly controlled in trophoblasts. Disturbance of this spatial and temporal regulation is thought to contribute to the rare formation of choriocarcinomas. Promoter hy...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:10.1186/s13046-017-0634-x

    authors: Huebner H,Strick R,Wachter DL,Kehl S,Strissel PL,Schneider-Stock R,Hartner A,Rascher W,Horn LC,Beckmann MW,Ruebner M,Fahlbusch FB

    更新日期:2017-11-23 00:00:00

  • A long-standing cystic lymph-node metastasis from occult thyroid carcinoma.

    abstract::Here we report the case of a patient with a soft tissue mass of the neck. For more than 10 years it was thought to be a branchial cyst and was later diagnosed to be a cystic lymph node metastasis from an occult thyroid carcinoma. ...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:

    authors: Appetecchia M,Meçule A,Sciarretta F

    更新日期:2002-03-01 00:00:00

  • Human mammaglobin mRNA is a reliable molecular marker for detecting occult breast cancer cells in peripheral blood.

    abstract::Occult carcinoma cells in peripheral blood of breast cancer (BC) patients is generally associated with poor disease prognosis. Reverse transcriptase-polymerase chain reaction (RT-PCR) is a sensitive method for revealing rare circulating cancer cells. The target mRNA must be carefully chosen, as it must be expressed on...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:

    authors: Roncella S,Ferro P,Bacigalupo B,Pronzato P,Tognoni A,Falco E,Gianquinto D,Ansaldo V,Dessanti P,Fais F,Rosai J,Fedeli F

    更新日期:2005-06-01 00:00:00

  • Correction to: Musashi2 promotes EGF-induced EMT in pancreatic cancer via ZEB1-ERK/MAPK signaling.

    abstract::An amendment to this paper has been published and can be accessed via the original article. ...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章,已发布勘误

    doi:10.1186/s13046-020-01663-2

    authors: Sheng W,Shi X,Lin Y,Tang J,Jia C,Cao R,Sun J,Wang G,Zhou L,Dong M

    更新日期:2020-08-24 00:00:00

  • Chemokine receptor CXCR7 regulates the invasion, angiogenesis and tumor growth of human hepatocellular carcinoma cells.

    abstract:BACKGROUND:In spite of recent advances in diagnostic and therapeutic measures, the prognosis of hepatocellular carcinoma (HCC) patients remains poor. Therefore, it is crucial to understand what factors are involved in promoting development of HCC. Evidence is accumulating that members of the chemokine receptor family a...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:10.1186/1756-9966-29-31

    authors: Zheng K,Li HY,Su XL,Wang XY,Tian T,Li F,Ren GS

    更新日期:2010-04-11 00:00:00

  • Co-administration of 20(S)-protopanaxatriol (g-PPT) and EGFR-TKI overcomes EGFR-TKI resistance by decreasing SCD1 induced lipid accumulation in non-small cell lung cancer.

    abstract:BACKGROUND:Non-small cell lung cancer (NSCLC) patients with sensitive epidermal growth factor receptor (EGFR) mutations are successfully treated with EGFR tyrosine kinase inhibitors (EGFR-TKIs); however, resistance to treatment inevitably occurs. Given lipid metabolic reprogramming is widely known as a hallmark of canc...

    journal_title:Journal of experimental & clinical cancer research : CR

    pub_type: 杂志文章

    doi:10.1186/s13046-019-1120-4

    authors: Huang Q,Wang Q,Li D,Wei X,Jia Y,Zhang Z,Ai B,Cao X,Guo T,Liao Y

    更新日期:2019-03-15 00:00:00