Effects of miRNA-140 on the Growth and Clinical Prognosis of SMMC-7721 Hepatocellular Carcinoma Cell Line.

BACKGROUND: A growing number of studies have suggested that microRNAs exert an essential role in the development and occurrence of multiple tumours and act as crucial regulators in various biological processes. However, the expression and function of miRNA-140 in hepatocellular carcinoma (HCC) cells are not yet adequately identified and manifested. METHODS: The expression of miRNA-140 was determined in HCC tissues and adjacent nontumour tissues by quantitative real-time polymerase chain reaction (qRT-PCR). Kaplan-Meier survival analysis and Cox regression analysis were performed to explore the correlation between miRNA-140 expression level and the survival rate of patients with HCC. Additionally, overexpression experiments were conducted to investigate the biological role of miRNA-140 in HCC cells. Bioinformatics was used to predict the related target genes and pathways of miRNA-140. RESULTS: QRT-PCR results signified that the expression level of miRNA-140 in HCC was lower than that of adjacent normal tissues (P < 0.0001). Compared with the control group, the SMMC-7721 HCC cells in the miRNA-140 mimic group had a decrease in proliferation, migration, and invasion (P < 0.05), whereas those in the miRNA-140 inhibitor group had an increase in proliferation, migration, and invasion (P < 0.05). Cell cycle arrest occurred in the G0/1 phase. Prognosis analysis showed that the expression level of miRNA-140 was not related to the prognosis of HCC. Furthermore, the Kaplan-Meier test revealed that patients with lower miRNA-140 expression levels in liver cancer tissue had significantly shorter disease-free survival (DFS, P = 0.004) and overall survival (OS) times (P = 0.010) after hepatectomy. Cox regression analysis further indicated that miRNA-140 was an independent risk factor that may affect the DFS (P = 0.004) and OS times (P = 0.014) of patients after hepatectomy. Our results suggested that miRNA-140 might be a crucial regulator involved in the HCC progression and is thus considered a potential prognostic biomarker and therapeutic target for HCC.

The differential magnetic relaxation behaviours of slightly distorted triangular dodecahedral dysprosium analogues in a type of cyano-bridged 3d-4f zig-zag chain compounds.

A class of cyano-bridged 3d-4f zig-zag chain compounds, {RE[TM(CN)6] (PNO)2(H2O)4}·(H2O) {RE = YIII, TM = [FeIII]LS (1); RE = DyIII, TM = [FeIII]LS (2), CoIII (3)}, have been synthesized and characterized by single-crystal X-ray diffraction. The rare earth ions in these compounds are situated in a slightly distorted triangular dodecahedral (D2d) coordination environment. The magnetic properties of compounds 1-3 have been comparatively studied in detail. Under a zero dc field, the temperature dependence of ac susceptibility measurements for YFe (1) indicates the absence of magnetic relaxation stemming from the single anisotropic [FeIII]LS ion. The dysprosium analogue DyFe (2) shows only magnetic relaxation behavior with a prominent QTM effect, while DyCo (3) exhibits SIM properties not completely covered by QTM, with an extracted energy barrier of 73 K under a zero dc field. The ab initio calculations indicate that both compounds 2 and 3 are SMMs with well-behaved magnetic relaxation properties primarily from the individual DyIII ion. Therefore, the different magnetic behaviors exhibited by compound 2 compared to 3 may be ascribed to the stronger QTM effect caused by the extra weak interaction of [FeIII]LS ions in 2 as a fluctuating transverse field around the DyIII ion. The QTM effect for both 2 and 3 is suppressed under an applied dc field with an effective energy barrier of 134 and 150 K, respectively. Compared with compound 2, the higher extracted Ueff/kB and χ''(T) peak temperature for 3 should be further attributed to its slightly higher single-ion axiality as calculated and the elimination of the transverse field from the [FeIII]LS ion.

Hypoxia-induced USP22-BMI1 axis promotes the stemness and malignancy of glioma stem cells via regulation of HIF-1α.

AIMS: This study intends to investigate the mechanisms of ubiqutin-specific protease 22 (USP22)/B cell-specific Moloney murine leukemia virus integration site 1 (BMI1) on the biological phenotypes of glioma stem cells (GSCs) under hypoxia. MAIN METHODS: Western blot, Cell Counting Kit-8, colony formation and flow cytometry assays were preformed to evaluate cells biological behaviors. Luciferase assay was utilized to identify the associations among USP22, HIF-1α and BMI1. KEY FINDINGS: Silencing USP22 reduced the stemness and proliferation of GSCs, and increased its apoptosis in response to hypoxia. Whilst, overexpression of BMI1 reversed these phenomena. Whilst, a significant decrease in proliferation and stemness of GSCs caused by HIF-1α exhaustion were inversed by overexpression of USP22 or BMI1. SIGNIFICANCE: Function of USP22-BMI1 on biological behaviors of GSCs was regulated by HIF-1α in response to hypoxia.

Ubiquitin-specific protease 22 acts as an oncoprotein to maintain glioma malignancy through deubiquitinating B cell-specific Moloney murine leukemia virus integration site 1 for stabilization.

Ubiquitin-specific protease 22 (USP22) is a member of the "death-from-cancer" signature, which plays a key role in cancer progression. Previous evidence has shown that USP22 is overexpressed and correlates with poor prognosis in glioma. The effect and mechanism of USP22 in glioma malignancy, especially cancer stemness, remain elusive. Herein, we find USP22 is more enriched in stem-like tumorspheres than differentiated glioma cells. USP22 knockdown inhibits cancer stemness in glioma cell lines. With a cell-penetrating TAT-tag protein, B cell-specific Moloney murine leukemia virus integration site 1 (BMI1), a robust glioma stem-cell marker, is found to mediate the effect of USP22 on glioma stemness. By immunofluorescence, USP22 and BMI1 are found to share similar intranuclear expression in glioma cells. By analysis with immunohistochemistry and bioinformatics, USP22 is found to positively correlate with BMI1 at the post-translational level only rather than at the transcriptional level. By immunoprecipitation and in vivo deubiquitination assay, USP22 is found to interact with and deubiquitinate BMI1 for protein stabilization. Microarray analysis shows that USP22 and BMI1 mutually regulate a series of genes involved in glioma stemness such as POSTN, HEY2, PDGFRA and ATF3. In vivo study with nude mice confirms the role of USP22 in promoting glioma tumorigenesis by regulating BMI1. All these findings indicate USP22 as a novel deubiquitinase of BMI1 in glioma. We propose a working model of the USP22-BMI1 axis, which promotes glioma stemness and tumorigenesis through oncogenic activation. Thus, targeting USP22 might be an effective strategy to treat glioma especially in those with elevated BMI1 expression.

Isolation and identification of Penicillium chrysogenum strain Y5 and its copper extraction characterization from waste printed circuit boards.

Biohydrometallurgy is generally considered as a green technology for the recycling of industrial solid waste. In this study, an indigenous fungal strain named Y5 with the ability of high-yielding organic acids was isolated and applied in bioleaching of waste printed circuit boards (PCBs). The strain Y5 was identified as Penicillium chrysogenum by morphological and molecular identification. Meanwhile, we investigated that an optimal set of culturing conditions for the fungal growth and acids secretion was 15 g/L glucose with initial pH 5.0, temperature 25°C and shaking speed 120 rpm in shaken flasks culture. Moreover, three bioleaching processes such as one-step, two-step and spent medium processes were conducted to extract copper from waste PCBs. Spent medium bioleaching showed higher copper extraction percentage and it was 47% under 5%(w/v) pulp density. Transmission electron microscope (TEM) observation combining with energy dispersive analysis of X-rays (EDAX) showed that the leached metal ions did not obviously damage the hypha cells. All above results indicated that P.chrysogenum strain Y5 has the tolerance to metal ions, suggesting its potential in recycling of metals from waste PCBs in industry.

Organoids: An intermediate modeling platform in precision oncology.

Cancer harbors variable heterogeneity and plasticity. Thus far, our comprehension is greatly based on cell lines, organoids, and patient-derived tumor xenografts (PDTXs). Organoids are a three-dimensional in vitro culture platform constructed from self-organizing stem cells. They can almost accurately recapitulate tumor heterogeneity and microenvironment "in a dish," which surpass established cell lines and are not as expensive and time-consuming as PDTXs. As an intermediate model, tumor organoids are also used to study the fundamental issues of tumorigenesis and metastasis. They are specifically applied for drug testing and stored as "living biobanks." In this review, we highlight the translational applications of organoid technologies in tumor research and precision medicine, discuss the advantages and limitations compared with other mentioned methods, and provide our outlook on its future.

Reprogramming of the Tumor in the Hypoxic Niche: The Emerging Concept and Associated Therapeutic Strategies.

Hypoxia exerts a profound impact on diverse aspects of cancer biology. Increasing evidence has revealed novel functions of hypoxia in cancer cell epigenomics, epitranscriptomics, metabolism, and intercellular communication, all hotspots of cancer research. Several drugs have been developed to target intratumoral hypoxia and have entered clinical trials to treat refractory tumors. However, direct targeting of hypoxia signaling still has limitations in the clinic with regard to cancer progression and resistance to therapy. Comprehensive understanding of the molecular mechanisms by which hypoxia reshapes tumors and their microenvironment, as well as how tumor cells adapt to and thrive in hypoxic conditions, will therefore continue to be a focus of cancer research and will provide new directions for hypoxic tumor treatment.

The bad seed gardener: Deubiquitinases in the cancer stem-cell signaling network and therapeutic resistance.

Tumors are comprised of highly heterogeneous populations of cells, of which only a small subset of stem-like cells possess the ability to regenerate tumors in vivo. These rare cancer stem cells (CSCs) have been regarded as the "bad seeds" accounted for tumor initiation, progression, metastasis, relapse and therapeutic resistance. CSC-targeted therapy seems to be a better avenue for radical cure of cancer. Deubiquitinases (DUBs), specifically disassembling ubiquitin chains, have been demonstrated to play an important role in rigidly maintaining the balance between ubiquitination and deubiquitination for protein quality control and homeostasis in normal circumstances. Dysfunction or deregulation of DUBs always leads to a series of disorders, even malignant transformation. Despite the accumulative evidence that DUB inhibitors in cancer remedy mainly target the tumor bulk, side effects like toxicity and resistance are still hard nuts to crack. In this article, we review the concept of ubiquitin proteasome system (UPS) and hallmarks of CSCs related to tumor obstinacy. We primarily summarize the CSC-related factors and signaling pathways and focus on the function of DUBs on biological traits of CSCs. We also illustrate the opportunities and challenges for the application of DUB inhibitors in the CSC-targeted therapy. Finally, we discuss the complexity of cancer stem cell hierarchy complexity and argue that a combination therapy for both CSCs and non-CSCs should be a desirable option.

Targeting Deubiquitinating Enzymes in Glioblastoma Multiforme: Expectations and Challenges.

Glioblastoma (GBM) is regarded as the most common primary intracranial neoplasm. Despite standard treatment with tumor resection and radiochemotherapy, the outcome remains gloomy. It is evident that a combination of oncogenic gain of function and tumor-suppressive loss of function has been attributed to glioma initiation and progression. The ubiquitin-proteasome system is a well-orchestrated system that controls the fate of most proteins by striking a dynamic balance between ubiquitination and deubiquitination of substrates, having a profound influence on the modulation of oncoproteins, tumor suppressors, and cellular signaling pathways. In recent years, deubiquitinating enzymes (DUBs) have emerged as potential anti-cancer targets due to their targeting several key proteins involved in the regulation of tumorigenesis, apoptosis, senescence, and autophagy. This review attempts to summarize recent studies of GBM-associated DUBs, their roles in various cellular processes, and discuss the relation between DUBs deregulation and gliomagenesis, especially how DUBs regulate glioma stem cells pluripotency, microenvironment, and resistance of radiation and chemotherapy through core stem-cell transcriptional factors. We also review recent achievements and progress in the development of potent and selective reversible inhibitors of DUBs, and attempted to find a potential GBM treatment by DUBs intervention.

Evidence of cell surface iron speciation of acidophilic iron-oxidizing microorganisms in indirect bioleaching process.

While indirect model has been widely accepted in bioleaching, but the evidence of cell surface iron speciation has not been reported. In the present work the iron speciation on the cell surfaces of four typically acidophilic iron-oxidizing microorganism (mesophilic Acidithiobacillus ferrooxidans ATCC 23270, moderately thermophilic Leptospirillum ferriphilum YSK and Sulfobacillus thermosulfidooxidans St, and extremely thermophilic Acidianus manzaensis YN25) grown on different energy substrates (chalcopyrite, pyrite, ferrous sulfate and elemental sulfur (S(0))) were studied in situ firstly by using synchrotron-based micro- X-ray fluorescence analysis and X-ray absorption near-edge structure spectroscopy. Results showed that the cells grown on iron-containing substrates had apparently higher surface iron content than the cells grown on S(0). Both ferrous iron and ferric iron were detected on the cell surface of all tested AIOMs, and the Fe(II)/Fe(III) ratios of the same microorganism were affected by different energy substrates. The iron distribution and bonding state of single cell of A. manzaensis were then studied in situ by scanning transmission soft X-ray microscopy based on dual-energy contrast analysis and stack analysis. Results showed that the iron species distributed evenly on the cell surface and bonded with amino, carboxyl and hydroxyl groups.

The transducible TAT-RIZ1-PR protein exerts histone methyltransferase activity and tumor-suppressive functions in human malignant meningiomas.

Malignant meningiomas are a rare meningioma subtype and tend to have post-surgical recurrence. Significant endeavors have been taken to identify functional therapeutic targets to halt the growth of this aggressive cancer. We have recently discovered that RIZ1 is downregulated in high-grade meningiomas, and RIZ1 overexpression inhibits proliferation while promoting cell apoptosis of the IOMM-Lee malignant meningioma cell line. In this report, we show that the N-terminal PR domain of RIZ1 alone possessed growth-inhibitory activity and anticancer activity in primary human meningioma cells. Interestingly, the effects seem to be dependent on differential RIZ1 protein levels. Transducible TAT-RIZ1-PR protein could also inhibit meningioma tumor growth in nude mice models. We further demonstrate that PR protein exerts histone methyltransferase activity. A microarray analysis of TAT-RIZ1-PR-treated human malignant meningioma cells reveals 969 differentially expressed genes and 848 alternative splicing exons. Moreover, c-Myc and TXNIP, two putative downstream targets of H3K9 methylation, may be involved in regulating RIZ1 tumor-suppressive effects. The reciprocal relationship between RIZ1 and c-Myc was then validated in primary meningioma cells and human tumor samples. These findings provide insights into RIZ1 tumor suppression mechanisms and suggest that TAT-RIZ1-PR protein is a potential new epigenetic therapeutic agent for advanced meningiomas.

Differential utilization and transformation of sulfur allotropes, µ-S and α-S8, by moderate thermoacidophile Sulfobacillus thermosulfidooxidans.

The utilization of amorphous µ-S and orthorhombic α-S8 by thermoacidophile Sulfobacillus thermosulfidooxidans was firstly investigated in terms of cell growth and sulfur oxidation behavior. The morphology and surface sulfur speciation transformation were evaluated by using scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform-infrared spectroscopy (FT-IR), Raman spectroscopy and sulfur K-edge X-ray absorption near edge structure (XANES) spectroscopy. The results showed that the strain grown on µ-S entered slower (about 1 day later) into the exponential phase, while grew faster in exponential phase and attained higher maximal cell density and lower pH than on α-S8. After bio-corrosion, both sulfur samples were evidently eroded, but only µ-S surface presented much porosity, while α-S8 maintained glabrous. µ-S began to be gradually converted into α-S8 from day 2 when the bacterial cells entered the exponential phase, with a final composition of 62.3% µ-S and 37.7% α-S8 on day 4 at the stationary phase. α-S8 was not found to transform into other species in the experiments with or without bacteria. These data indicated S. thermosulfidooxidans oxidized amorphous µ-S faster than orthorhombic α-S8, but the chain-like µ-S was transformed into cyclic α-S8 by S. thermosulfidooxidans.

Cone beam CT quality assure procedure and the analysis of results / 中华放射肿瘤学杂志

Objective To study the image quality control system to ensure that equipment meet clinical needs.Methods It was scanning the Catphan504 phantom with models of high quality head,standard dose head and pelvis,we could get the results of CT numbers linearity,uniformity,spatial resolution,contrast resolution.Using T test to compare different scanning technique results.Results The standard dose head scanning technique was better than the pelvis scanning technique in CT numbers linearity test,and gets the best result in uniformity test.The result of CT numbers uniformity was higher in the standard dose head scanning than the high quality head and the pelvis scanning (9.7 ±3.9 vs.17.9 ±5.3,P =0.00 and 9.5 ± 4.0 vs.31.1 ± 5.7,P =0.00).The result of contrast resolution was higher in the pelvis scanning than the high quality head and the pelvis scanning (5.6 ± 0.1 vs.1.3 ± 0.5,P =0.00 and 6.0 ± 1.0 vs.1.3 ± 0.5,P =0.00).The result of spatial linear distance was very accurate,the range was 4.98 -5.06 cm.Conclusions The results of spatial linearity test are stable and accuracy,but CT numbers linearity and uniformity test are affected by the scanning technique significantly for device.To spatial resolution test and contrast resolution test,we need to set the standard and tolerance according to each linear accelerator specialty.

Attachment of Acidithiobacillus ferrooxidans onto different solid substrates and fitting through Langmuir and Freundlich equations.

Attachments of Acidithiobacillus ferrooxidans ATCC 23270 onto elemental sulfur, quartz and complex chalcopyrite were investigated by analysis of its extracellular polymeric substances as well as applying Langmuir and Freundlich equations. The two equations fitted the adsorption equilibrium data with significant correlation coefficient over 0.9. This indicated that bacterial attachment is complicated and involves Langmuir and Freundlich characterizations. Sulfur-grown cells showed the highest affinity for the three solid substrates. The investigated complex chalcopyrite possessed a higher maximum adsorption capacity for A. ferrooxidans than elemental sulfur or quartz. The Freundlich fitting parameters suggested that quartz had a weaker adsorption capacity and smaller adsorption areas than elemental sulfur or the complex chalcopyrite. It is not the content of total carbohydrates or proteins in EPS but their ratios that determine the affinity differences between cells and substrates.

Thermophilic archaeal community succession and function change associated with the leaching rate in bioleaching of chalcopyrite.

The community succession and function change of thermophilic archaea Acidianus brierleyi, Metallosphaera sedula, Acidianus manzaensis and Sulfolobus metallicus were studied by denaturing gradient gel electrophoresis (DGGE) analysis of amplifying 16S rRNA genes fragments and real-time qPCR analysis of amplifying sulfur-oxidizing soxB gene associated with chalcopyrite bioleaching rate at different temperatures and initial pH values. The analysis results of the community succession indicated that temperature and initial pH value had a significant effect on the consortium, and S. metallicus was most sensitive to the environmental change, A. brierleyi showed the best adaptability and sulfur oxidation ability and predominated in various leaching systems. Meanwhile, the leaching rate of chalcopyrite closely related to the consortium function embodied by soxB gene, which could prove a desirable way for revealing microbial sulfur oxidation difference and tracking the function change of the consortium, and for optimizing the leaching parameters and improving the recovery of valuable metals.

A daily quality assure procedure for the on board imager and analysis of the results / 中华放射肿瘤学杂志

Objective Performing a daily quality assure (QA) program to get variation and error range of on board imager (OBI) system,so that the OBI system can meet the needs of clinical treatment.Methods The daily QA program including: mechanical accuracy,2D/2D Shift calculations accuracy,couch motion accuracy.Results The max deviation was-0.7 mm in lcft-right (LR) dircction and 0.8 mm in superior-inferior (S1) direction in Linac& OBI isocenter accuracy check.The max deviations in 4 blades (x1,x2,y2,y1) position accuracy check were:-2.1 mm,2.2 mm,± 1.7 mm,-2.1 mm.In OBI mechanical arms position accuracy check,31％ standard data was 85.2 cm with 0 mm deviation; 69％ standard data was 85.1 cm with 1 mm deviation.In LR,SI and anterior-posterior direction,2D/2D shift calculations accuracy was 0.46 mm,1.35 mm,-0.04 mm and couch motion accuracy was-0.1 mm,0.3 mm,0.2 mm,respectively.Conclusions By performing the daily QA program,it could be found whether OBI works properly and satisfies the clinical use.The physicist can pay more attention to the parameters which change frequently,and adjust the frequency of the parameters which are stable,so that working efficiently.

Changes of rectal volume and location and their influence on rectal dose during the course of intensity-modulated radiotherapy of cervical cancer / 中华放射肿瘤学杂志

ObjectiveTo investigate the anatomical changes and dose variation of rectum during radiotherapy in patients with cervical cancer.Methods Ten patients with cervical cancer underwent intensity-modulated radiotherapy using online cone beam computed tomography (CBCT) before each fraction.Rectum was contoured on each CBCT and projected onto the planning CT to analyze the changes of the rectal volume and position.The rectal volume receiving ≥ 45 Gy ( V45 ) was evaluated accordingly.Results227 CBCT images in 10 patients were collected.The rectal volume changed from ( 35.0 ± 7.3)cm3 to (97.7±14.7) cm3.The shift of rectal center was (0.14 ±0.06) cm in left and right direction,(0.24±0.10) cm in anterior and posterior direction,and (0.55±0.28) cm in superior and inferior direction.The V45 of rectum varied from (9.19±2.46)％ to (60.54 ±11.67)％.In7 of the 10 patients,rectal volume and V45 of the rectum had significant positive correlation (r =0.582 - 0.743,all P ＜ 0.01 ).Among the 227 images,the V45 of rectum was ≤50％ in 68 images (30.0％ ).ConclusionsSignificant changes in rectal volume and position occurred during fractionated radiotherapy in patients with cervical cancer,which resuhs in variations in the dose rectum received.For most patients,rectal volume and the V45of rectum had significant positive correlation.

Effects of copper exposure on expression of glutathione-related genes in Acidithiobacillus ferrooxidans.

The response of Acidithiobacillus ferrooxidans to variations in extracellular Cu exposure was investigated in terms of glutathione-related genes expression profiling based on reverse-transcription quantitative PCR analysis. The results show that the higher concentration of Cu would induce the expression of glutathione-related enzymes and cells elicited specific transcriptional responses when challenged with environmental Cu (0.08 mol l(-1)) conditions over a 60-min period. In comparison to the control, glutathione S-transferases (GST) and glutathione reductase (GR) were highly expressed when the cells were grown in the medium with copper, and the increase of glutathione and glutathione-related enzymes makes the cells acclimate to oxidative stress induced by Cu and protects the cells from toxicity caused by Cu exposure. It suggests that in order for Acidithiobacillus ferrooxidans to counteract the conditions of external Cu exposure, it modulated its expression level of GST, GR, glutathione hydrolase, and glutathione synthetase, which may protect organisms from oxidative damage. These parameters may be used to assess the biological impact of Cu in mining activities.

Sulfur oxidation activities of pure and mixed thermophiles and sulfur speciation in bioleaching of chalcopyrite.

The sulfur oxidation activities of four pure thermophilic archaea Acidianus brierleyi (JCM 8954), Metallosphaera sedula (YN 23), Acidianus manzaensis (YN 25) and Sulfolobus metallicus (YN 24) and their mixture in bioleaching chalcopyrite were compared. Meanwhile, the relevant surface sulfur speciation of chalcopyrite leached with the mixed thermophilic archaea was investigated. The results showed that the mixed culture, with contributing significantly to the raising of leaching rate and accelerating the formation of leaching products, may have a higher sulfur oxidation activity than the pure cultures, and jarosite was the main passivation component hindering the dissolution of chalcopyrite, while elemental sulfur seemed to have no influence on the dissolution of chalcopyrite. In addition, the present results supported the former speculation, i.e., covellite might be converted from chalcocite during the leaching experiments, and the elemental sulfur may partially be the derivation of covellite and chalcocite.

Bladder anatomical changes and dose variation during the course of intensity-modulated radiation therapy of cervical cancer / 中华放射肿瘤学杂志

Objective To investigate bladder anatomical changes and dose variation in patients with cervical cancer.Methods We analyzed 20 patients,undergoing external beam radiotherapy scanning cone beam CT(CBCT)before each fraction.Bladder was contoured on each CBCT,was projected onto the planning CT and assesses anatomical changes and dose variation.Results A total 451 CBCT images,for 20 patients were collected for analysis,show more change in bladder volume and position.In 15 cases bladder volume and V45 had no significant correlation(r=0.225 -0.473,all P>0.05),4 cases shows negative correlation(r=-0.564,P<0.05;r=-0.597,P<0.01;r=-0.942,P<0.01;r=-0.816,P<0.01),1 case shows positive correlation(r=0.662,P<0.01).Have more than the criteria(V45≤50%)number is 64/451(14.2%)in whole treatment.Conclusions For most patients by filling adequacy bladder,bladder dose variation is acceptable:CTV lager for individual patients should be closely observed its regression,implementation of the offline or online calibration.