METTL3 promotes the progression of osteosarcoma through the N6-methyladenosine modification of MCAM via IGF2BP1.

BACKGROUND: The molecular mechanisms of osteosarcoma (OS) are complex. In this study, we focused on the functions of melanoma cell adhesion molecule (MCAM), methyltransferase 3 (METTL3) and insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) in OS development. METHODS: qRT-PCR assay and western blot assay were performed to determine mRNA and protein expression of MCAM, METTL3, IGF2BP1 and YY1. MTT assay and colony formation assay were conducted to assess cell proliferation. Cell apoptosis, invasion and migration were evaluated by flow cytometry analysis, transwell assay and wound-healing assay, respectively. Methylated RNA Immunoprecipitation (MeRIP), dual-luciferase reporter, Co-IP, RIP and ChIP assays were performed to analyze the relationships of MCAM, METTL3, IGF2BP1 and YY1. The functions of METTL3 and MCAM in tumor growth were explored through in vivo experiments. RESULTS: MCAM was upregulated in OS, and MCAM overexpression promoted OS cell growth, invasion and migration and inhibited apoptosis. METTL3 and IGF2BP1 were demonstrated to mediate the m6A methylation of MCAM. Functionally, METTL3 or IGF2BP1 silencing inhibited OS cell progression, while MCAM overexpression ameliorated the effects. Transcription factor YY1 promoted the transcription level of METTL3 and regulated METTL3 expression in OS cells. Additionally, METTL3 deficiency suppressed tumor growth in vivo, while MCAM overexpression abated the effect. CONCLUSION: YY1/METTL3/IGF2BP1/MCAM axis aggravated OS development, which might provide novel therapy targets for OS.

Calcium-binding protein 39 overexpression promotes macrophages from 'M1' into 'M2' phenotype and improves chondrocyte damage in osteoarthritis by activating the AMP-activated protein kinase/sirtuin 1 axis.

Osteoarthritis (OA) is a degenerative joint disease that affects cartilage and its peripheral tissues. Up-regulation of Calcium-binding protein 39 (CAB39) has a significant protective effect on osteoblasts, but the role and related molecular mechanisms of CAB39 in OA have not yet been reported. CAB39 overexpression and knockdown models were set up in chondrocytes (ATDC5) and macrophages (RAW264.7). The OA cell model was induced in ATDC5 cells with IL-1ß (10 ng/mL). Cell viability was tested by the cell counting kit-8 assay, apoptosis was checked by flow cytometry. Western blot was applied for checking the expression of MMP3, MMP13, Aggrecan, the AMPK/Sirt-1 pathway, apoptosis-related proteins (Bax, Bcl-2, and Caspase-3), and macrophage phenotypic markers (CD86, iNOS, CD206, and Arg1). An OA model was constructed in mice, and CAB39 overexpression plasmids were administered to the knee cavity of the OA model mice. As a result, CAB39 was down-regulated in IL-1ß-treated chondrocytes and OA mice. Overexpressing CAB39 enhanced ATDC5 cell viability and choked IL-1ß-mediated apoptosis. Overexpression of CAB39 boosted the polarization of macrophages from M1-phenotype into M2 phenotype. In addition, overexpressing CAB39 facilitated the AMPK/Sirt-1 pathway activation, and AMPK inhibitors reversed the protective effect of CAB39 overexpression on chondrocytes. Moreover, CAB39 exhibited anti-inflammatory effects in OA mice by activating the AMPK/Sirt-1 pathway. Collectively, overexpressing CAB39 heightened macrophages' M2 polarization and declined chondrocyte injury in OA by activating the AMPK/Sirt-1 pathway.Abbreviations AMPK: AMP-activated protein kinaseArg1: arginase 1Bax: Bcl-2-associated X proteinBcl-2: B-cell lymphoma-2CAB39: Calcium-binding protein 39CM: Conditioned mediumDMM: destabilization of the medial meniscusECM: extracellular matrixELISA: enzyme-linked immunosorbent assayFCM: Flow cytometryIL-1ß: interleukin-1ßIL-4: interleukin-4IL-6: interleukin-6IL-10: interleukin-10IFN - γ: Interferon-gammaIHC: ImmunohistochemistryiNOS: Inducible nitric oxide synthaseLKB1: liver kinase B1MMP3: Matrix metalloproteinase3MMP13:Matrix metalloproteinase13NF-κB: NF-kappaBOA: OsteoarthritisqRT-PCR: Quantitative reverse transcription-polymerase chain reactionRT: room temperatureSirt-1: sirtuin 1STRAD: STE20-related adaptor alphaWB: Western blot.

The influence of prior arthroscopy on outcomes of primary total lower extremity arthroplasty: A systematic review and meta-analysis.

PURPOSE: The primary purpose of this systematic review and meta-analysis was to investigate the impact of prior arthroscopy on postoperative revisions, complications, and other clinical outcomes after conversion total lower extremity arthroplasty. METHODS: Two individual researchers conducted the platform searches on the Embase, PubMed, Cochrane Central, and Google Scholar electronic databases from inception to June 02, 2021. We identified cohort trials that compared the outcomes of patients who underwent primary THA or TKA in the prior arthroscopy or control groups. The primary outcome was revision, and secondary outcomes included reoperation, patient-reported outcomes, and postoperative complications. A modified version of the Downs and Black tool was used to assess the methodological quality of the non-randomized cohort studies. RESULTS: Of the 23 included studies with 319946 cases, 18 were matched retrospectively and five were non-matched retrospectively. Methodological quality was high in ten studies and moderate in thirteen studies. Our analysis demonstrated that TKA or THA patients with prior arthroscopy were associated with an increased risk of revision, reoperation, infection, and aseptic loosening. THA patients with prior arthroscopy were also associated with an increased risk of dislocation. Furthermore, there were no significant intergroup differences in periprosthetic fracture, range of motion, Harris Hip Score, or Knee Society Score. CONCLUSION: Arthroscopy performed before total lower extremity arthroplasty substantially increased the revision, reoperation, infection, and aseptic loosening rates. THA patients with prior arthroscopy were also associated with an increased risk of dislocation. Patients should be counseled on the potential increased risks associated with conversion total lower extremity arthroplasty after prior arthroscopy. Further research is needed to better characterize these findings.

Effectiveness and safety of ultrasound-guided hydrostatic reduction for children with acute intussusception.

OBJECTIVE: This study aims to explore the effectiveness and safety of the new-type ultrasound-guided hydrostatic reduction for children with acute intussusception. METHODS: The clinical data of 364 children with primary acute intussusception who underwent nonsurgical reduction in our hospital between January 2016 and May 2019 were retrospectively analyzed. Among the 364 children, 119 formed the hydrostatic reduction group. There were 89 males and 30 females, and the average age of admission was 25.13 ± 1.43 months. Among the pneumatic reduction group of 245 patients, there were 163 males and 82 females. The average age of admission was 22.47 ± 1.52 months. The reduction rate, length of stay, and perforation rate were compared between the two groups. RESULTS: Univariate analysis showed that the reduction rate in the hydrostatic group (94.96%) was higher than in the pneumatic group (85.31%) (p = 0.007), and the hospital stay (2.76 ± 0.15 days) of the hydrostatic reduction group was shorter than that of the pneumatic reduction group (3.56 ± 0.35 days) (p = 0.038). In children with intussusception time >48 h, the reduction rate was 95.45% in the hydrostatic reduction group and 86.20% in the pneumatic reduction group. CONCLUSION: The new-type ultrasound-guided hydrostatic reduction has a higher reduction rate in the treatment of acute intussusception in children results in a shortened hospital stay, It is effective, safe, and avoids radiation exposure.

Lysosomal dysfunction and autophagy blockade contribute to autophagy-related cancer suppressing peptide-induced cytotoxic death of cervical cancer cells through the AMPK/mTOR pathway.

BACKGROUND: Autophagy is an intracellular process through which intracellular components are recycled in response to nutrient or growth factor deficiency to maintain homeostasis. We identified the peptide autophagy-related cancer-suppressing peptide (ARCSP), a potential antitumor peptide that disrupts intracellular homeostasis by blocking autophagic flux and causes cytotoxic death. METHODS: The proliferative ability of ARCSP-treated cervical cancer cells was examined by the CCK8, EdU, and colony formation assays. The TUNEL assay was used to detect apoptosis. Mitochondrial function was evaluated based on the mitochondrial membrane potential. Autophagic flux was detected by immunofluorescence and confocal microscopy. The autophagy-related proteins AMPK, Raptor, mTOR, p62, LC3B, atg7, Rab7, LAMP1, LAMP2, and cathepsin D were detected by Immunoblotting. The antitumor effect of ARCSP was explored in vivo by establishing a transplant tumor model in nude mice. RESULTS: The results demonstrated that ARCSP induced cell death and inhibited proliferation. ARCSP induced AMPK/mTOR activation, resulting in the accumulation of the proteins LC3B, p62 and Atg7. ARCSP also blocked autophagosome-lysosome fusion by inhibiting endosomal maturation and increasing the lysosomal pH. The accumulation of nonfused autophagosomes exacerbated cytotoxic death, whereas knocking down Atg7 reversed the cytotoxic death induced by ARCSP. ARCSP-treated cells exhibited increased cytotoxic death after cotreatment with an autophagy inhibitor (Chloroquine CQ). Furthermore, the tumors of ARCSP-treated nude mice were significantly smaller than those of untreated mice. CONCLUSIONS: Our findings demonstrate that ARCSP, a novel lethal nonfused autophagosome inducer, might cause mitochondrial dysfunction and autophagy-related cytotoxic death and is thus a prospective agent for cancer therapy.

Integration of metabolomic and transcriptomic profiles to identify biomarkers in serum of lung cancer.

We used blood serum samples collected from 31 lung cancer (LC) patients and 29 healthy volunteers in this study. Levels of serum metabolites were qualitative quantified with gas chromatography-mass spectrometry (GC-MS), and the data were analyzed by partial least-squares discrimination analysis (PLS-DA). Based on the Kyoto Encyclopedia of Genes and Genomes database, we performed pathway-based analysis utilizing metabolites presented at differential abundance between the LC serum samples and the normal healthy serum samples for systematical investigation on the metabolic alterations associated with LC pathogenesis. Finally, we analyzed the significantly enriched pathways as well as their relevant differentially expressed messenger RNAs, and drawn a correlation network plot to identify the serum metabolic biomarkers and the significantly altered metabolic pathways for LC. GC-MS analysis showed that 23 of the 169 metabolites identified were significantly different. PLS-DA model revealed that 13 of these metabolites were with variable importance > 1, and particularly five were with area under curve > 0.9. Pathway-based analysis demonstrated that five of eight enriched metabolic pathways were statistically significant with false discovery rate < 0.05. Lastly, the correlation networks between these pathways and their related genes suggested that 29 genes had correlation degree > 10, which were mainly engaged in the purine metabolism. In conclusion, we identified indole-3-lactate, erythritol, adenosine-5-phosphate, paracetamol and threitol as serum metabolic biomarkers for LC through metabolomics analysis. Besides, we identified the purine metabolism as the significantly altered metabolic pathway in LC with the help of transcriptomics analysis.

Hypermethylated CD36 gene affected the progression of lung cancer.

OBJECTIVE: Our study aimed to explore the relationship between CD36 methylation and the development of lung cancer and investigate the effect of combine treatment of Decitabine and Chidamide in lung cancer. METHODS: The differentially expression genes in tumor samples and normal samples were determined by microarray analysis. Weighted gene co-expression network analysis (WGCNA) was utilized to analyze gene expression data of lung cancer and the hub genes were screened in the modules. Methylation-specific PCR (MSP) was conducted to detect the methylation level of CD36 in lung cancer cells. QRT-PCR analysis and western blotting were performed to explore the relative mRNA expression and protein level. MTT assays, wound healing assay, Transwell assays and flow cytometry were conducted to clarify the cell proliferation, migration, invasion and cell cycle of lung carcinoma cell lines in vitro respectively. By xenograft and immunohistochemistry, the effect of co-treatment of Decitabine and Chidamide was further verified in vivo. RESULTS: The heatmap displayed that the top 20 differential mRNA-expression gene in lung cancer tissues and normal tissues in which CD36 was low expressed in the tumor samples and high expressed in the normal samples. By WGCNA, CD36 was selected to be the hub gene in the brown module. And then CD36 was confirmed to be differential expressed and hypermethylated in lung cancer through qRT-PCR and western blotting. CD36 inhibited the lung cancer cell proliferation, promoted cell apoptosis, blocked cell cycle at G0/G1 phase, and inhibited cell migration. What's more, we found that Decitabine (DCTB) and Chidamide (CDM) co-treatment induced de-methylation and re-expression of silenced CD36 by conducting the vivo experiments. DCTB + CDM co-treatment synergistically suppressed tumor growth. CONCLUSION: Our results showed that the high methylation of CD36 in lung cancer played an important role in the procession of lung cancer and Decitabine joint Chidamide had obvious effect of inhibiting the growth of lung tumor.

A network meta-analysis of the short-term efficacy of five chemotherapy regimens based on cisplatin and fluorouracil for esophagogastric junctional adenocarcinoma.

The primary purpose of this study was to explore the short-term efficacy of different cisplatin and fluorouracil-based chemotherapy regimens in the treatment of patients with esophagogastric junctional adenocarcinoma (EGJA) using a network meta-analysis (NMA). Randomized controlled trials (RCTs) related to chemotherapy regimens based on cisplatin and fluorouracil for EGJA were included from the PubMed, EMBASE and Cochrane Library electronic databases (from inception to June 2016). Direct and indirect evidence were combined to calculate the pooled odds ratio (OR) and its 95% confidence interval (95% CI) as well as to draw the surface under the cumulative ranking (SUCRA) curves. This NMA finally enrolled ten eligible RCTs with the following five regimens: cisplatin plus fluorouracil (cisplatin+fluorouracil), cisplatin+fluorouracil-based chemotherapy (cisplatin+fluorouracil+docetaxel/epirubicin/irinotecan), fluorouracil-based chemotherapy (fluorouracil+docetaxel/doxorubicin/methotrexate/irinotecan), cisplatin-based chemotherapy (cisplatin+docetaxel/epirubicin/irinotecan/capecitabine/s-1) and other drug-based chemotherapy (docetaxel/irinotecan/capecitabine). These results revealed that compared with a cisplatin+ fluorouracil-based chemotherapy regimen, the fluorouracil-based chemotherapy regimen had a lower overall response rate (ORR) and partial response (PR) for EGJA patients (ORR: OR=0.43, 95% CI=0.22-0.86; PR: OR=0.46, 95% CI=0.23-0.91). Cluster analyses suggested that the cisplatin+fluorouracil-based chemotherapy regimen had the best short-term efficacy for EGJA in terms of the complete response (CR), PR, ORR, stable disease (SD) and progression disease (PD). Our results indicated that cisplatin+fluorouracil-based chemotherapy regimens may have the best short-term efficacy in the treatment of EGJA.

Detection and identification of serum protein peak at 6648 m/z as a novel indicator in breast cancer based on mass spectrometry.

Breast cancer (BC) is the second-leading cause of cancer mortality after lung cancer in women owing partly to a lack of specific and sensitive tests for early screening and monitoring. The detection of novel specific BC serum indicators for screening purposes is an essential clinical need. A total of 437 serum specimens from 310 BC patients that were divided into mining and testing sets were collected in this study. In contrast with the conventional BC indicators through receiver operating characteristic, survival and hazard function curves, and multivariate Cox regression analyses, we intended to hunt for stable protein indicators from serum specimens and identify their diagnostic and prognostic potential for BC. We identified a unique serum peptide located at 6648 Da originated from apoC-III with a validated correlation with BC tumorigenesis with confirmation in a substantive testing set and minimization of systematic bias by pre-analytical parameters. We found that the diagnostic efficacy of this peptide is better than the present conventional BC diagnostic indicators either alone or in combination with conventional indicators in distinguishing BC patients from control volunteers. Moreover, this peptide denotes a stronger prognostic factor for BC patients than conventional indicators. In light of these findings, we speculate that this peptide is a potential diagnostic and prognostic indicator and a supplement to conventional indicators in monitoring BC. The detection of this peptide located at 6648 Da in sera could enhance early screening and assessment of the postoperative survival opportunity for BC patients.

Clinical application of multidisciplinary teams in tumor therapy.

Multidisciplinary team (MDT) model is a diagnostic and treatment model characterized by interdisciplinarity, integration, centralism, individualization, and precision and is becoming more common in the management of complex malignancies. MDT emphasizes team spirit and a personalized treatment strategy according to the actual condition of each patient. A cooperative and effective multidisciplinary team is an important guarantee for delivering high-quality services to patients. Under the guidance of a medical humanistic concept, MDT provides reasonable, effective, convenient, and a full range of excellent quality medical service to patients. The MDT maximizes patient benefits, and it is the developmental direction for large-scale general hospitals. At the same time, the MDT is also an important measure to strengthen the core competitiveness of hospitals. Here, we introduce the clinical application of the model in tumor therapy as well as the current state and development in our hospital.

Transcription factor ZFP38 is essential for meiosis prophase I in male mice.

The production of haploid gametes by meiosis is a cornerstone of sexual reproduction and maintenance of genome integrity. Zfp38 mRNA is expressed in spermatocytes, indicating that transcription factor ZFP38 has the potential to regulate transcription during meiosis. In this study, we generated Zfp38 conditional knockout mice (Zfp38(flox/flox), Stra8-Cre, hereafter called Zfp38 cKO) and found that spermatogenesis did not progress beyond meiosis prophase I in Zfp38 cKO mice. Using a chromosomal spread technique, we observed that Zfp38 cKO spermatocytes exhibited a failure in chromosomal synapsis observed by SYCP1/SYCP3 double staining. Progression of DNA double-strand breaks (DSB) repair is disrupted in Zfp38 cKO spermatocytes, as revealed by γ-H2AX, RAD51 and MLH1 staining. Furthermore, the mRNA and protein levels of DSB repair enzymes and factors that guide their loading onto sites of DSBs, such as RAD51, DMC1, RAD51, TEX15 and PALB2, were significantly reduced in Zfp38 cKO spermatocytes. Taken together, our data suggest that ZFP38 is critical for the chromosomal synapsis and DSB repairs partially via its regulation of DSB repair-associated protein expression during meiotic progression in mouse.

Diagnostic and prognostic significance of serum apolipoprotein C-I in triple-negative breast cancer based on mass spectrometry.

Women with triple-negative breast cancer (TNBC) have poor prognosis because of the aggressive nature of the tumor, delayed diagnosis and non-specific symptoms in the early stages. Identification of novel specific TNBC serum biomarkers for screening and therapeutic purposes therefore remains an urgent clinical requirement.We obtained serum samples from a total of 380 recruited individuals split into mining and testing sets, with the aim of screening for reliable protein biomarkers from TNBC and non-TNBC (NTNBC) sera. Samples were assessed using mass spectrometry, followed by receiver operating characteristic (ROC), survival and hazard function curve as well as multivariate Cox regression analyses to ascertain the potential of the protein constituents as diagnostic and prognostic biomarkers for TNBC.We identified upregulated apolipoprotein C-I (apoC-I) with a validated positive effect on TNBC tumorigenesis, with confirmation in an independent test set and minimization of systematic bias by pre-analytical parameters. The apoC-I protein had superior diagnostic ability in distinguishing between TNBC and NTNBC cases. Moreover, the protein presented a more robust potential prognostic factor for TNBC than NTNBC. The apoC-I protein identified in this study presents an effective novel diagnostic and prognostic biomarker for TNBC, indicating that measurement of the peak intensity at 7785 Da in serum samples could facilitate improved early detection and estimation of postoperative survival prognosis for TNBC.

Diagnostic and prognostic role of serum protein peak at 6449 m/z in gastric adenocarcinoma based on mass spectrometry.

BACKGROUND: Gastric cancer (GC) is a highly aggressive cancer type associated with significant mortality owing to delayed diagnosis and non-specific symptoms observed in the early stages. Therefore, identification of novel specific GC serum biomarkers for screening purposes is an urgent clinical requirement. METHODS: This study recruited a total of 432 serum samples from 296 GC patients split into the mining and testing sets. We aimed to screen for reliable protein biomarkers from matched serum samples based on mass spectrometry, followed by comparison with three representative conventional markers using receiver operating characteristic and survival curve analyses to ascertain their potential values as diagnostic and prognostic biomarkers for GC. RESULTS: We identified an apoC-III fragment with confirmation in an independent test set from a second hospital. We found that the diagnostic ability of this fragment performed better than current standard GC diagnostic biomarkers both individually and in combination in distinguishing patients with GC from healthy individuals. Moreover, we found that this apoC-III protein fragment represents a more robust potential prognostic factor for GC than the three conventional markers. CONCLUSIONS: In view of these findings, we suggest that apoC-III protein fragment is a novel diagnostic and prognostic biomarker, a complement to conventional biomarkers in detecting GC.

Identification of proteins associated with pediatric bilateral Wilms tumor.

Wilms tumor (WT) is the most common cancer that primarily develops in abdominal solid organ of children. It has no incipient symptom, and the most frequent symptoms are a painless, palpable abdominal mass. Proteomics technology was used to select the differentially expressed proteins of bilateral Wilms tumor (BWT). Ten serum samples of children with BWT were chosen, 20 serum samples of children with unilateral WT (UWT) and 20 serum samples of healthy children were selected, and proteomics technology was used to detect and collect data. Using bioinformatics, the data were analyzed and 10 difference peaks were obtained (P<0.01). Non-linear support vector machine was used to classify and to select the composite pattern with the highest Youdens index, and one differentially expressed protein with m/z of 5,648 kDa was obtained. A significantly high expression in children with BWT was obtained, and the expression intensity was also significantly (3,889.36±1,796.83) higher for children with BWT compared to those with UWT (2,886.81±1,404.65) and healthy children (432.21±730.42). Matrix-assisted laser desorption ionization/time-of-flight ionization/time-of-flight mass spectrometry was used for identification of the peak, and the peak was further identified as apolipoprotein C-III (APO C-III) by western blot analysis. In conclusion, to the best of our knowledge, a differentially expressed protein of APO C-III of BWT was obtained through proteomics technology for the first time, and it is expected to be a new marker for the early diagnosis and prognosis of BWT.

Evaluation of promoter hypomethylation and expression of p73 as a diagnostic and prognostic biomarker in Wilms' tumour.

AIMS: A member of the p53 family, the p73 gene is essential for the maintenance of genomic stability, DNA repair and apoptosis regulation. This study was designed to evaluate the utility of expression and DNA methylation patterns of the p73 gene in the early diagnosis and prognosis of Wilms' tumour (WT). METHODS: Methylation-specific PCR, semi-quantitative (sq-PCR), real-time quantitative PCR (qRT-PCR), receiver operating characteristic (ROC), and survival and hazard function curve analyses were utilised to measure the expression and DNA methylation patterns of p73 in WT tissue samples with a view to assessing diagnostic and prognostic value. RESULTS: The relative expression of p73 mRNA was higher, while the promoter methylation level was lower in the WT than the control group (p<0.05) and closely associated with poor survival prognosis in children with WT (p<0.05). Increased expression and decreased methylation of p73 were correlated with increasing tumour size, clinical stage and unfavourable histological differentiation (p<0.05). ROC curve analysis showed areas under the curve of 0.544 for methylation and 0.939 for expression in WT venous blood, indicating the higher diagnostic yield of preoperative p73 expression. CONCLUSIONS: Preoperative venous blood p73 level serves as an underlying biomarker for the early diagnosis of WT. p73 overexpression and concomitantly decreased promoter methylation are significantly associated with poor survival in children with WT.

Assessment of promoter methylation and expression of SIX2 as a diagnostic and prognostic biomarker in Wilms' tumor.

This study was designed to evaluate the utility of expression and DNA methylation patterns of the sine oculis homeobox homolog 2 (SIX2) gene in early diagnosis and prognosis of Wilms' tumor (WT). Methylation-specific polymerase chain reaction (MSP), real-time quantitative polymerase chain reaction (qRT-PCR), receiver operating characteristic (ROC), and survival curve analyses were utilized to measure the expression and DNA methylation patterns of SIX2 in a cohort of WT tissues, with a view to assessing their diagnostic and prognostic value. Relative expression of SIX2 mRNA was higher, while the promoter methylation level was lower in the WT than control group (P < 0.05) and closely associated with poor survival prognosis of WT children (P < 0.05). Increased expression and decreased methylation of SIX2 were correlated with increasing tumor size, clinical stage, vascular invasion, and unfavorable histological differentiation (P < 0.05). ROC curve analysis showed areas under the curve (AUCs) of 0.579 for methylation and 0.917 for expression in WT venous blood, indicating higher diagnostic yield of preoperative SIX2 expression. The preoperative venous blood SIX2 expression level serves as an underlying biomarker for early diagnosis of WT. SIX2 overexpression and concomitantly decreased promoter methylation are significantly associated with poor survival of WT children.

[Expression and promoter methylation of SIX2 gene in peripheral blood of pediatric patients with nephroblastoma].

OBJECTIVE: To explore the transcriptional expression and promoter methylation status of SIX2 gene in peripheral blood of pediatric children with nephroblastoma and discuss their clinicopathological correlations. METHODS: Approved by the hospital ethics committee, peripheral blood samples were collected from 45 children with Wilms' tumor(case group) at the Department of Pediatric Surgery, First Affiliated Hospital, Zhengzhou University from October 2008 to January 2012. And another 15 pediatric cases gender-and-age matched, were selected as the control group (excluding cancer and other malignant diseases). The real-time quantitative (qRT)-PCR and methylation-specific PCR (MSP) were used to detect the mRNA expression level and methylation status of SIX2 gene.t or χ(2) test were used. Then analyzed their clinicopathological correlations in the case group and how SIX2 gene methylation affected its transcription. RESULTS: Relative quantity(RQ) of SIX2 mRNA in the case group was higher than that of the control group (1.93 ± 1.10 vs 0.57 ± 0.39, t = 5.354, P = 0.000). There were 8 SIX2 gene methylation-positive cases in the case group versus 12 cases in the control group. And the methylation positive ratio was extremely lower in the case group (χ(2) = 19.600, P = 0.000). RQ values in the case group was associated with tumor size, clinical stage, pathological type, lymph node metastasis, treatment and outcome (all P < 0.05). RQ values in the methylated group was lower than that of the unmethylated group both in case and control group (1.35 ± 0.44 vs 1.95 ± 1.15, 0.43 ± 0.29 vs 1.13 ± 0.20, t = 2.459 and 3.896, P = 0.020 and 0.002) . RQ values of case group was higher than that of the control group in methylated group (t = 5.624, P = 0.000) . No statistical significance existed in RQ values between the case and control groups of unmethylated group (t = 1.222, P = 0.229) . CONCLUSIONS: A close correlation between SIX2 low methylation and high mRNA expression in blood suggests that aberrant promoter methylation is possibly one of gene expression regulations, and may be correlated with the occurrence and development of Wilms' tumor. And SIX2 gene in methylated Wilms' tumor may play the role of oncogenes. A negative correlation exists between the overexpression in transcriptional level and its methylation status.

[Relationship between mRNA expression and promoter methylation status of p73 gene in peripheral blood among children with Wilms' tumor].

OBJECTIVE: To investigate the mRNA expression and promoter methylation status of p73 gene in the peripheral blood of children with Wilms' tumor (WT), and their relationship. METHODS: Forty-five children with WT were selected as the case group, and 15 sex- and age- matched children (without malignancies) who visited the hospital for physical examination or other reasons were selected as the control group. Peripheral blood was collected from both groups. Real-time quantitative PCR and methylation-specific PCR were used to determine the mRNA expression level and promoter methylation status of p73 gene. Their relationship with clinicopathological features and the effect of promoter methylation on mRNA expression of p73 gene were analyzed in the case group. RESULTS: The relative quantity (RQ) of p73 mRNA in the case group was significantly higher than in the control group (3.2 ± 0.9 vs 1.6 ± 1.1; P<0.01). The positive rate of p73 gene promoter methylation in the case group was significantly lower than in the control group (20% vs 73%; P<0.01). In the case group, the RQ of p73 mRNA was significantly higher in children with methylated p73 gene promoter than in those with unmethylated p73 gene promoter (P<0.01). In children with methylated p73 gene promoter, the RQ of p73 mRNA was significantly higher in the case group than in the control group (P<0.01). In children with unmethylated p73 gene promoter, there was no significant difference in RQ of p73 mRNA between the case and control groups (P=0.810). CONCLUSIONS: Aberrant promoter methylation of p73 gene in peripheral blood is one of the gene expression regulations in children with WT, and it is related to the onset and development of WT. The p73 gene may play a role as oncogene in WT patients with p73 gene promoter methylation and mRNA overexpression is associated with promoter methylation status of p73 gene.