Intraoperative dexmedetomidine on postoperative sleep disturbance in older patients undergoing major abdominal surgery: A randomized controlled trial protocol.

Background: Postoperative sleep disturbance (PSD) occurs frequently in patients who undergo major abdominal surgical procedures. Dexmedetomidine is a promising agent to improve the quality of sleep for surgical patients. We designed this trial to investigate the effects of two different doses of intraoperative dexmedetomidine on the occurrence of PSD in elderly patients who have major abdominal surgery. Methods: In this randomized, double-blind, controlled trial, 210 elderly patients aged ≥65 years will be randomized, with an allocation ratio of 1:1:1, to two dexmedetomidine groups (intraoperative infusion of 0.3 or 0.6 µg/kg/h) and a normal saline placebo group. The primary endpoint is the occurrence of PSD on the first night after surgery, assessed using the Athens Insomnia Scale. The secondary endpoints are (1) the incidence of PSD during the 2nd, 3rd, 5th, 7th, and 30th nights postoperatively; (2) pain at rest and on movement at 24 and 48 h postoperatively, assessed using the Numerical Rating Scale; (3) the incidence of postoperative delirium during 0-7 days postoperatively or until hospital discharge, assessed using the 3-min Confusion Assessment Method; (4) depressive symptoms during 0-7 days postoperatively or until hospital discharge, assessed using the 15-items Geriatric Depression Scale; and (5) quality of recovery on postoperative days 1, 2, and 3, assessed using the 15-items Quality of Recovery Scale. Patients' sleep data will also be collected by Xiaomi Mi Band 7 for further analysis. Discussion: The findings of this trial will provide clinical evidence for improving the quality of sleep among elderly patients undergoing major abdominal surgery. Ethics and dissemination: This trial was approved by the Ethics Committee of the First Affiliated Hospital of Soochow University (No. 2023-160). The results will be published in a peer-reviewed journal. Trial registration: Chinese Clinical Trial Registry (ChiCTR2300073163).

[Expression and Clinical Significance of Exosome Component 4 in Newly Diagnosed Patients with Diffuse Large B-Cell Lymphoma].

OBJECTIVE: To explore the expression of Exosome Component 4（EXOSC4） in the tissues of newly diagnosed patients with diffuse large B-cell lymphoma (DLBCL) and its clinical significance. METHODS: The expression of EXOSC4 protein in the tissues of 181 newly diagnosed DLBCL patients was analyzed by immunohistochemical staining. Clinical data were collected. The correlation between EXOSC4 protein expression in the tissues of newly diagnosed DLBCL patients and clinical features were analyzed and its prognostic significance. RESULTS: The positive rate of EXOSC4 protein expression was 68.51% in the tissues of 181 newly diagnosed DLBCL patients. These patients were divided into two groups, with 44 cases in high expression group and 137 cases in low expression group. There were no significant differences in age, gender, B symptoms, serum lactate dehydrogenase (LDH) level, Eastern Cooperative Oncology Group (ECOG) score, Ann Arbor stage, extranodal disease, International Prognostic Index (IPI) score, National Comprehensive Cancer Network IPI (NCCN-IPI) score, and cell origin between the two groups (P>0.05). Cox multivariate regression analysis showed that high EXOSC4 protein expression in tissues was an independent poor prognostic factor for OS and PFS in newly diagnosed DLBCL patients (all P<0.05). K-M survival analysis showed that newly diagnosed DLBCL patients with high EXOSC4 protein expression had significantly shorter overall survival (OS) and progression free survival (PFS) than those patients with low EXOSC4 protein expression (all P<0.05). CONCLUSION: High EXOSC4 protein expression in tissues of newly diagnosed DLBCL patients is an independent poor prognostic factor for survival.

[Release of Exosomes Derived from Leukocyte-Depleted Red Cell Suspension and Its Regulation on Hematological Tumor Cells].

OBJECTIVE: To investigate the release of exosome (Exo) from leukocyte-depleted red cell suspension (LDRCS) at different storage time and its regulation on proliferation of hematological tumor cells and possible mechanism. METHODS: The Exo (RBC-Exo) in LDRCS at different storage time was obtained by ultracentrifugation, and the morphology and immunological marker of RBC-Exo were detected by transmission electron microscopy and Western blot, respectively. The particle size distribution of RBC-Exo in LDRCS at different storage time was detected by Dynamic Light Scattering. CCK-8 assay was used to explore the effect of RBC-Exo on hematological tumor cell proliferation. Western blot was used to detect the expression of proliferation-related proteins in hematological tumor cells after co-culture with RBC-Exo. RESULTS: RBC-Exo was isolated, which was characterized by cup-like shape, particle size distribution ranged from 20 to 200 nm, CD63/TSG101 enriched, Calnexin negative, CD235a positive and CD41 negative. The particle size distribution of RBC-Exo from LDRCS between middle was not significantly different and late stored stage. But the particle size distribution of RBC-Exo at middle-late stored stage(>14 d) was larger than that at early stored stage (≤14 days). Compared with the control group, RBC-Exo could significantly promote the proliferation of HBL1, U2932 and Jurkat cells. Compared with the control group, the cycle-related protein P21 was significantly down-regulated in HBL1, U2932 and Jurkat cells after co-culture with RBC-Exo for 3 days, while the anti-apoptotic protein BCL-2 was not changed significantly. CONCLUSION: The morphology of RBC-Exo from LDRCS at middle-late stored stage was different from that at early stored stage. RBC-Exo could promote the proliferation of hematological tumor cells, possibly by regulating the expression of cycle-associated protein P21.

Machine learning-based Radiomics analysis for differentiation degree and lymphatic node metastasis of extrahepatic cholangiocarcinoma.

BACKGROUND: Radiomics may provide more objective and accurate predictions for extrahepatic cholangiocarcinoma (ECC). In this study, we developed radiomics models based on magnetic resonance imaging (MRI) and machine learning to preoperatively predict differentiation degree (DD) and lymph node metastasis (LNM) of ECC. METHODS: A group of 100 patients diagnosed with ECC was included. The ECC status of all patients was confirmed by pathology. A total of 1200 radiomics features were extracted from axial T1 weighted imaging (T1WI), T2-weighted imaging (T2WI), diffusion weighted imaging (DWI), and apparent diffusion coefficient (ADC) images. A systematical framework considering combinations of five feature selection methods and ten machine learning classification algorithms (classifiers) was developed and investigated. The predictive capabilities for DD and LNM were evaluated in terms of area under precision recall curve (AUPRC), area under the receiver operating characteristic (ROC) curve (AUC), negative predictive value (NPV), accuracy (ACC), sensitivity, and specificity. The prediction performance among models was statistically compared using DeLong test. RESULTS: For DD prediction, the feature selection method joint mutual information (JMI) and Bagging Classifier achieved the best performance (AUPRC = 0.65, AUC = 0.90 (95% CI 0.75-1.00), ACC = 0.85 (95% CI 0.69-1.00), sensitivity = 0.75 (95% CI 0.30-0.95), and specificity = 0.88 (95% CI 0.64-0.97)), and the radiomics signature was composed of 5 selected features. For LNM prediction, the feature selection method minimum redundancy maximum relevance and classifier eXtreme Gradient Boosting achieved the best performance (AUPRC = 0.95, AUC = 0.98 (95% CI 0.94-1.00), ACC = 0.90 (95% CI 0.77-1.00), sensitivity = 0.75 (95% CI 0.30-0.95), and specificity = 0.94 (95% CI 0.72-0.99)), and the radiomics signature was composed of 30 selected features. However, these two chosen models were not significantly different to other models of higher AUC values in DeLong test, though they were significantly different to most of all models. CONCLUSION: MRI radiomics analysis based on machine learning demonstrated good predictive accuracies for DD and LNM of ECC. This shed new light on the noninvasive diagnosis of ECC.

AIDA directly connects sympathetic innervation to adaptive thermogenesis by UCP1.

The sympathetic nervous system-catecholamine-uncoupling protein 1 (UCP1) axis plays an essential role in non-shivering adaptive thermogenesis. However, whether there exists a direct effector that physically connects catecholamine signalling to UCP1 in response to acute cold is unknown. Here we report that outer mitochondrial membrane-located AIDA is phosphorylated at S161 by the catecholamine-activated protein kinase A (PKA). Phosphorylated AIDA translocates to the intermembrane space, where it binds to and activates the uncoupling activity of UCP1 by promoting cysteine oxidation of UCP1. Adipocyte-specific depletion of AIDA abrogates UCP1-dependent thermogenesis, resulting in hypothermia during acute cold exposure. Re-expression of S161A-AIDA, unlike wild-type AIDA, fails to restore the acute cold response in Aida-knockout mice. The PKA-AIDA-UCP1 axis is highly conserved in mammals, including hibernators. Denervation of the sympathetic postganglionic fibres abolishes cold-induced AIDA-dependent thermogenesis. These findings uncover a direct mechanistic link between sympathetic input and UCP1-mediated adaptive thermogenesis.

[Expression of Ribonucleotide Reductase M2 in Patients with Multiple Myeloma and Its Mechanism Inhibiting Tumor Cell Proliferation].

OBJECTIVE: To investigate the expression of ribonucleotide reductase M2 (RRM2) in patients with multiple myeloma and its mechanism of inhibiting human multiple myeloma cell proliferation. METHODS: Thirty-Six patients with multiple myeloma in our hospital from July 2016 to September 2018 were selected as MM group, at the same time simple iron deficiency anemia patients were taken as the control group. RT-qPCR and Western blot techniques were used to determine the mRNA and protein expression of RRM2 in bone marrow mononuclear cells, respectively. siRNAs oligos targeting RRM2 was transfected into RPMI8226 cells to establish the RRM2 silence model. CCK-8 and flow cytometry were used to analyze the cell proliferation and the cell cycle, and Western blot was used to detect the expression of cell cycle related proteins. RESULTS: The expression level of RRM2 mRNA and the expression level of RRM2 protein in multiple myeloma group were significantly higher than those in control group (P＜0.05). Further analysis indicated that the level of RRM2 expression closely correlated with ISS staging, bone destruction and extramedullary infiltration (P＜0.05). Transfection with siRNAs targeting RRM2 could significantly down regulate the RRM2 expression. And the RRM2 down regulation inhibited the cell proliferation and arrested the cell cycle at S stage (P＜0.05). Further study indicated that RRM2 silencing influenced cell cycle-related proteins expression. CONCLUSION: RRM2 overexpressies in bone marrow mononuclear cells of MM patients, moreover significantly relates with the degree of malignancy. The silencing RRM2 in multiple myeloma cells can inhibit cell proliferation through arresting cell cycle at S phase. RRM2 may be a marker to predict the prognosis of patients with multiple myeloma and novel target for new drug development.

Bioinformatics analysis reveals novel core genes associated with nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is the most frequent liver disease and associated with a wide spectrum of hepatic disorders ranging from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma (HCC). NASH is projected to become the most common indication for liver transplantation, and the annual incidence rate of NASH-related HCC is 5.29 cases per 1000 person-years. Owing to the epidemics of NAFLD and the unclear mechanism of NAFLD progression, it is important to elucidate the underlying NAFLD mechanisms in detail. NASH is mainly caused by the development of NAFL Therefore, it is also of great significance to understand the mechanism of progression from NAFL to NASH. Gene expression chip data for NAFLD and NASH were downloaded from the Gene Expression Omnibus database to identify differentially expressed genes (DEGs) between NAFLD and normal controls (called DEGs for NAFLD), as well as between NASH and normal tissue (called DEGs for NASH-Normal), and between NASH and NAFL tissue (called DEGs for NASH-NAFL). For DEGs for the NAFLD group, key genes were identified by studying the form of intersection. Potential functions of DEGs for NASH were then analyzed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. A protein-protein interaction network (PPI) was constructed using the STRING database. A total of 249 DEGs and one key gene for NAFLD were identified. For NASH-Normal, 514 DEGs and 11 hub genes were identified, three of which were closely related to the survival analysis of HCC, and potentially closely related to progression from NASH to HCC. One key gene for NASH-NAFL (AKR1B10) was identified. These genes appear to mediate the molecular mechanism underlying NAFLD and may be promising biomarkers for the presence of NASH.

Long non-coding RNA CRNDE enhances cervical cancer progression by suppressing PUMA expression.

Cervical cancer is the second most common gynecological malignancy, and it remains a leading cause of tumor-related death among female in the world. Long non-coding RNAs (lncRNAs) have been indicated to play essential roles in tumorigenesis, and the lncRNA colorectal neoplasia differentially expressed (CRNDE) is increased in several tumors. Nevertheless, little is known about the effects of lncRNA CRNDE on human cervical cancer. The aim of the study was to explore the clinical significance of lncRNA CRNDE expression in human cervical cancer. Our results indicated that CRNDE expression was increased in cervical cancer tissues and several cervical cancer cell lines. Through loss-of-function and gain-of-function approaches, we found that CRNDE knockdown markedly reduced cervical cancer cell proliferation, while CRNDE overexpression significantly promoted cervical cancer cell growth. Consistently, CRNDE decreasing obviously inhibited tumorigenicity of cervical cancer cells in vivo, whereas CRNDE increasing markedly promoted cervical cancer progression. Mechanistically, we verified that CRNDE bond to p53 upregulated modulator of apoptosis (PUMA), and PUMA was required for CRNDE to enhance cervical cancer cell growth. Our study demonstrated that CRNDE, combined with PUMA, could be utilized as factor for the clinical diagnosis and prognosis of cervical cancer, and might be potential target for developing effective therapeutic strategy to prevent cervical cancer progression.

Clinical, biochemical, neuroimaging and molecular findings of X-linked Adrenoleukodystrophy patients in South China.

X-linked adrenoleukodystrophy is a common X-linked recessive peroxisomal disorder caused by the mutations in the ABCD1 gene. In this study, we analyzed 19 male patients and 9 female carriers with X-linked adrenoleukodystrophy in South China. By sequencing the ABCD1 gene, 13 different mutations were identified, including 7 novel mutations, and 6 known mutations, and 1 reported polymorphism. Mutation c.1180delG was demonstrated to be de novo mutation. 26.3 % (5/19) patients carried the deletion c.1415_16delAG, which may be the mutational hot spot in South China population. In addition, 73.7 % (14/19) patients were type of childhood cerebral adrenoleukodystrophy, 26.3 %(5/19) were in Addison only. Half of the childhood cerebral adrenoleukodystrophy patients had the adrenocortical insufficiency preceded the onset of neurological symptoms. Furthermore, 5 of 19 cases underwent hematopoietic stem cell transplantation. Our data showed that hematopoietic stem cell transplantation performed at an advanced stage of the cerebral X- linked adrenoleukodystrophy would accelerate the progression of the disease. Good clinical outcome achieved when hematopoietic stem cell transplantation performed at the very early stage of the disease.

[Analysis of the efficiency and influence factors of PBSC collection with AutoPBSC and MNC procedure of cell separator].

This study was aimed to analyze the efficiency and influence factors of PBSC collection by an automatic (AutoPBSC procedure) and a semiautomatic apheresis procedure ( MNC procedure) of COBE Spectra cell separators. According to the different objects, A total of 109 apheresis cases were divided into autologous cohort (patient) and allogeneic cohort (donor). The quantity and quality of the collections and the characteristics of apheresis procedure were compared, the yields and influence factors of two cohorts with two kinds of procedures were analyzed respectively. The results showed that the collections of two procedure in patients and donors which processed the similar blood volumes were insignificantly different in MNC%, CD34⁺ %, CD34⁺ cell counts and Hb concentration (P > 0.05) ; the collections by AutoPBSC procedure had got fewer platelets, less product volumes whereas more ACD-A used, longer apheresis time in comparison with MNC procedure (P < 0.05). Correlation analysis indicated that MNC (r = 0.314,P = 0.015) , CD34⁺ cell counts (r = 0.922, P = 0.000) in collections were positively correlated with preahperesis in the autologus cohort by two procedures, CD34⁺ cell counts were correlated with WBC (r = 0.369, P = 0.004) and MNC (r = 0.495,P = 0.000) in collections; MNC (r = 0.896, P = 0.000) was positive correlated with preahperesis by AutoPBSC procedures and CD34⁺ cell counts also (r = 0.666,P = 0.000) by MNC procedure in the allogeneic cohort. Male had got more MNC and CD34⁺ cell counts than female (P < 0.05), age ≤ 40 had got more MNC and CD34⁺ cell counts than age>40 (P < 0.05) in patients by AutoPBSC procedure; age > 40 had got more CD34⁺ cell counts than age ≤ 40 by MNC procedure(P < 0.05). Only male had got more MNC and CD34⁺ cell counts than female (P < 0.05) by MNC procedure in donors. It is concluded that with same amount of blood processing, the PBSC collections from autologous patients and allogeneic donors had got a high degree of uniformly in purity of MNC and purity and concentration of CD34(+) cell counts by two procedure, whereas sex and age imposed more influence on PBSC collection in autologous.

[MicroRNA-193b expression in newly diagnosed leukemia patients and its significance].

This study was aimed to quantitatively detect the levels of microRNA-193b (miR-193b) in leukemia patients and explore its significance. Real time fluorescent quantitative PCR was used to detect the relative expression level of miR-193b. The expression changes of miR-193b in various types of leukemia were analyzed. Then the relationship among miR-193b expression, parts of laboratory index and the response to chemotherapy was analyzed as well. The results showed that miR-193b expression level in acute promyelocytic leukemia (APL) and chronic myeloid leukemia (CML) patients was not lower than that in normal group (P > 0.05). Except for APL, miR-193b expression level in acute myeloid leukemia (AML) patients was lower than that in normal group (P < 0.05). In AML (except for APL) patients, there was no correlation between white blood cell count (P > 0.05), the expression of CD34 (P > 0.05) and miR-193b expression level, but there was negative correlation between chemotherapy response and miR-193b expression level (P < 0.05). It is concluded that miR-193b expression level may be correlated with susceptibility of cells to chemotherapy in AML (except for APL) patients. miR-193b maybe become a new target in AML (except for APL) therapy.

[Expression of MCL-1 and microRNA-29a in extranodal NK/T-cell lymphoma tissue].

This study was aimed to explore the expression level and correlation of MCL-1 and miR-29a in extranodal NK/T-cell lymphoma (ENKTCL) tissue. Maxvision immunohistochemistry technique and real time fluorescent quantitative PCR were used to detect the expression level of MCL-1 and miR-29a in tissue of 20 patients with ENKTCL and 10 patients with proliferative lymphadenitis, respectively. The results showed that the expression of MCL-1 protein were higher in patients with ENKTCL than that in patients with proliferative lymphadenitis, but there were no significant correlation between MCL-1 overexpression and age, sex, Ann Arbor stage and International Prognostic Index (IPI), respectively. Correlation analysis indicated that there was significant negative correlation between miR-29a expression and MCL-1 expression (r = -0.59, P = 0.016). It is concluded that miR-29a may target MCL-1 gene, regulate its expression, then participate in tumorigenesis and development of ENKTCL.

[Enzymatic diagnosis and clinical characteristics of 52 children with mucopolysaccharidosis].

OBJECTIVE: To explore the incidence of various types of mucopolysaccharidosis (MPS) and their clinical characteristics. METHODS: A total of 75 children highly suspected as having MPS underwent quantitative and electrophoretic analysis of urinary glycosaminoglycans (GAGs) and enzymatic analysis of seven types of MPS from January 2009 to December 2011. Fluorescence assay was used to measure the activities of α-L-iduronidase, iduronate-2-sulfatase, α-N-acetylglucosaminidase, galactosamine-6-sulfatase, ß-galactosidase, arylsulfatase B and ß-glucuronidase in the white blood cells. RESULTS: A total of 52 cases were confirmed with MPS based on clinical, radiological, and enzymatic examinations. The 52 cases, with a mean age of 4.0 ± 2.2 years, included 5 cases of MPS I (10%), 20 cases of MPS II (38%), 20 cases of MPS IVA (38%), 6 cases of MPS VI (12%) and 1 case of MPS VII (2%). No MPS IV B cases or MPS IIIB cases were found. Compared with healthy children of the same age, the GAG/Cr ratio was significantly elevated in 50 confirmed cases of MPS (two MPS IVA cases having no increased ratio). All children with increased urinary GAGs had a confirmed diagnosis of MPS. The age of onset was between 1 and 2 years after birth in most cases, and often complicated by hernia and valvular heart disease. Children with MPS I, MPS II, and MPS VI presented with ugly and unsmooth face, short stature, joint stiffness, and limitation of motion, while children with MPS IVA presented with short stature, skeletal dysplasia, and joint laxity. CONCLUSIONS: Type IVA and type II are the most common in MPS cases, followed by type VI and type I. MPS children are characterized by special appearances including ugly and unsmooth facial appearance, short stature and skeletal dysplasia. Quantitative analysis of urinary GAG, as a simple, rapid, and reliable method, is recommended for screening of MPS.

Methylation of secreted frizzled related protein gene in acute leukemia patients in China.

BACKGROUND: DNA methylation of CpG islands within the promoters of specific genes may play roles in tumor initiation and progression. It has been suggested such events may serve as critical check points. METHODS: The present study analyzed the methylation status of CpG islands within the promoters of secreted frizzled-related proteins (SFRPs) in 87 acute leukemia (AL) patients, 20 normal controls, and four AL cell lines. 5-aza-2'- deoxycytidine (5-Aza-CdR), an inhibitor of DNA methylation, was employed to determine its effect on SFRP expression. RESULT: Methylation of at least one SFRP promoter was observed in 69% of the AL patients analyzed. In addition, methylation of all four SFRP promoters was observed in Molt-4, Jurkat, HL60 and NB4 cells. In Jurkat cells, methylation levels of four SFRP promoters decreased in a dose-dependent manner upon treatment with 5-Aza-CdR, which coincided with increased mRNA expression. With increasing 5-Aza-CdR concentrations, the expression of DNA methyltransferases, DNMT3A and DNMT3B, significantly decreased in a dose-dependent manner. CONCLUSION: The present study demonstrated that SFRP gene methylation may be involved in AL progression, with a possible epigenetic mechanism influencing Wnt signaling.

[april mRNA expression in newly diagnosed leukemia patients].

This study was aimed to quantitatively detect the levels of april mRNA expression in leukemia patients so as to provide theoretical basis for the target therapy directing at april in leukemia. Real time fluorescent quantitative PCR was used to detect the relative expression level of april mRNA in newly diagnosed leukemia patients and to analyze the changes of its expression level in various type of leukemia. The results showed that the april mRNA expression level in acute leukemia (AL) patients was significantly higher than that in normal controls, there was statistical difference between them (p < 0.05); april mRNA expression level in acute myeloid leukemia (AML) patients was significantly higher than that in normal controls (p < 0.05) and positively correlated with white blood cell count ≥ 20.0 × 10(9)/L (p < 0.05), but not related with extramedullary infiltration and the expression of CD34. Except for acute promyelocytic leukemia (APL), april mRNA expression level was negatively correlated with sensitivity of patients to chemotherapy. april mRNA expression levels in acute lymphoid leukemia (ALL) and chronic myeloid leukemia (CML) patients were not higher than that in normal controls, there was no statistical difference between them (p > 0.05). It is concluded that april gene overexpression exits in AML patients. APRIL protein produced by AML cells probably plays an important role in abnormal proliferation and drug-resistance of AML cells.

[Expression of Maf-b mRNA in de novo leukemia patients and its clinical significance].

This study was aimed to quantitatively detect the level of maf-b mRNA in leukemia patients and evaluate its clinical significance. Real-time fluorescence quantitative PCR was used to detect the relative expression level of maf-b mRNA. The expression change of maf-b mRNA in various types of leukemia was analyzed. Then, the relationship of maf-b mRNA expression with laboratory index and the response to chemotherapy was analyzed. The results showed that maf-b mRNA expression level in acute myeloid leukemia (AML) patients was lower than that in normal group (p<0.01) and positively correlated with white blood cell count (p<0.01) and the expression of CD34 (p<0.01). There was no correlation between maf-b mRNA expression level and chemotherapy response in AML patients except for acute promyelocytic leukemia (APL). Maf-b mRNA expression levels in acute lymphoid leukemia (ALL) and chronic myeloid leukemia (CML) patients were also lower than that in normal group (p<0.01). It is concluded that there is low expression of maf-b gene in AML patients. Abnormal expression of maf-b correlates with abnormal proliferation of AML cells, which may be a new prognostic factor for AML.

[Effect of triptolide on reversing hypermethylation of apc gene in Jurkat cells and its possible mechanisms].

This study was aimed to investigate the effect of traditional Chinese medicine, Triptolide (TPL) on reversing hypermethylation of antioncogene (apc gene) in acute lymphoblastic leukemia cell line Jurkat in vitro and to explore its mechanisms. The effects of TPL on cell growth, proliferation and cell cycle were detected by growth curve, MTT assay, colony formation test and flow cytometry, respectively. The effect of TPL on apc gene methylation of Jurkat cells was analyzed by nested methylation specific PCR; the expressions of apc gene, dnnt3a, dnmt3b mRNA were measured by RT-PCR; the protein expression of apc gene was detected by Western blot. The results showed that as compared with untreated control cells, the TPL of different concentrations could significantly inhibit growth and proliferation of Jurkat cells in dose-and time-dependent manners with IC50 19.7 ng/ml at 48 hours. All cytosines in CpG dinucleotides in untreated Jurkat cells had no changed, while all cytosines in Jurkat cells treated with TPL had been converted to thymidine suggesting the methylation of apc gene in Jurkat cells. The TPL could reverse hypermethylation of apc gene and induced the mRNA and protein expression of apc gene in dose-dependent manner. It is concluded that the small dose of TPL can obviously suppress the proliferation of Jurkat cells, activate and up-regulate the expression of apc gene through demethylation of apc gene resulting from DNMT and/or direct action, thereby inhibit the proliferation rate of Jurkat cells.

Arsenic trioxide inhibits DNA methyltransferase and restores expression of methylation-silenced CDKN2B/CDKN2A genes in human hematologic malignant cells.

Cyclin-dependent kinase inhibitors CDKN2B and CDKN2A are tumor suppressor genes that are frequently dysregulated in a variety of cancers. Aberrant regulation via DNA hypermethylation causes gene silencing. Arsenic trioxide has been successfully used to treat malignant, hematopoietic diseases and is known to act by induction of apoptosis and inhibition of cellular proliferation. However, arsenic trioxide has been recently reported to act via inhibition of DNA hypermethylation in some solid tumors. The goal of this study was to explore the mechanism of arsenic trioxide induced demethylation of the CDKN2B and CDKN2A promoters in the hematologic malignant cell lines Molt4, MUTZ-1, U937, U266 and CA46. We used bisulphate modification and nested-methylation specific PCR to determine the levels of methylated and unmethylated promoter sequences in untreated and As2O3-treated cells. We used semi-quantitative RT-PCR and immunoblotting to quantify CDKN2B and CDKN2A mRNA and protein levels, respectively. We measured DNMT activity in nuclear extracts of untreated and treated cells using radiolabeled SAM as a methyl donor. The CDKN2B promoter was hypermethylated in Molt4 and MUTZ-1 cells, while the CDKN2A promoter was hypermethylated in U937, U266 and CA46 cells. As2O3 treatment caused demethylation associated with an increase in mRNA levels of the CDKN2B and CDKN2A genes. We also demonstrated a concomitant inhibition in DNMT activity and DNMT mRNA levels in As2O3-treated cells. In summary, As2O3 restored expression levels of tumor suppressor genes in hematologic malignant cells by causing promoter demethylation along with an inhibition of DNMTs 1, 3a and 3b.

[Effect of epigallocatechin-3-galate on human acute monocytic leukemia cell line U937 and its relevant mechanism].

The purpose of this study was to explore the effect of epigallocatechin-3-galate (EGCG) on acute monocytic leukemia cell line U937 and its relevant mechanism. The viability of U937 cells were assayed by SRB method. The cell cycle of U937 cells was analyzed by flow cytometry. The mRNA and protein expression of p16 gene were detected by RT-PCR and Western blot, respectively. Methylation level of U937 cells was analyzed by n-MSP. The mRNA expression of DNA methyltransferase 1 (DNMT1), DNMT3A and DNMT3B genes were analyzed by RT-PCR. The results showed that EGCG could inhibit the growth of U937 cells significantly in dose-and time-dependent manners (r=0.71), and induce the G0/G1 arrest of U937 cells in dose-dependent manner. EGCG could up-regulate the mRNA and protein expression of P16 gene in U937 cells in dose-dependent manner. EGCG could down-regulate the methylation level of p16 gene in U937 cells in dose-dependent manner. EGCG could down-regulate the mRNA expression of DNMT3A, DNMT3B genes, while did not influence the mRNA expression of DNMT1 gene. It is concluded that EGCG can up-regulate the mRNA and protein expression of p16 gene by demethylation or/and by inhibiting DNMT3A and DNMT3B genes, leading, in turn, to G0/G1 arrest and growth inhibition of U937 cells.

[Arsenic trioxide reverses hypermethylation of p16 and activates its transcription in malignant lymphoma cell line CA46].

This study was aimed to investigate the reversing effect of arsenic trioxide (As2O3) on methylation status and the regulatory effect on transcription of malignant lymphoma cell line CA46 p16 gene as well as their possibe mechanisms. The hypermethylated malignant lymphoma cell line CA46 was used as a subject of experiment for studying relation of gene methylation with expression. The effect of As2O3 on the proliferation and viability of CA46 was detected by SRB method, the change of p16 methylation status after exposure to As2O3 was determined by nMSP, the expressions of p16, DNMT1, DNMT3A, DNMT3B mRNA were assayed by RT-PCR, the influence of As2O3 on CA46 cell cycle was analyzed by flow cytometry using analytical method for DNA ploidy. The results showed that the methylation level of p16 gene was obviously reduced after treatment with As2O3 for 72 hours and the hypermethylation of p16 gene was successfully reversed; the expression of p16 gene in untreated (control) group was low while it was enhanced in treated groups; the gray scale ratios of p16 gene to beta-actin in groups treated with As2O3 of concentration 0.5, 1.0 and 2.0 micromol/L were 0.33+/-0.10, 0.57+/-0.11 and 0.67+/-0.09 respectively, exhibiting a significant difference in comparison with 0.73+/-0.13 of positive control (p<0.01); as compared with the untreated group, the expression of DNMT3A and DNMT3B in treated groups was obviously down-regulated in a concentration-dependent manner, while expression of DNMT1 was nearly unchanged; as compared with control, all the 3 different concentrations of As2O3 could inhibit the proliferation of CA46 cells and increase the cell number in G0/G1 phase. It is concluded that the As2O3 may up-regulate the expression of p16 gene, recover the activity of p16 gene, thereby promote the regulatory function on cell cycle resul-ting in arrest of cells in G0/G1 phase and inhibit growth of tumor cells through depressing the expression of DNMT3A and DNMT3B and/or directly reversing the methylation status of p16 gene.