Research Progress of DNA Methylation in Cisplatin Resistance in Lung Cancer / 中国肺癌杂志

As one of the most common malignant tumors, lung cancer poses a serious threat to human life and health. The platinum-based drug cisplatin (DDP) is used as the first-line treatment for lung cancer. The poor prognosis of lung cancer is mostly due to developed resistance to cisplatin, which poses a serious treatment challenge. The mechanism of cisplatin resistance is complex and unclear. Numerous studies have shown that DNA methylation plays a crucial role in the emergence of lung cancer cisplatin resistance. DNA hypermethylation results in the deactivation of numerous drug resistance genes and tumor suppressor genes through a change in chromatin conformation. Finding new therapeutic targets and indicators to predict the therapeutic effect can be aided by elucidating the complex mechanism. In order to discover novel strategies to overcome cisplatin resistance in lung cancer, this paper discusses DNA methylation-mediated cisplatin resistance and offers an overview of current demethylation procedures.
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Prevention and treatment of high-risk acute myeloid leukemia recurrence after allogeneic hematopoietic stem cell transplantation / 器官移植

Acute myeloid leukemia (AML) is a group of highly-heterogeneous clonal diseases. Chemotherapy and hematopoietic stem cell transplantation are considered as effective treatment for AML. For high-risk AML patients, allogeneic hematopoietic stem cell transplantation is an effective therapeutic option. However, some AML patients may still face the problem of disease recurrence after hematopoietic stem cell transplantation. A majority of recurrent patients cannot be effectively treated by chemotherapy or secondary transplantation, which is the main cause of death after allogeneic hematopoietic stem cell transplantation. Therefore, it is of significance to strengthen follow-up of AML patients after allogeneic hematopoietic stem cell transplantation and implement appropriate measures to prevent postoperative recurrence. In this article, the monitoring, drug prevention and cell therapy of recurrence after allogeneic hematopoietic stem cell transplantation in high-risk AML patients were reviewed, aiming to provide reference for improving clinical prognosis of high-risk AML patients undergoing allogeneic hematopoietic stem cell transplantation.

Prognostic predictive value of detecting minimal residual disease in acute myeloid leukemia after 2 courses of hypomethylating agents combined with low-dose induction chemotherapy / 白血病·淋巴瘤

Objective:To explore the prognostic predictive value of detecting minimal residual disease (MRD) after 2 courses of hypomethylating agents (HMA) combined with low-dose induction chemotherapy in patients with acute myeloid leukemia (AML).Methods:The data of 43 newly diagnosed AML patients treated by HMA combined with low-dose induction chemotherapy in Jingjiang People's Hospital of Jiangsu Province from January 2016 to January 2021 were retrospectively analyzed, and the bone marrow MRD levels were detected by multiparametric 10-color flow cytometry (MFC) after 1 course and 2 courses of chemotherapy. Patients were divided into three groups according to MRD levels: the group with negative MRD after 1 course of induction chemotherapy (MRD-1 group), the group with negative MRD after 2 courses of induction chemotherapy (MRD-2 group), and the group without negative MRD after 2 courses of induction chemotherapy (MRD+ group). Kaplan-Meier method was used to draw the progression-free survival (PFS) and overall survival (OS) curves of all patients and each group, and log-rank test was performed to compare them; the influencing factors for OS were analyzed using univariate and multivariate Cox proportional hazards models.Results:Among the 43 patients, 17 patients (39.5%) were in the MRD-1 group, 14 patients (32.6%) were in the MRD-2 group, and 12 patients (27.9%) were in the MRD+ group. There were no statistical differences among the 3 groups in gender, age, hemoglobin level at initial diagnosis, white blood cell count, platelet count, lactate dehydrogenase level, disease subtype, WT1 expression, karyotype, and genetic risk stratification (all P > 0.05). The median follow-up was 15 months (1-67 months). Survival analysis showed a median OS time of 21 months (95% CI 15 months -not reached) in 43 patients and a median PFS time of 12 months (95% CI 9-18 months) in 29 patients included in the PFS analysis; PFS and OS in the MRD-1 and MRD-2 groups were better than those in the MRD+ group (all P < 0.01), and the differences in PFS and OS between the MRD-1 and MRD-2 groups were not statistically significant (both P > 0.05); the median PFS time was 5 months (95% CI 2 months-not reached) in the MRD+ group, the median PFS time was 15 months (95% CI 7 months-not reached) in the MRD-1 group, and the median PFS time was 18 months (95% CI 11 months-not reached) in the MRD-2 group; the median OS time in the MRD+ group was 9 months (95% CI 7 months-not reached), the median OS time was not reached in the MRD-1 group, and the median OS time was 38 months (95% CI 38 months-not reached) in the MRD-2 group. Multivariate Cox regression analysis showed that age ( HR = 1.080, 95% CI 1.004-1.160, P = 0.038), MRD status (MRD-1 vs. MRD+: HR = 0.125, 95% CI 0.031-0.507, P = 0.004; MRD-2 vs. MRD+: HR = 0.146, 95% CI 0.037-0.577, P = 0.006) were independent influencing factors for OS in AML patients. Conclusions:The survival is good in AML patients with MRD negative conversion after both 1 course and 2 courses of HMA combined with low-dose induction chemotherapy, and both are better than that in patients with positive MRD after 2 courses of chemotherapy.

Levels of base excision repair proteins in CD4+ T cells in patients with systemic lupus erythematosus / 中南大学学报(医学版)

OBJECTIVES@#Systemic lupus erythematosus (SLE) is a multi-systemic disease with the unknown pathogenic mechanism. DNA demethylation is involved in SLE pathogenesis. Growth arrest and DNA damage inducible 45 alpha (Gadd45a) takes part in the process of DNA demethylation. Gadd45a is a DNA repair-related protein. This study aims to investigate the expressions of some proteins [including activation-induced cytidine deaminase (AID), thymine DNA glycosylase (TDG), and methyl-CpG-binding domain protein 4 (MBD4)] involving in base excision repair (BER) process in CD4+ T cells in patients with SLE, and to analyze the correlations between the above BER proteins and lupus disease.@*METHODS@#From January 2019 to September 2020, 12 SLE patients and 12 healthy controls were recruited from Second Xiangya Hospital of Central South University. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque density gradient centrifugation and then CD4+ T cells were isolated via positive selection using Miltenyi beads. We measured the messenger RNA (mRNA) and protein expressions of AID, TDG, and MBD4 by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively.@*RESULTS@#In contrast to controls, in SLE CD4+ T cells, the mRNA and protein expressions of AID were elevated (P=0.003, P=0.022, respectively); TDG protein expression was increased (P=0.017); and MBD4 protein level was reduced (P<0.001). No visible distinctions was found in the mRNA expressions of either TDG or MBD4 between the 2 groups (both P>0.05). The mRNA and protein expressions of AID and the protein levels of TDG were positively correlated with SLE disease activity index (SLEDAI). And the mRNA and protein expressions of MBD4 were negatively correlated with SLEDAI.@*CONCLUSIONS@#In SLE CD4+ T cells, the increased expressions of AID and TDG and the decreased MBD4 expression may participate in SLE pathogenic mechanism.

Detailed resume of RNA m6A demethylases

N6-Methyladenosine (m6A) is the most abundant internal modification in eukaryotic mRNA, playing critical role in various bioprocesses. Like other epigenetic modifications, m6A modification can be catalyzed by the methyltransferase complex and erased dynamically to maintain cells homeostasis. Up to now, only two m6A demethylases have been reported, fat mass and obesity-associated protein (FTO) and alkylation protein AlkB homolog 5 (ALKBH5), involving in a wide range of mRNA biological progress, including mRNA shearing, export, metabolism and stability. Furthermore, they participate in many significantly biological signaling pathway, and contribute to the progress and development of cancer along with other diseases. In this review, we focus on the studies about structure, inhibitors development and biological function of FTO and ALKBH5.

The function and regulation of TET2 in innate immunity and inflammation

TET2, a member of ten-eleven translocation (TET) family as α-ketoglutarate- and Fe

Effects of 5-azacytidine on DNA methylation and index components in patchouliol-type Pogostemon cablin / 中国中药杂志

This study aims to explore the relationship of DNA methylation with the contents of the index components as well as the growth and development of Pogostemon cablin. The demethylation reagent 5-azacytidine(5-azaC) was used to treat the tissue culture seedlings of patchouliol-type P. cablin. High performance liquid chromatography was employed to evaluate the changes of DNA methy-lation in P. cablin, and GC-MS to detect the contents of index components in P.cablin. The agronomic characters of P.cablin were measured using the common methods. The results showcased that DNA methylation of P.cablin was significantly reduced by 5-azaC in a concentration-dependent manner. Thirty days after treatment with 5-azaC at different concentrations, the content of patchouli alcohol changed slightly; compared with that in the control group, the content of pogostone in 50 μmol·L~(-1) and 100 μmol·L~(-1) 5-azaC groups was significantly up-regulated. The 100 μmol·L~(-1) 5-azaC group had the largest differences in contents of pogostone and patchouli alcohol compared with the control group, followed by the 50 μmol·L~(-1) 5-azaC group. Ninety days after disinhibition, the content of pogostone in the treatment group was significantly increased and the content of patchouli alcohol was significantly decreased. In addition, 5-azaC significantly inhibited the growth and development of P.cablin in a dose-dependent manner. These results indicate that DNA methylation regulates the biosynthesis of the index components in patchouliol-type P.cablin and proper demethylation can directly promote the synthesis of pogostone and indirectly affect the accumulation of patchouli alcohol.

Effects of different treatment regimens before allogeneic-hematopoietic stem cell transplantation on the outcome after transplantation of patients with myelodysplastic syndrome: a meta analysis / 白血病·淋巴瘤

Objective:To systematically evaluate effects of different treatment schemes before allogeneic-hematopoietic stem cell transplantation (allo-HSCT) on the long-term relapse and survival of patients with myelodysplastic syndrome (MDS) after transplantation.Methods:The related literatures were searched from databases of Ovid, Cochrane Library, PubMed, Embase, CNKI, VIP, WanFang and CBM from inception to December 2019. And then 2 reviewers independently extracted data, assessed methodological quality and crosschecked on the included literatures. According to the treatment methods, the cases included in the literatures were divided into demethylation drug (decitabine or azacytidine) treatment (demethylation treatment group) and traditional treatment regimen (including chemotherapy and support treatment) (traditional treatment group). RevMan 5.3 software was used to analyze overall survival (OS), recurrence, non-relapse mortality (NRM) and relapse free survival (RFS).Results:Finally, 10 articles were included. The results of meta-analysis showed that in the traditional treatment group, the differences of 3-year OS rate [44.6% (146/327) vs. 35.5%(138/389); OR = 0.93, 95% CI 0.38-2.27, P = 0.87], the recurrence rate [32.4% (106/327) vs. 37.3% (145/389); OR = 1.00, 95% CI 0.49-2.05, P = 0.99], NRM [26.3% (86/327) vs. 27.0% (105/389); OR = 1.05, 95% CI 0.75-1.49, P = 0.77], RFS rate [9.2% (30/327) vs. 12.6% (49/389); OR = 0.74, 95% CI 0.26-2.10, P = 0.57] between the chemotherapy group and the support treatment group were not statistically significant. The differences of 3-year OS rate [40.7% (165/405) vs. 45.9% (290/632); OR = 0.98, 95% CI 0.71-1.36, P = 0.28], recurrence rate [32.6% (132/405) vs. 38.3% (242/632); OR = 1.05, 95% CI 0.79-2.05, P = 0.25], NRM [27.2% (110/405) vs. 24.8% (157/632); OR = 0.81, 95% CI 0.59-1.11, P = 0.68], RFS rate [46.7% (189/405) vs. 42.2 (267/632); OR = 0.84, 95% CI 0.63-1.12, P = 0.85] between demethylation treatment group and traditional treatment group were not statistically significant. There were no significant differences in 3-year OS rate, recurrence rate, NRM and RFS rate between demethylation treatment group and chemotherapy group, demethylation treatment group and support treatment group (all P > 0.05). Conclusion:Different treatment regimens before allo-HSCT have no significant effect on survival or recurrence after transplantation for patients with MDS.

Clinical characteristics of acute myeloid leukemia patients with TET2 gene mutation and effects of TET2 mutation on therapeutic efficacy and prognosis / 白血病·淋巴瘤

Objective:To investigate clinical features of adult patients with acute myeloid leukemia (AML) with TET2 gene mutation and effects of TET2 mutation on therapeutic efficacy and prognosis.Methods:A total of 123 newly diagnosed adult AML patients (except for acute promyelocytic leukemia) admitted to Jining No.1 People's Hospital from March 2017 to April 2021 were selected. Mutations of 24 AML-related genes including TET2 mutation were detected by using second-generation sequencing technology. Patients were divided into two groups according to the presence of TET2 mutation: TET2 mutation group and TET2 wild type group. The differences in clinicopathological characteristics, short-term efficacy and survival of both groups were compared.Results:Among 123 patients, TET2 mutation was detected in 28 cases (22.8%). Compared with TET2 wild type group, the patients were older [(59±15) years vs.(49±16) years, t = 2.984, P = 0.003], French-American-British (FAB) Corporative Group M 4 and M 5 subtypes were more common [75.0% (21/28) vs. 51.6% (49/95), χ2 = 4.838, P = 0.028], and the positive rate of CD34 in AML patients was lower in TET2 mutation group [46.4% (13/28) vs.72.6% (69/95), χ2 = 6.685, P = 0.010]. Moreover, TET2 mutation was more likely to be accompanied with ZRSR2 mutation [10.7% (3/28) vs. 1.1% (1/95), P = 0.037] and NPM1 mutation [35.7% (10/28) vs.17.9% (17/95), χ2 = 4.008, P = 0.045], but less likely to be accompanied with IDH1/2 mutation [0 vs.17.9% (17/95), P = 0.012]. However, there were no statistically significant differences in gender, peripheral blood leukocyte count at initial diagnosis, hemoglobin level, platelet count, bone marrow blasts ratio, cytogenetics and the European LeukemiaNet (ELN) risk stratification between the two groups (all P>0.05). In addition, there were no significant differences in the overall response rate (ORR) of 1 cycle chemotherapy [75.0% (12/16) vs. 66.7% (42/63), χ2 = 0.410, P = 0.522] and demethylation therapy [66.7% (4/6) vs. 44.4% (8/18), P = 0.640]. The difference in overall survival (OS) of both groups was not statistically significant [median OS time: 23 months (95% CI 5-41 months) vs. 35 months (95% CI 18-52 months, P = 0.498]. Conclusions:In AML patients, TET2 mutation is associated with advanced age, M 4 and M 5 subtypes, and low expression of CD34 on AML blasts. TET2 mutation is commonly accompanied by ZRSR2 and NPM1 mutation, but not IDH1 or IDH2 mutation. TET2 mutation may have no significant effects on therapeutic efficacy and survival in the whole cohort of AML patients without risk stratification.

Expression and characterization of a novel cytochrome P450 enzyme from Variovorax paradoxus S110 / 生物工程学报

Cytochrome P450 monooxygenases as powerful biocatalysts catalyze a wide range of chemical reactions to facilitate exogenous substances metabolism and biosynthesis of natural products. In order to explore new catalytic reactions and increase the number of P450 biocatalysts used in synthetic biology, a new self-sufficient cytochrome P450 monooxygenase (P450(VpMO)), belongs to CYP116B class, was mined from Variovorax paradoxus S110 genome and expressed in Escherichia coli. Based on characterization of the enzymatic properties, it shows that the optimal pH and temperature for P450(VpMO) reaction activity are 8.0 and 45 °C, respectively. P450(VpMO) is relatively stable at temperatures below 35 °C. The Km and kcat of P450(VpMO) toward 4-Methoxyacetophenone are 0.458 mmol/L and 2.438 min⁻¹, respectively. Importantly, P450(VpMO) was able to catalyze the demethylation reaction for a range of substrates containing methoxy group. Its demethylation reactivity is reasonably better than other P450s belongs to CYP116B class, particularly, for 4-methoxyacetophenone with a great conversion efficiency at 91%, showing that P450(VpMO) could be used as a great biocatalyst candidate for further analysis.

Effect of Qinghuang Powder () Combined with Bupi Yishen Decoction () in Treating Patients with Refractory Cytopenia with Multilineage Dysplasia through Regulating DNA Methylation / 中国结合医学杂志

OBJECTIVE@#To explore the effect of Qinghuang Powder (QHP,()combined with Bupi Yishen Decoction (BPYS, ) on myelodysplastic syndromes (MDS) patients with refractory cytopenia with multilineage dysplasia (RCMD) and determine the change of DNA methylation in MDS-RCMD patients after the treatment of Chinese medicine formula.@*METHODS@#All 308 MDS-RCMD patients were treated with QHP combined with BPYS for 2 months at least, absolute neutrophil count (ANC), hemoglobin (Hb), platelets (PLT), primitive bone marrow cells and chromosome karyotype were chosen as the main evaluation indexes to analyze the treatment effect according to criteria from the MDS International Working Group. Then 43 bone marrow samples from 15 MDS-RCMD patients and 28 healthy donors were obtained for the examination of DNA methylation. Gene Ontology (GO) and Pathway analysis were applied to analyze the methylation data.@*RESULTS@#The overall MDS response rate to QHP was 61.68% (190/360) including hematologic improvement-neutrophil (HI-N) or hematologic improvement-erythroid (HI-E) or hematologic improvement-platelet (HI-P). Patients with anemia had a better response rate than patients with neutropenia or thrombocypenia (55.88% vs 31.54% or 55.88% vs. 36.9%). The DNA methylation microarray analysis disclosed that 4,257 hypermethylated genes were demethylated upon the treatment with QHP and BPYS. GO analysis and Pathway analysis showed that these demethylated genes were involved in a lot of tumor-related pathways and functions.@*CONCLUSIONS@#QHP combined with BPYS could effectively treat MDS-RCMD patients through hematologic improvement (HI-N, HI-P or HI-E) and PLT and RBC transfusion independence due to the demethylation, thereby providing another choice for the treatment of patients with MDS-RCMD.

Research progress of histone lysine demethylase KDM5A in tumors / 肿瘤

Histone methylation is one of the important research contents of epigenetics. Methylation of histones is dynamically regulated by histone methylase and histone demethylase. Histone lysine (K) demethylase 5A (KDM5A) is an important member in the histone demethylase family, which can specifically remove the dimethyl and trimethyl from the 4th lysine of histone H3 (H3K4me2/3), thus mediating gene silencing and regulating cell function. KDM5A can directly or indirectly maintain tumor cell dryness, inhibit cell metabolism and differentiation, promote tumor cell proliferation, metastasis and drug resistance, so it is closely related to the occurrence, development and drug resistance of various tumors. KDM5A may become a new potential target for the diagnosis and treatment of tumors.

Role of TET3-induced DNA demethylation in methane-induced up-regulation of Nrf2 expression in rat spinal cord neurons subjected to oxygen-glucose deprivation and restoration injury / 中华麻醉学杂志

Objective To evaluate the role of TET3-induced DNA demethylation in methane-in-duced up-regulation of nuclear factor-erythroid 2-related factor 2 ( Nrf2) expression in rat spinal cord neu-rons subjected to oxygen-glucose deprivation and restoration ( OGD∕R) injury. Methods The primarily cultured spinal cord neurons of rats were seeded in 6-well plates at a density of 1×105 cells∕ml and divided into 5 groups ( n=48 each) using a random number table method: control group ( group C) , group OGD∕R, methane group (group M), methane plus TET3-siRNA group (group M+siTET3) and methane plus negative siRNA group (group M+siCon). The medium was replaced with glucose- and serum-free Earle's salt solution, and the neurons were exposed to 5％ CO2-95％N2 in an incubator for 2 h followed by routine culture to establish the model of OGD∕R. In group M, 200μl methane-saturated saline ( final concentration of methane 1. 8 mmol∕L) was added at oxygen-glucose restoration. TET3-siRNA 100 pmol∕L and negative siRNA 100 pmol∕L were added at 24 h before oxygen-glucose restoration to perform transfection in M+siTET3 and M+siCon groups, respectively. At 12 h of oxygen-glucose restoration, the neuronal survival rate, release rate of lactic dehydrogenase ( LDH) and apoptotic rate of neurons were measured, and the ex-pression of TET3 and Nrf2 protein and mRNA was detected by Western blot and fluorescent quantitative re-al-time polymerase chain reaction, respectively, and contents of superoxide dismutase (SOD), catalase ( CAT) and malonaldehyde ( MDA) were measured by enzyme-linked immunosorbent assay. Neuronal DNA was extracted for determination of methylation and hydroxymethylation rates of DNA ( by enzyme-linked im-munosorbent assay) and methylation of CpG island in Nrf2 gene promoter ( by fluorescent real-time methyla-tion specific polymerase chain reaction). Results Compared with group C, the survival rate of neurons was significantly decreased, the release rate of LDH and apoptotic rate were increased in group OGD∕R ( P<0. 01) . Compared with OGD∕R, the survival rate of neurons was significantly increased, the release rate of LDH and apoptotic rate were decreased, the expression of TET3 and Nrf2 protein and mRNA was up-regula-ted, DNA hydroxymethylation rate and contents of SOD and CAT were increased, and the DNA and Nrf2 promoter methylation rates and MDA content were decreased in group M ( P<0. 05 or 0. 01) . Compared with group M, the neuronal survival rate was significantly decreased, the release rate of LDH and apoptotic rate were increased, the expression of TET3 and Nrf2 protein and mRNA was down-regulated, the DNA hydroxymethylation rate and contents of SOD and CAT were decreased, and the DNA and Nrf2 promoter methylation rates and MDA content were increased in group M+siTET3 ( P<0. 05 or 0. 01) , and no signifi-cant change was found in the parameters mentioned above in group M+siCon ( P>0. 05) . Conclusion The mechanism by which methane up-regulates Nrf2 expression in rat spinal cord neurons subjected to OGD∕R injury is related to activating TET3 and promoting DNA demethylation in Nrf2 promoter.

YTH Domain: A Family of N-methyladenosine (mA) Readers / 基因组蛋白质组与生物信息学报·英文版

Like protein and DNA, different types of RNA molecules undergo various modifications. Accumulating evidence suggests that these RNA modifications serve as sophisticated codes to mediate RNA behaviors and many important biological functions. N-methyladenosine (mA) is the most abundant internal RNA modification found in a variety of eukaryotic RNAs, including but not limited to mRNAs, tRNAs, rRNAs, and long non-coding RNAs (lncRNAs). In mammalian cells, mA can be incorporated by a methyltransferase complex and removed by demethylases, which ensures that the mA modification is reversible and dynamic. Moreover, mA is recognized by the YT521-B homology (YTH) domain-containing proteins, which subsequently direct different complexes to regulate RNA signaling pathways, such as RNA metabolism, RNA splicing, RNA folding, and protein translation. Herein, we summarize the recent progresses made in understanding the molecular mechanisms underlying the mA recognition by YTH domain-containing proteins, which would shed new light on mA-specific recognition and provide clues to the future identification of reader proteins of many other RNA modifications.

Construction and identification of LSD1 overexpression and demethylation disfunction plasmids / 国际生物医学工程杂志

Objective To construct rat lysine-specific histone demethylase 1 (LSD1) overexpression plasmids and LSD 1 demethylation fragment disfunction plasmids,and to evaluate their expression levels in HEK293T cells.Methods LSD1 fragments were amplified by PCR,and LSD1 demethylation disfunction fragments were amplified by overlap PCR.Rat-specific LSD1 overexpression plasmids and LSD1 demethylation disfunction plasmids were constructed and verified by agarose gel electrophoresis and sequencing.HEK293T cells were infected using the validated recombinant plasmids and blank vectors,and the stably expressed cells were selected by puromycin.The expression of LSD1 in the stably expressed HEK293T was detected by Western Blot and real-time quantitative PCR.Results The results of agarose gel electrophoresis and sequencing showed that LSD1 overexpression plasmids and LSD1 demethylation disfunction plasmids were successfully constructed.The Western Blot and real-time quantitative PCR results showed that compared with the blank group,the relative expression of LSD1 and mRNA in the LSD1 overexpression group and the LSD1 demethylation disfunction group were up-regulated,and the differences were statistically significant(all P＜0.01).Conclusions The constructed LSD1 overexpression plasmids and LSD1 demethylationi disfunction plasmids can achieve overexpression of LSD1 gene in HEK293T cells.This paper lays a foundation for further study of the relationship between LSD 1 gene and related diseases and demethylation of LSD 1.

The relationship and clinical significance of TMS1,MBD2 and DNMT1 in hepatocellular carcinoma / 国际检验医学杂志

Objective To observe the expression of methylation related silent target 1 (TMS1),DNA methyltransferase 1 (DNM T1)and methyl CpG binding domain protein 2 (MBD2),and the methylation of the TMS1 gene promoter,in hepatocellular carcinoma (HCC),and to investigate the relationship between the three genes and the clinical diagnostic significance.Methods The protein expression of TMS1,MBD2 and DNMT1 in 48 cases of HCC tissues and 48 case of normal tissues were detected by immunohistochemistry (IHC);the methylation status of TMS1 gene in serum of 34 patients with HCC,26 patients with hepatitis B and 23 healthy controls were analyzed by methylation specific polymerase chain reaction (MSP).Results The posi-tive rates of TMS1 expression in HCC tissues and normal tissues were 26.08% and 97.92%,respectively;the positive rate of MBD2 expression were 18.75% and 80.00%,respectively;the positive rate of expression were 77.08 % and 32.25%,respectively.The positive rates of TMS1 and MBD2 expression in HCC tissues were significantly lower than those in the normal tissues,and DNM T1 expression was higer than that in the normal tissues,and the difference was statistically significant (P<0.05).The expression TMS1 and MBD2 were neg-atively correlated with DNMT1 (P< 0.05).TMS1 protein expression was associated with TNM stage and tumor differentiation but not associated with the age and gender (P<0.05).The methylation detection rates of TMS1 gene in HCC group,hepatitis B group and healthy group were 70.6%,50.0% and 0.0%,respective-ly,and the differences were statistically significant (P<0.05).The methylation detection rate of HCC group and hepatitis b group was obviously higher than the normal group,which aggravated with the increase of tumor grade and stage.Conclusion TMS1 hypermethylation may be an early event in the carcinogenesis of liver tissue,the abnormal expression of TMS1,MBD2 and DNM T1 proteins plays an important role in the oc-currence and the development of HCC,w hich can be used as new molecular markers for the early diagnosis and prognosis of HCC,and become the new targets for the treatment of HCC.

Effect of HPV-16 LCR internal meCpG site demethylation on HPV positive head and neck tumor cells / 中国耳鼻咽喉头颈外科

OBJECTIVE To investigate the effect of HPV-16 LCR internal meCpG site demethylation on HPV positive head and neck tumor cells. METHODS After 5-azoxy-2-deoxycytidine(5-Aza-2'-dc) demethylation treatment, the methylation status of CpG sites in the long regulatory region of UM-SCC47, CaSki and SiHa tumor cells was detected by BSP assay. The expression of HPV 16 E6 and E7 mRNA was detected by real-time PCR. The effects of 5-Aza-2'-dc on cell growth and apoptosis were detected. Using Lipolnfectamine RNAiMAX to transfect siRNA, the expression of E6 and E7 was silenced, and then the changes of biological state of cell proliferate inhibition, apoptosis and cycle were detected. RESULTS Cells treated with 5-Aza-2, -dc at a concentration of 0.5 μmol/L for 96 h had the best demethylation. After demethylation of 5-Aza-2'-dc, the expression levels of E6 and E7 mRNA in UM-SCC47 cells and CaSki cells were significantly decreased compared with the control group(t=1.356, 2.623, P=0.031, 0.005; t=1.798, 2.015, P=0.011, 0.009). The expression levels of E6 and E7 mRNA in SiHa cells were not significantly differences compared with the control group(t=1.591, 1.153, P=1.105, 0.753); After treated with 5-Aza-2'-dc, cells proliferation were significantly inhibited. After E6 and E7 expression was silenced, the growth of all three kinds of cells was inhibited. The apoptosis of all three kinds of cells were significantly increased, cells arrested in S phase and G2/M phase were significantly increased compared with the control groups, the difference were statistically significant. CONCLUSION After demethylation, the response of HPV-16 positive tumor cells with different methylation status in the long regulatory region is completely different, indicating that in the HPV-16 positive tumors, there may be some information about the methylation status in the long regulatory region Different tumor characteristics and mechanism of cancer.

Preliminary Study on the Effect of 5-Aza-CdR on the Demethylation of RNF180 in Prostate Cancer Cell Line DU145 / 中国医科大学学报

Objective To investigate the mechanism and cause of the inactivation of tumor suppressor gene RNF180 in prostate cancer cell line by observing the effect of 5-Aza-CdR on the RNF180 gene in prostate cancer cell line DU145. Methods MTT method was adopted to study the effect of 5-Aza-CdR(0,1,2,5,10,15 and 20μmoI/L)on the proliferation of prostate cancer cells. Western blotting,real-time PCR,and methyla-tion specific PCR(MSP)were separately used to detect the expression of RNF180 in prostate cancer cells before and after the treatment of the most suitable drug concentration(5μmoI/L). Results In a certain range,the effect of 5-Aza-CdR on the proliferation of prostate cancer cell line DU145 was increased with the increase of drug concentration and the time of drug treatment(P<0.05). After the treatment of the most suitable drug concentration,the protein and mRNA expression of RNF180 in prostate cancer cells was significantly increased(P<0.05),but the methyla-tion of the promoter region was obviously decreased. Conclusion 5-Aza-CdR can reverse the methylation status of RNF180 gene in DU145 pros-tate cancer cell line,and relieve the silencing status of RNF180gene expression.

Tet: novel anti-tumor drug target based on DNA demethylation / 上海交通大学学报（医学版）

Tet (ten-eleven translocation) proteins belong to α-ketoglutaric acid (α-KG or 2-OG) and Fe2+ dependent dioxygenases. Tets are found to be involved in the unique mammalian DNA active demethylation process by specifically oxidizing the methyl group of 5-methylcytosine (5mC) in mammalian genome, and play critical roles in gene regulation in early embryonic development and stem cell differentiation via regulating the dynamic balance distribution of 5mC. Abnormal expression and function of Tets are closely associated with various hematological malignances, including myelodysplastic syndrome, chronic myelomonocytic leukemia, and acute lymphoblastic leukemia, as well as solid tumors. Hence, Tets and Tets-mediated DNA demethylation are novel anti-tumor drug targets. Investigation of biological function and catalytic mechanism of Tets is helpful for further understanding mechanisms of tumor incidence and development relevant to DNA demethylation pathway and can provide reference for developing new anti-tumor targeted drugs.

Progress in TET enzymes and their intermediate products / 中国病理生理杂志

DNA methylation is an important epigenetic modification mode , which plays a crucial role in gene expression , genome stability and development .DNA methylation is catalyzed and maintained in cell proliferation by the family of DNA methyltransferases.The ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC).Here, we briefly describe the TET enzymes and their role in cancer , and the distribution , the role and detection method of those three oxidation products of cytosine in genome .