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N 6-methyladenosine (m 6A) is the most abundant epigenetic modification in eukaryotic messenger RNA (mRNA), which could be catalyzed by m 6A methyltransferase (Writers), recognized by methylation recognition enzymes (Readers), and removed by demethylase (Erasers). RNA splicing, translation, and stability could be modulated by m 6A methylation modification. The m 6A methylation modification is involved in the biological regulation of a variety of important functional genes in cellular activities. Importantly, abnormal m 6A modification affects the occurrence, development, metastasis and recurrence of tumors. Ionizing radiation can affect the level of m 6A and m 6A methylation-related enzymes. Recently, m 6A methylation is reported to regulate the efficacy of tumor radiotherapy by affecting DNA damage and radiosensitivity of tumor cells. In addition, ionizing radiation can also affect the level of m 6A modification in normal cells to regulate the progress of radiation-induced injuries. This review summarizes the research progress on the roles of m 6A methylation in tumor radiosensitivity and radiation-induced injuries, with the aim of providing novel strategies for the development of clinical tumor radiosensitizers and radioprotective agents.
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Background Chemical modification of RNA is a recent hotspot in the field of epigenetics, but the specific mechanism of chemical modification of RNA in aluminum neurotoxicity has not been fully reported. Objective To investigate the alterations of fat mass and obesity-associated protein (FTO), that demethylates N6-methyladenosine (m6A), and brain-derived neurotrophic factor (BDNF) in different brain regions of rats and rat adrenal pheochromocytoma differentiated cells (PC12 cells) following aluminum exposure. Methods Animal experiment: Twenty-four healthy male SD rats were randomly divided into a control group (normal saline) and 10, 20, and 40 μmol·kg−1 exposure groups according to body weight, with 6 rats in each group. Maltol aluminum [Al(mal)3] was injected intraperitoneally every other day for 3 months. Cell experiment: PC12 cells were divided into a control group and 100, 200, and 400 μmol·L−1 exposure groups exposed to Al(mal)3 for 24 h. After exposure, the learning and memory ability of rats was measured by water maze experiment, and the protein expression levels of FTO and BDNF in rat cortex (n=6) and hippocampus (n=6) samples as well as in PC12 cells (n=5) were determined by Western blotting. Results The results of water maze test showed that the escape latency of the 40 μmol·kg−1Al(mal)3 group was higher than those of the control group, the 10 μmol·kg−1Al(mal)3 group, and the 20 μmol·kg−1Al(mal)3 group on day 3, 4, and 5 of training (P<0.05). The retention time of the target quadrant of the 40 μmol·kg−1Al(mal)3 group was also reduced compared with that of the control group (P<0.05), indicating that aluminum exposure damaged the learning and memory ability of the rats. The Western blotting results showed that in the cortex, compared with the control group, the protein expression levels of FTO and BDNF in the aluminum treated groups were decreased (P<0.05). In the hippocampus, compared with the control group, the protein expression levels of FTO and BDNF in the 20 μmol·kg−1 and the 40 μmol·kg−1Al(mal)3 groups were decreased (P<0.05). In PC12 cells, compared with the control group, the protein expression levels of FTO and BDNF in the aluminum treated groups were decreased (P<0.05). Conclusion Aluminum-induced learning and memory impairment is related to a simultaneous reduction of FTO and BDNF protein expressions, suggesting that m6A methylation may be involved.
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Background Environmental pollutants can affect N6-methyladenosine (m6A) level in the body, but the change of m6A level in kidney after being exposed to cadmium (Cd) and the molecular mechanism of renal injury need to be further studied. Objective To analyze the associations of m6A modification and methyltransferases/demethylases with microRNA-21 (miR-21) and transforming growth factor- β1 (TGF - β1) in kidney of rats exposed to Cd. Methods Twenty-four SPF male SD rats were divided into 4 groups, with 6 rats in each group, and were exposed to Cd by subcutaneous injection of 2.0, 1.0, and 0.5 mg·kg−1 cadmium chloride (CdCl2) and equal volume of normal saline for 2 weeks, 7 d a week, respectively. The levels of N-acetyl-β-D-glucosidase (UNAG) and albumin (UALB) in urine, and the levels of m6A methylation and TGF-β1 in kidney were detected by enzyme-linked immunosorbent assay (ELISA). The level of blood urea nitrogen (BUN) was measured by urease method. The levels of renal oxidative stress indicators such as malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were detected by total bile acid method, water-soluble tetrazolium asssay, and colorimetric method respectively. The relative levels of TGF-β1, methyltransferases, and demethylases in kidney were measured by reverse transcription-polymerase chain reaction. The expression of miR-21 in kidney was detected by fluorescent quantitative polymerase chain reaction. Results After 2 weeks of exposure to Cd, the body weights of rats in the 2.0 and 1.0 mg·kg−1 cadmium chloride groups decreased, and the ratio of kidney/body weight and the levels of BUN, UNAG, and TGF-β1 mRNA and protein increased in the 2.0 mg·kg−1 cadmium chloride group (P<0.05). The expression levels of m6A modification, methyltransferases METTL3, METTL14, Wilms’ tumor 1-associated protein (WTAP), and miR-21 were increased both in the 2.0 and 1.0 mg·kg−1 cadmium chloride groups, with significant differences compared with the control group (P<0.05). The results of correlation analysis showed that the m6A modification level was negatively correlated with SOD (r=−0.4489, P<0.05) and GSH-Px (r=−0.4874, P<0.05), METTL3 was negatively correlated with MDA (r=−0.5158, P<0.05), while there was a positive correlation between FTO and GSH-Px (r=0.4802, P<0.05). In addition, miR-21 was positively correlated with METTL3 (r=0.7491), METTL14 (r=0.6157), and WTAP (r=0.6660) (P<0.05), TGF-β1 was positively correlated with METTL3 (r=0.5025, P<0.05) but negatively correlated with FTO (r=−0.5634, P<0.05) . Conclusion Cd can induce m6A methylation and up-regulation of METTL3, METTL14, WTAP, and miR-21 expression levels in rat kidney tissues, indicating that m6A and miR-21 may be associated with Cd-induced renal fibrosis.
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Background Trichloroethylene (TCE) can enter human body through biological accumulation of polluted water or air, resulting in health hazards. The most commonly involved organs are the liver. Objective To observe potential polarization of M1 Kupffer cells (KCs) in mice liver exposed to TCE orally, and to investigate the relationship between histones lysin demethylase JMJD3 and M1 KCs polarization. Methods A total of 72 SPF BALB/c mice aged 6 to 8 weeks were randomly divided into a blank control group (n=18), a vehicle control group (n=18), a 2.5 mg·mL−1 TCE group (n=18), and a 5.0 mg·mL−1 TCE group (n=18) after adaptive feed for one week. A TCE transoral exposure model was established after eight weeks of administration according to previous research of the research group. In the 2nd, 4th, and 8th weeks, the mice were sacrificed and liver tissue samples were collected. Western blotting was used to detect the expression level of JMJD3 in the liver tissue samples. Immunofluorescence was used to co-locate the macrophage marker F4/80 and the surface marker CD11c of M1 macrophages. Immunohistochemistry was used to detect the expressions of CD16/32, a marker of M1 macrophages, and TNF-α, an inflammatory factor of M1 macrophages in mouse liver. Results In the 2nd, 4th, and 8th weeks, the mice in each group were generally in good condition, and no individual died due to TCE. There was no statistically significant difference in the amount of water consumed by each group, nor in the body weight gain and the liver coefficient of mice at each time point (P>0.05). The results of Western blotting analysis showed that there was no statistically significant difference in JMJD3 protein expression level between the blank control group and the vehicle control group at each time point, the expression levels of JMJD3 protein in the 2.5 mg·mL−1 TCE group and the 5.0 mg·mL−1 TCE group were higher than that in the control group , and the expression level of JMJD3 protein in the 5.0 mg·mL−1 TCE group was higher than that in the 2.5 mg·mL−1 TCE group (P<0.05). The results of immunofluorescence co-localization showed that the expressions of F4/80 and CD11c were low in the blank control group and the vehicle control group, while the expressions of F4/80 and CD11c were increased in the 2.5 mg·mL−1 and the 5.0 mg·mL−1 TCE groups. The results of immunohistochemistry showed that the expressions of CD16/32 and TNF-α in the blank control group and the vehicle control group were low, and there were large deposits in the 2.5 mg·mL−1 TCE group and the 5.0 mg·mL−1 TCE group. Conclusion The polarization of M1 KCs and the expression of proinflammatory factors may be related to an increased expression level of JMJD3 induced by oral TCE exposure.
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@#N6-methyladenine (m6A) modification, the most abundant and dynamic chemical modification on messenger RNA, plays an essential role in physiological and pathological progress.Recent studies have found that tumor progression can be affected by altering the m6A modification level of target genes. Therefore, small molecule targeted m6A demethylase can be used as a new anti-tumor strategy.This review focuses on the regulatory mechanism of m6A demethylases, including fat mass and obesity-associated protein (FTO) and AlkB homlog 5 (ALKBH5), as well as their biological functions in tumors, and summarizes the research progress of their small molecule inhibitors.
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Lysine specific demethylase 1 (LSD1), a transcriptional corepressor or coactivator that serves as a demethylase of histone 3 lysine 4 and 9, has become a potential therapeutic target for cancer therapy. LSD1 mediates many cellular signaling pathways and regulates cancer cell proliferation, invasion, migration, and differentiation. Recent research has focused on the exploration of its pharmacological inhibitors. Natural products are a major source of compounds with abundant scaffold diversity and structural complexity, which have made a major contribution to drug discovery, particularly anticancer agents. In this review, we briefly highlight recent advances in natural LSD1 inhibitors over the past decade. We present a comprehensive review on their discovery and identification process, natural plant sources, chemical structures, anticancer effects, and structure-activity relationships, and finally provide our perspective on the development of novel natural LSD1 inhibitors for cancer therapy.
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Humans , Antineoplastic Agents/therapeutic use , Enzyme Inhibitors/therapeutic use , Histone Demethylases/metabolism , Lysine/therapeutic use , Neoplasms/drug therapyABSTRACT
Colorectal cancer is a malignant tumor occurring in the colon or rectum, which has a high incidence rate. In order to improve the prognosis of colorectal cancer, the pathogenesis of colorectal cancer still needs to be further clarified. Epigenetics can directly affect the progression and metastasis of colorectal cancer, and histone methylation is an important means of histone modification, which can regulate the transcriptional activation and inhibition of downstream genes. A large number of studies have confirmed the effects of histone methylation on the progression of colorectal cancer, and inhibitors of related methylation and demethylation may play a role as potential therapeutic drugs for colorectal cancer. In this article, the colorectal cancer and its related methylation regulation were introduced, the types of histone methylation modifications and their regulation were summarized, and the regulation of histone methyltransferases and demethylases involved in the progression of colorectal cancer was demonstrated. In addition, the potential significance of histone methylation inhibitors for the treatment of colorectal cancer was summarized, and the possibility of related inhibitors as treatment drugs for colorectal cancer was explored.
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Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and has relatively high incidence and mortality rates. Abnormal modification of N6-methyladenosine (m6A) may promote the development and progression of HCC. This article describes the structure and function of m6A and summarizes the mechanism of action of methylase complexes which decide the function of m6A in HCC, including methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers). It is pointed out that more in-depth studies are needed to clarify the diverse and specific role of methylase complexes in HCC, so as to help them become the new targets for the prevention and treatment of HCC in the future.
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As a top leading cause of cancer death in many countries, colorectal cancer (CRC) has drawn increasing attention to the study of the pathological mechanism. According to the "cancer stem cell hypothesis", malignancies originate from a small fraction of cancer cells that show self-renewal properties to initiate and sustain tumor growth and tumor metastasis. Therefore, these cancer stem cells (CSC) probably play important roles in tumor recurrence, metastasis, and drug resistance. Previous research reported that lysine-specific histone demethylase 1 (LSD1) maintains cancer stemness through up-regulating stemness markers SOX2 and OCT4. CD133 is believed to be the most robust surface marker for CRC stem cells, however the regulatory effect of LSD1 on stemness of CD133+ CRC has never been reported. In this study, our objectives included: 1) to isolate pure CD133+ and CD133− cells from SW620 cell line; 2) to investigate the effect of LSD1 on the characteristics of CD133+ stem cancer cells by knocking down the target gene. Results suggested that the SW620 cell line had both CD133+ and CD133− subsets. The CD133+ subset exhibited more CSC-like characteristics compared with the CD133− subset with higher viability, colony formation rate, migration and invasion rate, resistance to anti-cancer drugs, and apoptosis in vitro. The CD133+ also induced faster tumor formation and larger tumors in vivo. In the LSD1-knockdown CD133+ cells, the CSC-like characteristics had been all weakened. We conclude that LSD1 was important for CSCs to maintain their "stemness" features, which could be a potential therapeutic target of CRC.
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Humans , Animals , Rats , Neoplastic Stem Cells/drug effects , Colorectal Neoplasms/pathology , Cell Movement/drug effects , Apoptosis/drug effects , Cell Proliferation/drug effects , Histone Demethylases/pharmacology , Neoplastic Stem Cells/metabolism , Gene Expression Regulation, Neoplastic , Blotting, Western , Colony-Forming Units Assay , Cell Line, TumorABSTRACT
@#[Abstract] Objective: To investigate the effect of histone demethylase JMJD3 (jumonji domain-containing protein 3) on the stemness of diffuse large B-cell lymphoma (DLBCL) cells. Methods: The relationship between the expression of JMJD3 and the overall survival of DLBCL patients was analyzed using the clinical data of DLBCL patients in TCGA database. The control plasmid (pCMV) and JMJD3 expression plasmid(pCMV-JMJD3)weretransfectedintoDLBCLcellsofABCandGCBsubtype via lipofectamine transfection. Then, the mRNAlevels of JMJD3,ALDH1, OCT4 and SOX2 were detected by RT-PCR and qPCR; the activity ofALDH1 enzyme was detected by Flow cytometry; the protein expressions of OCT4 and SOX2 were detected by Western blotting. Gene enrichment in DLBCL patients with high JMJD3 expression was analyzed by gene set enrichment analysis (GSEA). Results: The result of prognosis analysis showed that high expression of JMJD3 was related with poor prognosis in DLBCL patients (P<0.05); however, multivariate analysis showed that the expression of JMJD3 was not the independent factor affecting the prognosis of DLBCL patients (all P>0.05). The expression of JMJD3 was remarkably increased in DLBCL cells transfected with pCMV-JMJD3, which led to significantly increased mRNA level and enzyme activity of ALDH1 as well as up-regulated mRNA and protein expressions of OCT4 and SOX2 (P<0.05 or P<0.01). GSEA analysis showed that enrichment of Wnt/β-catenin signaling pathway related gene set was observed in DLBCL patients with high JMJD3 expression (P<0.05). Conclusion: JMJD3 promotes the stemness of DLBCL cells, which may be a potential therapeutic target for DLBCL patients.
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N6-methyladenosine (m6A) is the most prevalent and abundant post-transcriptional RNA modification in eukaryotes. m6A is a dy-namic and reversible process catalyzed by m6A methyltransferase complex and demethylase. m6A is involved in the regulations of gene expres-sion and biological processes. Recently, researches have discovered that m6A plays a vital role in the differentiation and regulation of immune cells, which could provide a new idea for the research and therapy of immune-related diseases. The present article reviews the recent progresses of m6A in immunoregulation.
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Abstract INTRODUCTION: Candidiasis is the most frequent opportunistic mycosis in humans and can cause mortality, particularly in immunodeficient patients. One major concern is the increasing number of infections caused by drug-resistant Candidas trains, as these cannot be efficiently treated with standard therapeutics. The most common mechanism of fluconazole resistance in Candida is mutation of ERG11, a gene involved in the biosynthesis of ergosterol, a compound essential for cell integrity and membrane function. METHODS: Based on this knowledge, we investigated polymorphisms in the ERG11 gene of 3 Candida species isolated from immunocompromised and immunocompetent patients. In addition, we correlated the genetic data with the fluconazole susceptibility profile of the Candida isolates. RESULTS: A total of 80 Candida albicans, 8 Candida tropicalis and 6 Candida glabrata isolates were obtained from the saliva of diabetic, kidney transplant and immunocompetent patients. Isolates were considered susceptible to fluconazole if the minimum inhibitory concentration was lower than 8 μg/mL. The amino acid mutations F105L, D116E, K119N, S137L, and K128T were observed in C. albicans isolates, and T224C and G263A were found in C. tropicalis isolates. CONCLUSIONS: Despite the high number of polymorphisms observed, the mutations occurred in regions that are not predicted to interfere with ergosterol synthesis, and therefore are not related to fluconazole resistance.
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Humans , Male , Female , Adult , Aged , Polymorphism, Genetic/drug effects , Candida/drug effects , Candida/genetics , Fluconazole/pharmacology , Kidney Transplantation , Diabetes Mellitus/microbiology , Antifungal Agents/pharmacology , Reference Values , Saliva/microbiology , Candida/isolation & purification , DNA, Fungal/genetics , Microbial Sensitivity Tests , Polymerase Chain Reaction , Drug Resistance, Fungal/genetics , Immunocompetence , Middle Aged , Mutation/drug effectsABSTRACT
Objective@#To investigate the expressions of histone lysine-specific demethylase 1 (LSD1), O6-methylguanine DNA methyltransferase (MGMT) and cell proliferation-associated antigen Ki-67 in high-grade glioma and their influences on prognosis.@*Methods@#Sixty-five cases of grade Ⅲ and Ⅳ glioma confirmed by pathology from January 2011 to June 2017 in the First Affiliated Hospital of Xinjiang Medical University were selected. Immunohistochemistry (SP method) was used to detect the expressions of LSD1, MGMT and Ki-67 in pathological specimens. The therapeutic effect was evaluated by long-term follow-up. The relationships between the three markers and pathological grade, progression-free survival (PFS) and overall survival (OS) were analyzed.@*Results@#The overall positive rates of LSD1, MGMT and Ki-67 in the 65 high-grade glioma specimens were 70.8% (46/65), 60.0% (39/65) and 100.0% (65/65), respectively. There were no significant differences in the expressions of LSD1 and MGMT in grade Ⅲ and Ⅳ glioma (χ2=1.588, P=0.208, χ2=0.013, P=0.908). Ki-67 expression (+ ), (+ + ), (+ + + ) in grade Ⅳ glioma were observed in 18, 19 and 11 cases, respectively. Ki-67 expression (+ ), (+ + ) in grade Ⅲ glioma were observed in 11, 5 cases, and 1 case was (+ + + ), and the difference in expression intensity between the two groups was statistically significant (Z=-2.083, P=0.037). Log-rank test showed that the positive expressions of LSD1, MGMT and Ki-67 were negatively correlated with the PFS of patients with high-grade glioma (χ2=12.217, P=0.007; χ2=4.446, P=0.035; χ2=12.536, P=0.002), also were negatively correlated with OS (χ2=11.708, P=0.008; χ2=6.637, P=0.010; χ2=11.807, P=0.003). Grade Ⅳ patients were more likely to have relapse progression than grade Ⅲ patients (χ2=6.573, P=0.010), and OS was shorter (χ2=3.974, P=0.046). Cox proportional hazards model analysis showed that the expressions of LSD1 (HR=1.361, 95%CI: 1.094-1.694, P=0.006; HR=1.406, 95%CI: 1.117-1.771, P=0.004) and Ki-67 (HR=1.703, 95%CI: 1.175-2.468, P=0.005; HR=1.778, 95%CI: 1.209-2.616, P=0.003) were the independent prognostic risk factors for PFS and OS of patients with high-grade glioma. Correlation analysis results showed that the expression of MGMT was positively correlated with the expression of LSD1 (r=0.406, P=0.001).@*Conclusion@#LSD1, MGMT and Ki-67 have higher positive expression rates in high-grade glioma. MGMT is a prognostic factor for high-grade glioma, and LSD1 and Ki-67 can be used as independent predictors of prognosis for high-grade gliomas.
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@#【Objective】To investigate the impact of long- term storage time on epigenetic modification of histone in human cleavage stage embryos.【Methods】According to the length of storage time，donated embryos after slow-freezing were divided into 3 groups ：6-year group ，9-year group ，and 12-year group ，while the control group consisted of donated fresh embryos. Immunocytochemistry was performed to compare the expression levels of HDAC1， H3K9ac， H3K4me3 ，and H3K9me3 among 4 groups. Transcription levels of HDAC1 ，SUV39H1 ，SETDB1 ，and KDM5A were analyzed through Single-Cell qRT-PCR.【Results】The relative abundances of HDAC1 and SUV39H1 mRNA showed no significant differences among 4 groups（P > 0.05）. SETDB1 exhibited a climbing pattern as storage time increased，but no significant difference was observed（P > 0.05）. There were no differences in H3K9 trimethylation and H3K9 methylation among 4 groups. However ，the expression level of KDM5A increased with the increasing storage time（P < 0.05）.【Conclusions】 Storage time did not affect the expression of deacetylase HDAC1，methylase SUV39H1 and SETDB1. H3K9ac/me3 and H3K4me3 also exhibited no significant difference as the storage time increases. However ，the increasing storage length might induce the elevating expression of demethylase KDM5A，which may be associated with inhibition of embryonic transcription.
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Lysine demethylase 6 (KDM6) is involved in the demethylation regulation of histone H3 as an important modification enzyme in epigenetic modification,and plays an important role in embryonic development,inflammation and disease development.Current researches indicate that KDM6 is involved in the occurrence and development of various tumors (pancreatic cancer,colon cancer,gastric cancer,breast cancer,bladder cancer,etc.),affects proliferation,metastasis,prognosis and chemotherapy resistance of tumors,and plays different roles due to different tumor backgrounds.
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Codeine-O-demethylase (CODM) is a key enzyme in the biosynthesis of codeine and morphine. In this study, CODM gene sequences were cloned from Papaver somniferum and Papaver rhoeas, and were compared with each other by sequence alignment and bioinformatics analysis. The results showed that there were three genotypes of CODM in Papaver somniferum and five genotypes of CODM in Papaver rhoeas. Bioinformatics analysis showed that all CODM proteins had no signal peptide sequence, and these proteins were predicted to be non-secretory proteins, belonging to the Pcbc supergene family. Although the amino acid sequences of CODM in poppies are the same, the expression levels of CODM in different poppy resources are significantly different. It is speculated that the variation of transcription level of CODM may be related to its non-coding region sequence, which lays a foundation for further research on the synthesis and regulation mechanism of alkaloids in poppies.
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Objective To investigate the expressions of histone lysine-specific demethylase 1 (LSD1),O6-methylguanine DNA methyltransferase (MGMT) and cell proliferation-associated antigen Ki-67 in high-grade glioma and their influences on prognosis.Methods Sixty-five cases of grade Ⅲ and Ⅳ glioma confirmed by pathology from January 2011 to June 2017 in the First Affiliated Hospital of Xinjiang Medical University were selected.Immunohistochemistry (SP method) was used to detect the expressions of LSD1,MGMT and Ki-67 in pathological specimens.The therapeutic effect was evaluated by long-term follow-up.The relationships between the three markers and pathological grade,progression-free survival (PFS) and overall survival (OS) were analyzed.Results The overall positive rates of LSD1,MGMT and Ki-67 in the 65 high-grade glioma specimens were 70.8％ (46/65),60.0％ (39/65) and 100.0％ (65/65),respectively.There were no significant differences in the expressions of LSD1 and MGMT in grade Ⅲ and Ⅳ glioma (x2 =1.588,P =0.208,x2 =0.013,P=0.908).Ki-67 expression (+),(++),(+++) in grade Ⅳ glioma were observed in 18,19 and 11 cases,respectively.Ki-67 expression (+),(++) in grade Ⅲ glioma were observed in 11,5 cases,and 1 case was (+++),and the difference in expression intensity between the two groups was statistically significant (Z =-2.083,P =0.037).Log-rank test showed that the positive expressions of LSD1,MGMT and Ki-67 were negatively correlated with the PFS of patients with high-grade glioma (x2 =12.217,P =0.007;x2=4.446,P =0.035;x2=12.536,P =0.002),also were negatively correlated with OS (x2 =11.708,P =O.008;x2 =6.637,P =0.010;x2 =11.807,P =0.003).Grade Ⅳ patients were more likely to have relapse progression than grade Ⅲ patients (x2 =6.573,P =0.010),and OS was shorter (x2 =3.974,P=0.046).Cox proportional hazards model analysis showed that the expressions of LSD1 (HR =1.361,95％CI:1.094-1.694,P=0.006;HR=1.406,95％CI:1.117-1.771,P =0.004) and Ki-67 (HR=1.703,95％ CI:1.175-2.468,P =0.005;HR =1.778,95％ CI:1.209-2.616,P =0.003) were the independent prognostic risk factors for PFS and OS of patients with high-grade glioma.Correlation analysis results showed that the expression of MGMT was positively correlated with the expression of LSD1 (r =0.406,P =0.001).Conclusion LSD1,MGMT and Ki-67 have higher positive expression rates in high-grade glioma.MGMT is a prognostic factor for high-grade glioma,and LSD1 and Ki-67 can be used as independent predictors of prognosis for high-grade gliomas.
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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.
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Objective Lysine-specific demethylase 1 (LSD1) is a flavin adenine dinucleotide-dependent oxidase, which participates in many biological processes , such as cell proliferation and differentiation and gene activation and repression .The aim of this study was to investigate LSD1 acetylation by histone deacetylase inhib -itor trichostatin A ( TSA) and its effect on TSA-induced apoptosis of ovarian cancer cells . Methods LSD1 shRNA was synthesized and implanted into the pLKO-Tet-On lentiviral vector , which was transfected into HO8910 and SKOV3 ovarian cancer cell lines , and then the transfected cells were screened with 1.5μg/mL puromycin for one week until stable clones were established .The cells were treated for 48 hours with methanol (2 mg/mL, control), TSA (200 nmol/L), TCP (100μmol/L), or TSA+TCP.And in the experiment of RNA interfering the LSD1 expression, the cells were also treated for 48 hours with methanol (2 mg/mL, control), TSA (200 nmol/L), Dox (100 ng/mL), or TSA +Dox.The levels of LSD1 acetylation and its substrate histone H3 lysine 4 dimethylation (H3K4me2) were de-tected by immunoprecipitation (IP) and Western blot.The apoptosis of the cells was determined by Annexin Ⅴ/PI staining and flow cytometry, the transcription levels of the Bax and p21 genes detected by real-time quantitative PCR, and the H3K4me2levels in the promoter regions of Bax and p21 measured by chromatin immunoprecipitation ( ChIP ) .Results In comparison with the methanol control, the TSA group showed significantly increased levels of LSD 1 acetylation in the HO8910(1.00±0.29 vs 5.83±0.46, P<0.01) and SKOV3 cells ( 1.00±0.24 vs 5.07±0.35, P<0.01) as well as that of H3K4me2 ( P<0.01);the total apoptosis rates of HO 8910 and SKOV3 cells were remarkably increased in the TSA, TCP, and TSA+TCP groups (P<0.05), even more significantly in the TSA+TCP than in the TSA and TCP groups ( P<0.05) .The mRNA expressions of Bax and p21 in the HO8910 cells were markedly upregulated in the TSA, Dox, and TSA+Dox groups (P<0.05), even more significantly in the latter than in the former two groups (P<0.05).The TSA group exhibited a higher level of H 3K4me2 than the methanol control in the promoters of Bax(2 .92±0.26 vs 0.68±0.19, P<0.01) and p21 (3.07±0.29 vs 0.93±0.17, P<0.01). Conclusion TSA induces the LSD1 acetylation, while suppression of LSD1 expres-sion and activity may enhance the antitumor activity of TSA .
ABSTRACT
Objective Lysine-specific demethylase 1 (LSD1) is a flavin adenine dinucleotide-dependent oxidase, which participates in many biological processes , such as cell proliferation and differentiation and gene activation and repression .The aim of this study was to investigate LSD1 acetylation by histone deacetylase inhib -itor trichostatin A ( TSA) and its effect on TSA-induced apoptosis of ovarian cancer cells . Methods LSD1 shRNA was synthesized and implanted into the pLKO-Tet-On lentiviral vector , which was transfected into HO8910 and SKOV3 ovarian cancer cell lines , and then the transfected cells were screened with 1.5μg/mL puromycin for one week until stable clones were established .The cells were treated for 48 hours with methanol (2 mg/mL, control), TSA (200 nmol/L), TCP (100μmol/L), or TSA+TCP.And in the experiment of RNA interfering the LSD1 expression, the cells were also treated for 48 hours with methanol (2 mg/mL, control), TSA (200 nmol/L), Dox (100 ng/mL), or TSA +Dox.The levels of LSD1 acetylation and its substrate histone H3 lysine 4 dimethylation (H3K4me2) were de-tected by immunoprecipitation (IP) and Western blot.The apoptosis of the cells was determined by Annexin Ⅴ/PI staining and flow cytometry, the transcription levels of the Bax and p21 genes detected by real-time quantitative PCR, and the H3K4me2levels in the promoter regions of Bax and p21 measured by chromatin immunoprecipitation ( ChIP ) .Results In comparison with the methanol control, the TSA group showed significantly increased levels of LSD 1 acetylation in the HO8910(1.00±0.29 vs 5.83±0.46, P<0.01) and SKOV3 cells ( 1.00±0.24 vs 5.07±0.35, P<0.01) as well as that of H3K4me2 ( P<0.01);the total apoptosis rates of HO 8910 and SKOV3 cells were remarkably increased in the TSA, TCP, and TSA+TCP groups (P<0.05), even more significantly in the TSA+TCP than in the TSA and TCP groups ( P<0.05) .The mRNA expressions of Bax and p21 in the HO8910 cells were markedly upregulated in the TSA, Dox, and TSA+Dox groups (P<0.05), even more significantly in the latter than in the former two groups (P<0.05).The TSA group exhibited a higher level of H 3K4me2 than the methanol control in the promoters of Bax(2 .92±0.26 vs 0.68±0.19, P<0.01) and p21 (3.07±0.29 vs 0.93±0.17, P<0.01). Conclusion TSA induces the LSD1 acetylation, while suppression of LSD1 expres-sion and activity may enhance the antitumor activity of TSA .