Research progress on m5C methylation and its prospects in tumor immunotherapy / 口腔疾病防治

@#Epigenetic modification plays an important role in the biological regulatory process of eukaryotic cells. Tumor immunotherapy is an important means and clinical strategy for the treatment of some cancers. 5-Methylcytosine (m5C) is an important component of the epigenetic regulatory network discovered after m6A and has become a new topic for life science research in recent years. The m5C methylation of RNA can affect the fate of the modified RNA molecules and play an important role in various biological processes, including RNA stability, protein synthesis and transcriptional regulation. Recent studies have shown that m5C writers, erasers and readers are related to a variety of cellular biological processes and systemic diseases, including the occurrence, metastasis and tumor immune microenvironment. m5C methylation can widely affect gene expression and the biological process of tumorigenesis and development at multiple levels, but its specific mechanism and potential interaction with other epigenetic modifications in tumor immunotherapy are still unclear, and its regulatory mechanism, risk assessment and role in targeted therapy for malignant tumors need to be further studied. This article will review the dynamic regulatory network of m5C, the biological role of m5C modification in solid tumors and potential targets in tumor immunotherapy.

Inhibiting DNA methylation alleviates cisplatin-induced hearing loss by decreasing oxidative stress-induced mitochondria-dependent apoptosis via the LRP1-PI3K/AKT pathway

Cisplatin-related ototoxicity is a critical side effect of chemotherapy and can lead to irreversible hearing loss. This study aimed to assess the potential effect of the DNA methyltransferase (DNMT) inhibitor RG108 on cisplatin-induced ototoxicity. Immunohistochemistry, apoptosis assay, and auditory brainstem response (ABR) were employed to determine the impacts of RG108 on cisplatin-induced injury in murine hair cells (HCs) and spiral ganglion neurons (SGNs). Rhodamine 123 and TMRM were utilized for mitochondrial membrane potential (MMP) assessment. Reactive oxygen species (ROS) amounts were evaluated by Cellrox green and Mitosox-red probes. Mitochondrial respiratory function evaluation was performed by determining oxygen consumption rates (OCRs). The results showed that RG108 can markedly reduce cisplatin induced damage in HCs and SGNs, and alleviate apoptotic rate by protecting mitochondrial function through preventing ROS accumulation. Furthermore, RG108 upregulated BCL-2 and downregulated APAF1, BAX, and BAD in HEI-OC1 cells, and triggered the PI3K/AKT pathway. Decreased expression of low-density lipoprotein receptor-related protein 1 (LRP1) and high methylation of the LRP1 promoter were observed after cisplatin treatment. RG108 treatment can increase LRP1 expression and decrease LRP1 promoter methylation. In conclusion, RG108 might represent a new potential agent for preventing hearing loss induced by cisplatin via activating the LRP1-PI3K/AKT pathway.

Evaluación de la metilación del DNA en tejidos de cáncer colorrectal fijados en formalina y embebidos en parafina (FFPE) / Evaluation of DNA methylation in formalin-fixed, paraffin embedded colorectal cancer tissues (FFPE)

Resumen Las alteraciones en la metilación de dinucleótidos CpG en regiones promotoras es uno de los mecanismos epigenéticos implicados en cáncer que tiene uso potencial como biomarcador. Su evaluación, a partir de tejidos fijados en formalina y embebidos en parafina (FFPE), representa un gran desafío dadas la degradación parcial, el entrecruzamiento y las bajas cantidades del DNA obtenido. En esta nota técnica, describimos un protocolo para el estudio del estado de metilación del promotor distal del proto-oncogén K-RAS, a partir de varias muestras obtenidas de dos tejidos FFPE de cáncer colorrectal con antigüedad de 11 años. Se empleó un protocolo de conversión con bisulfito alternativo al usual; se usó una DNA polimerasa modificada y una PCR anidada y se optimizó la secuenciación directa del DNA convertido con bisulfito. Este protocolo podría ser aplicado para determinar estados de metilación en otros genes y tipos de cáncer en tejidos FFPE.

Abstract Alterations in the methylation of CpG dinucleotides in promoter regions is one of the epigenetic mechanisms involved in cancer that has potential use as a biomarker. Its evaluation from formalin-fixed and paraffin-embedded (FFPE) tissues represents a great challenge given the partial degradation, crosslinking, and low amounts of the obtained DNA. In this technical note we describe a protocol for the study of the methylation status of the distal promoter of the K-RAS proto-oncogene from several samples obtained from two 11-years old FFPE tissues of colorectal cancer. An alternative bisulfite conversion protocol to the usual one was used; a modified DNA polymerase and a nested PCR were used and the direct sequencing of the converted DNA with bisulfite was optimized. This protocol could be applied to determine methylation states in other genes and types of cancer.

RNA methylation and neurovascular unit remodeling / 中南大学学报(医学版)

RNA methylation is of great significance in the regulation of gene expression, among which the more important methylation modifiers are N6-methyladenosine (m6A) and 5-methylcytosine (m5C). The methylation process is mainly regulated by 3 kinds of proteins: methyltransferase, demethylase, and reader. m6A, m5C, and their related proteins have high abundance in the brain, and they have important roles in the development of the nervous system and the repair and remodeling of the vascular system. The neurovascular unit (NVU) is a unit of brain structure and function composed of neurons, capillaries, astrocytes, supporting cells, and extracellular matrix. The local microenvironment for NVU has an important role in nerve cell function repair, and the remodeling of NVU is of great significance in the prognosis of various neurological diseases.

Identification of 5-methylcytosine-related signature for predicting prognosis in ovarian cancer

BACKGROUND: Ovarian cancer is one of the most common malignancies often resulting in a poor prognosis. 5-methylcytosine (m5C) is a common epigenetic modification with roles in eukaryotes. However, the expression and function of m5C regulatory factors in ovarian cancer remained unclear. RESULTS: Two molecular subtypes with different prognostic and clinicopathological features were identified based on m5C regulatory factors. Meanwhile, functional annotation showed that in the two subtypes, 452 differentially expressed genes were significantly related to the malignant progression of ovarian cancer. Subsequently, four m5C genes were screened to construct a risk marker predictive of overall survival and indicative of clinicopathological features of ovarian cancer, also the robustness of the risk marker was verified in external dataset and internal validation set. multifactorial cox regression analysis and nomogram demonstrated that risk score was an independent prognostic factor for ovarian cancer prognosis. CONCLUSIONS: In conclusion, our results revealed that m5C-related genes play a critical role in tumor progression in ovarian cancer. Further detection of m5C methylation could provide a novel targeted therapy for treating ovarian cancer.

The expression and clinical significance of 5-hmC and TETs in hepatocellular carcinoma / 中华普通外科杂志

Objective To investigate the levels of 5-hydroxymethylcytosine (5-hmC) and teneleven-translocation proteins (TET1/2/3) in diagnosis and prognosis prediction of hepatocellular carcinoma (HCC).Methods The expression of 5-hmC in 130 cases of HCC tissues were detected by immunohistochemical staining.Kaplan-Meier test was used for survival analysis.TET family plays critical role in the conversion of 5-methylcytosine (5-mC) to 5-hmC.The TET levels were detected by using immunohistochemical staining and RT-PCR,the correlation between 5-hmC and TET was analyzed.Results The level of 5-hmC decreased in HCC tissues,as compared with non-tumor tissues,the expression of TET1 was downregulated in HCC.There was significant difference in the expression between low and high grades of HCC tissues (x2 =10.611,P =0.001).Kaplan-Meier survival analysis showed that there was significant difference between the 5-hmC expression level and the survival rate of HCC patients (x2 =4.412,P =0.036).Conclusions In HCC tissues the expression of 5-hmC was specifically downregulated.Low 5-hmC level is significantly correlated with poor differentiation of the tumor and worse overall survival.Decreased expression of TET1 is likely one of the mechanisms underlying 5-hmC loss in HCC.

Effect of fluoride on the expression of 5-methylcytosine in blood, liver, kidney and brain of rats / 中华地方病学杂志

Objective To investigate the effects of fluoride exposure dose and exposure time on the expression of 5-methylcytosine (5-mC) in blood,liver,kidney and brain of rats;and to understand whether there is a difference in the effects of fluoride on DNA methylation levels in different tissues.Methods Eighty three-week old SPF male Wistar rats were randomly divided into four groups according to body weight [(82.34 ± 10.60) g],with 20 rats in each group.The rats of control group drank distilled water and the fluoride group's drank distilled water containing 25,50 and 100 mg/L of F ion,respectively.Rats were sacrificed after fed for 1 month and 3 months (n =10),and peripheral blood and tissue samples were collected.The incidence of dental fluorosis was observed in rats.Bone and urine fluoride content was detected by ion selective electrode method.The content of 5-mC in blood,liver,kidney and brain was detected by enzyme-linked immunosorbent assay (ELISA).The independent and interactive effects of fluoride exposure dose and exposure time on 5-mC in rat peripheral blood and different tissues were analyzed by factorial design anova.Results After feeding for 1 month and 3 months,all rats in the fluoride group had dental fluorosis with different severities,while none dental fluorosis was found in the control groups.Fluoride exposure dose and exposure time had a main effect on bone fluoride contents [1 month:(324.985 + 127.094),(846.148 ± 331.861),(1 886.601 + 250.140),(2 420.971 + 135.883) mg/kg;3 months:(417.591 ± 88.324),(1 582.243 ± 347.975),(2 163.519 ± 614.932),(2 755.434 ± 265.370)mg/kg;F =96.692,13.077,P ＜ 0.01],respectively,but there was no interaction effect (F =2.013,P ＞ 0.05);fluoride exposure dose had a main effect on urinary fluoride contents (F =62.358,P ＜ 0.01),the exposure time had no effect on it (F =0.862,P ＞ 0.05),and there was no interaction effect (F =0.081,P ＞ 0.05).Fluoride exposure dose had a main effect on the 5-mC content in the blood (F =8.446,P ＜ 0.01),the exposure time had no effect on it (F =0.095,P ＞0.05),and there had an interaction effect (F =4.676,P ＜ 0.01).Fluoride exposure dose and exposure time had a main effect on the 5-mC content in the liver,respectively (F =4.737,7.064,P ＜ 0.01 or ＜ 0.05),and an interaction effect was exist (F =8.302,P ＜ 0.01).Fluoride exposure time had a main effect on the 5-mC content in the kidney (F =6.340,P ＜ 0.05),the exposure dose had no effect on it (F =0.140,P ＞ 0.05),and there was no interaction effect (F =1.269,P ＞ 0.05).Fluoride exposure dose and exposure time had no effect on 5-mC content in the brain (F =0.633,2.065,P ＞ 0.05).Conclusion Fluoride exposure dose and exposure time have the different effect on the levels of 5-mC in blood,liver,kidney and brain,suggesting that there may be differences in the effects of fluoride on DNA methylation levels in different tissues.

Expression of TET and 5-HmC in Trophoblast Villi of Women with Normal Pregnancy and with Early Pregnancy Loss / 华中科技大学学报（医学）（英德文版）

Increasing evidence suggests that epigenetic dysfunction may influence the stability of normal pregnancy.The ten-eleven translocation (TET) family and 5-hydroxymethylcytosine (5-hmC) were found to be linked with epigenetic reprogramming.The present study aimed to examine the expression of the TET family and 5-hmC in the villi of human embryos and compared their expression between normal pregnancy and early pregnancy loss (EPL).Embryonic villi were collected from normal pregnant women (control) experiencing medical abortion and from EPL patients at gestation ages of 6,7 and 8 weeks.The mRNAs of TET family were analysed using quantitative polymerase chain reaction (qPCR),and TET proteins using Western blotting and immunohistochemical analysis.The MethylFlashTM Kit was used to quantify the absolute amount of 5-methylcytosine (5-mC) and 5-hmC.Our results showed that the expression of the TETs and 5-hmC in the normal villus decreased with increasing gestational age.Immunohistochemistry revealed that the TET proteins were expressed in the cytoplasm of trophoblasts and their expression was the highest in the 6-week tissue samples,which was consistent with the qPCR and Western blot results.The expression of TET1,TET2,and TET3 was lower in the villi in EPL group than in normal pregnancy group (P＜0.05 for all).It was concluded that the TET family and 5-hmC are critical in epigenetic reprogramming of human embryo.The findings also suggest that a deficiency of TETs in the villus might be associated with human EPL.

Correlation analysis between the expression of 5-hydroxymethyl-cytosine and invasion, metastasis and prognosis of melanoma / 中华皮肤科杂志

Objective To detect the level of 5-hydroxymethyl-cytosine (5-hmc)in melanoma tissues,and to analyze the correlation between 5-hmc and the invasion,metastasis and prognosis of melanoma.Methods A streptavidin-peroxidase immunohistochemical method was used to detect the level of 5-hmc in 67 melanoma tissues and 20 pigmented nevi tissues.Univariate and multivariate analyses were performed with the Cox's proportional hazards regression model to analyze the correlation between the expression of 5-hmc and the prognosis of melanoma.Results The expression rate of 5-hmc was significantly lower in melanoma tissues than in pigmented nevus tissues (40.30％ [27/67] vs.75％ [15/20],22 =7.428,P =0.006).According to American Joint Committee on Cancer (AJCC) TNM staging system,the expression level of 5-hmc was significantly lower in the stage Ⅳ melanoma tissues than in the stage Ⅱ and stage Ⅲ melanoma tissues (x2 =4.416,P =0.036).Patients with lymph node metastasis showed significantly lower expression of 5-hmc compared with those without lymph node metastasis (x2 =5.902,P =0.015),and the level of 5-hmc expression significantly decreased along with the increase of Clark grade (x2 =4.828,P =0.028).There were no significant differences in the level of 5-hmc expression between patients of different ages,genders or nationalities (P ＞ 0.05).Multivariate Cox regression analysis showed that distant lymph node metastasis (HR:2.67,95％ CI:1.22-5.84),not receiving surgical resection (HR:0.41,95％ CI:0.18-0.95),and low expression of 5-hmc (HR:3.54,95％ CI:1.09-11.43)were independent risk factors for poor prognosis of melanoma.Conclusion 5-Hmc may participate in the invasion and metastasis of melanoma,and be associated with the prognosis of melanoma.

Expression of TET and 5-HmC in Trophoblast Villi of Women with Normal Pregnancy and with Early Pregnancy Loss / 华中科技大学学报（医学）（英德文版）

Increasing evidence suggests that epigenetic dysfunction may influence the stability of normal pregnancy.The ten-eleven translocation (TET) family and 5-hydroxymethylcytosine (5-hmC) were found to be linked with epigenetic reprogramming.The present study aimed to examine the expression of the TET family and 5-hmC in the villi of human embryos and compared their expression between normal pregnancy and early pregnancy loss (EPL).Embryonic villi were collected from normal pregnant women (control) experiencing medical abortion and from EPL patients at gestation ages of 6,7 and 8 weeks.The mRNAs of TET family were analysed using quantitative polymerase chain reaction (qPCR),and TET proteins using Western blotting and immunohistochemical analysis.The MethylFlashTM Kit was used to quantify the absolute amount of 5-methylcytosine (5-mC) and 5-hmC.Our results showed that the expression of the TETs and 5-hmC in the normal villus decreased with increasing gestational age.Immunohistochemistry revealed that the TET proteins were expressed in the cytoplasm of trophoblasts and their expression was the highest in the 6-week tissue samples,which was consistent with the qPCR and Western blot results.The expression of TET1,TET2,and TET3 was lower in the villi in EPL group than in normal pregnancy group (P＜0.05 for all).It was concluded that the TET family and 5-hmC are critical in epigenetic reprogramming of human embryo.The findings also suggest that a deficiency of TETs in the villus might be associated with human EPL.

Global methylation status of sperm DNA in carriers of chromosome structural aberrations / 亚洲男科学杂志(英文版)

Male infertility might be clearly associated with aberrant DNA methylation patterns in human spermatozoa. An association between oxidative stress and the global methylation status of the sperm genome has also been suggested. The aim of the present study was to determine whether the global sperm DNA methylation status was affected in the spermatozoa of carriers of chromosome structural aberrations.

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 .

Determination of Global DNA Methylation Levels in Medicinal Plants by LC-MS/MS / 中国药房

OBJECTIVE:To develop a detection LC-MS/MS method for global DNA methylation in medicinal plants. METH-ODS:Genomic DNA was isolated using plant DNA extraction kit,and then hydrolyzed by 88% formic acid at 140 ℃. After dried with nitrogen,extracted DNA was dissolved again with mobile phase. LC separation was performed on HILIC column with mobile phase consisted of 7 mmol/L ammonium formate-acetonitrile (gradient elution) at flow rate of 0.3 ml/min. The analysis was con-ducted by tandem MS with positive ion electrospray ionization in multiple reaction monitoring(MRM)mode. The ratio of genomic DNA methylation in 10 commonly used medicinal plants was calculated. RESULTS:The linear ranges of Cyt and 5mC were 1-500 ng/ml(r=0.999 5)and 0.2-100 ng/ml(r=0.999 6). The relative standard deviations(RSDs)of accuracy were 1.12% and 3.68%(n=6). The RSDs of intra-day precision were 2.36% and 4.02% for Cyt and 5mC,respectively (n=5). The RSDs of inter-day precision were 1.04% and 3.54% for Cyt and 5mC,respectively (n=3). The RSDs of repeatability test were 1.53% and 3.27%for Cyt and 5mC,respectively(n=6). The recoveries of Cyt and 5mC were 98.7%-102.1% and 91.2%-103.5%. The percentages of global DNA methylation in 10 medicinal plants were ranged from 17.63% to 25.18%. CONCLUSIONS:LC-MS/MS method is simple,rapid,sensitive and precise,and can be used for the detection of global DNA methylation in medicinal plants.

5-Methylcytosine content and methylation status in six millet DNAs.

High performance liquid chromatographic analysis of the total nuclear DNAs of 6 millets plant species indicates that the 5-methylcytosine content ranges from 3% in barn yard millet to 9·6% in great millet while the fraction of cytosines methylated varies between 14% in little millet to 31 % in pearl millet. Digestion of millet DNAs with MspI/HpaII suggests that CpG methylation is more in great millet DNA while CpC methylation is more in the other 5 millet DNAs. Digestion of millet DNAs with MboI, Sau3AI and DpnI indicates that some of the 5’ GATC3’ sequences are methylated at adenine and/or cytosine residues except in little millet where adenine methylation of the 5’GATC3’ sequences is insignificant and there is a predominance of cytosine methylation in these sequences.

Age related changes in 5-methylcytosine content in human peripheral leukocytes by HPLC / 中国法医学杂志

Objective To investigate age-dependent variation of 5-methylcytosine (5-mC) content in human peripheral leukocytes.Methods 94 healthy individuals (50 males and 44 females) of ages varying from 4 to 87 years were dividied into six groups by age: age under 20 years, age between 20 to 30 years, age between 30 to 40 years, age between 40 to 50 years, age between 50 to 60 years and age beyond 60 years. Their 5-mC content in human peripheral leukocytes were analyzed by HPLC.Results The 5-mC content of age between 50 to 60 years (mean age 54.7) and age beyond 60 years (mean age 71.7) are statistically not significant, but statistically highly significant (P