Neuroprotective mechanisms of DNA methyltransferase in a mouse hippocampal neuronal cell line after hypoxic preconditioning

Hypoxic preconditioning has been shown to improve hypoxic tolerance in mice,accompanied by the downregulation of DNA methyltransferases(DNMTs)in the brain.However,the roles played by DNMTs in the multiple neuroprotective mechanisms associated with hypoxic preconditioning remain poorly understood.This study aimed to establish an in vitro model of hypoxic preconditioning,using a cultured mouse hippocampal neuronal cell line(HT22 cells),to examine the effects of DNMTs on the endogenous neuroprotective mechanisms that occur during hypoxic preconditioning.HT22 cells were divided into a control group,which received no exposure to hypoxia,a hypoxia group,which was exposed to hypoxia once,and a hypoxic preconditioning group,which was exposed to four cycles of hypoxia.To test the ability of hypoxic preadaptation to induce hypoxic tolerance,cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium assay.Cell viability improved in the hypoxic preconditioning group compared with that in the hypoxia group.The effects of hypoxic preconditioning on the cell cycle and apoptosis in HT22 cells were examined by western blot assay and flow cytometry.Compared with the hypoxia group,the expression levels of caspase-3 and spectrin,which are markers of early apoptosis and S-phase arrest,respectively,noticeably reduced in the hypoxic preconditioning group.Finally,enzyme-linked immunosorbent assay,real-time polymerase chain reaction,and western blot assay were used to investigate the changes in DNMT expression and activity during hypoxic preconditioning.The results showed that compared with the control group,hypoxic preconditioning downregulated the expression levels of DNMT3A and DNMT3B mRNA and protein in HT22 cells and decreased the activities of total DNMTs and DNMT3B.In conclusion,hypoxic preconditioning may exert anti-hypoxic neuroprotective effects,maintaining HT22 cell viability and inhibiting cell apoptosis.These neuroprotective mechanisms may be associated with the inhibition of DNMT3A and DNMT3B.

Potassium bisperoxo(1,10-phenanthroline) oxovanadate suppresses proliferation of hippocampal neuronal cell lines by increasing DNA methyltransferases

Bisperoxo(1,10-phenanthroline) oxovanadate(BpV) can reportedly block the cell cycle. The present study examined whether BpV alters gene expression by affecting DNA methyltransferases(DNMTs), which would impact the cell cycle. Immortalized mouse hippocampal neuronal precursor cells(HT_(22)) were treated with 0.3 or 3 μM BpV. Proliferation, morphology, and viability of HT_(22) cells were detected with an IncuCyte real-time video imaging system or inverted microscope and 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, respectively. mRNA and protein expression of DNMTs and p21 in HT_(22) cells was detected by real-time polymerase chain reaction and immunoblotting, respectively. In addition, DNMT activity was measured with an enzyme-linked immunosorbent assay. Effects of BpV on the cell cycle were analyzed using flow cytometry. Results demonstrated that treatment with 0.3 μM BpV did not affect cell proliferation, morphology, or viability; however, treatment with 3 μM BpV decreased cell viability, increased expression of both DNMT3B mRNA and protein, and inhibited the proliferation of HT_(22) cells; and 3 μM BpV also blocked the cell cycle and increased expression of the regulatory factor p21 by increasing DNMT expression in mouse hippocampal neurons.

Clinicopathological significance of human leukocyte antigen F-associated transcript 10 expression in colorectal cancer

BACKGROUND Colorectal cancer(CRC) is a common malignancy of the gastrointestinal tract. The worldwide mortality rate of CRC is about one half of its morbidity. Ubiquitin is a key regulatory factor in the cell cycle and widely exists in eukaryotes. Human leukocyte antigen F-associated transcript 10(FAT10), known as diubiquitin, is an 18 kDa protein with 29% and 36% homology with the N and C termini of ubiquitin. The function of FAT10 has not been fully elucidated, and some studies have shown that it plays an important role in various cell processes.AIM To examine FAT10 expression and to analyze the relationship between FAT10 expression and the clinicopathological parameters of CRC.METHODS FAT10 expression in 61 cases of CRC and para-cancer colorectal tissues was measured by immunohistochemistry and Western blotting. The relationship between FAT10 expression and clinicopathological parameters of CRC was statistically analyzed.RESULTS Immunohistochemical analysis showed that the positive rate of FAT10 expression in CRC(63.93%) was significantly higher than that in tumor-adjacent tissues(9.84%, P < 0.05) and normal colorectal mucosal tissue(1.64%, P < 0.05). Western blotting also indicated that FAT10 expression was significantly higher in CRC than in tumor-adjacent tissue(P < 0.05). FAT10 expression was closely associated with clinical stage and lymphatic spread of CRC. FAT10 expression also positively correlated with p53 expression.CONCLUSION FAT10 expression is highly upregulated in CRC. FAT10 expression is closely associated with clinical stage and lymphatic spread of CRC.

Copper-Free Click Chemistry-Mediated Assembly of Single Quantum Dot Nanosensor for Accurately Monitoring Locus-Specific m^(6)A in Cancer Cells

N6-methyladenosine(m^(6)A)plays an important role in embryogenesis,nuclear export,transcription splicing,and protein translation control.Herein,we demonstrate a copper-free click chemistry-mediated assembly of single quantum dot(QD)nanosensor for accurately monitoring locus-specific m^(6)A in cancer cells.The m^(6)A-sensitive endoribonuclease MazF can digest the unmethylated A-RNA,and the intact m^(6)A-RNA then hybridizes with DNA probes a and b to produce a sandwich hybrid,initiating the click chemistry to generate probe a–b ligation product via first tandem ligation detection reaction(LDR)cycle.Subsequently,DNA probes c and d can hybridize with the probe a–b ligation product to generate the probe c–d ligation product via second LDR cycle.Both LDR cycles can be repeated through denaturation and annealing reaction to generate abundant biotin-/fluorophore-modified probe c–d ligation products that can easily assemble on the QD surface to induce distinct fluorescence resonance energy transfer(FRET)between QD and Cy5.This assay can be homogenously performed without the involvement of copper catalyst,m^(6)A-specific antibody,radioactive labeling,ligase enzyme,enzymatic reverse transcription,and next-generation sequencing.Moreover,it can discriminate even 0.01% m^(6)A level in complex samples and accurately measure cellular m^(6)A-RNA expression,providing a promising avenue for clinical diagnostics and biomedical research.

Advances in single-particle detection for DNA sensing

Rapid, accurate and sensitive detection of particular DNA sequence is critical in fundamental biomedical research and clinical diagnostics. However, conventional approaches for DNA assay often suffer from cumbersome procedures, long analysis time and insufficient sensitivity. Recently, single-particle detection technology has emerged as a powerful tool in the biosensing area due to its significant advantages of ultrahigh sensitivity, low sample-consumption and rapid analysis time. Especially, the introduction of novel nanomaterials has greatly promoted the development of single-particle detection and its applications for DNA sensing. In this review, we summarize the recent advance in single-particle detection strategies for DNA sensing, and focus mainly on metallic nanoparticle-and semiconductor quantum dot-based single-particle detection. We highlight the emerging trends in this field as well.