Relationship between G9a and Slack channels in dorsal root ganglia of rats with neuropathic pain / 中华麻醉学杂志

Objective:To evaluate the relationship between the euchromatic histone-lysine N-methyltransferase (G9a) and sodium-dependent activation of potassium channel (Slack) in the dorsal root ganglia (DRG) of rats with neuropathic pain (NP).Methods:Forty-eight clean-grade healthy male Sprague-Dawley rats, aged 1 month, weighing 100-120 g, were divided into 4 groups ( n=12 each) by a random number table method: sham operation group (S group), vector plus sham operation group (VS group), vector plus NP group (VN group), and G9a CRISPR/Cas9 knockout plus NP group (GN group). Sham operation was performed at the age of 2 months in group S. In group VS, AAV5 1 μl was microinjected into L 4 and L 5 DRG at the age of 1 month, and sham operation was performed at the age of 2 months.In VN group and GN group, AAV5 and G9a CRISPR/Cas9 knockout plasmid 1 μl were microinjected into L 4 and L 5 DRG at the age of 1 month, and NP model was established by spinal nerve ligation (SNL) at the age of 2 months.Six rats in each group were selected to measure the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) before microinjection (T 0), before SNL (T 1), and at 3, 5 and 7 days after SNL (T 2-4). The animals were sacrificed after the last behavioral testing, the DRGs of lumbar segment (L 4, 5) were removed for determination of the expression of G9a, dimethylation of histone H3 at lysine 9(H3K9me2) and Slack (by Western blot). At 7 days after establishing the model, 6 rats from each group were selected to culture the primary DRG neurons.The frequency and amplitude of Slack current in DRG neurons and miniature excitatory post-synaptic currents (mEPSCs) in the spinal dorsal horn were measured by whole-cell patch-clamp technique. Results:Compared with group S, the TWL was significantly shortened and the MWT was decreased at T 2-4, the expression of G9a and H3K9me2 in the spinal dorsal horn was up-regulated, the expression of Slack was down-regulated, the amplitude and frequency of Slack currents in DRG neurons were decreased, and the frequency of mEPSCs was increased in group VN ( P<0.05), and no significant change was found in the parameters mentioned above in group VS ( P>0.05). Compared with group VN, the TWL was significantly prolonged and the MWT was increased at T 2-4, the expression of G9a and H3K9me2 in the spinal dorsal horn was down-regulated, the expression of Slack was up-regulated, the amplitude and frequency of Slack currents in DRG neurons were increased, and the frequency of mEPSCs was decreased in group GN ( P<0.05). Conclusion:The mechanism of NP is related to up-regulating the expression of G9a in DRG, thus inhibiting the expression and opening of Slack channels in rats.

Comprehensive Cancer Panel Sequencing Defines Genetic Diversity and Changes in the Mutational Characteristics of Pancreatic Cancer Patients Receiving Neoadjuvant Treatment

BACKGROUND/AIMS: Pancreatic ductal adenocarcinoma (PDA) is associated with an extremely poor prognosis. This study assessed the genetic diversity among patients with PDA and compared their mutational profiles before and after treatment. METHODS: Tumors and matched blood samples were obtained from 22 PDA patients treated with neoadjuvant chemoradiation therapy. The somatic mutations were analyzed with comprehensive cancer gene panel (CCP). In addition, the biopsy samples obtained at diagnosis and the surgically resected samples after treatment were compared for seven patients. The CCP provided formalin-fixed paraffin-embedded sample-compatible multiplexed target selection for 409 genes implicated in cancer. RESULTS: Assessments of the MLH1, MLH3, MSH2, and PMS2 genes showed that the four patients with the highest relative burdens of mutations harbored somatic mutations in at least three of these genes. Genes in the histone-lysine N-methyltransferase 2 (KMT2) family, such as KMT2D, KMT2A, and KMT2C, were frequently mutated in tumor samples. Survival was worse in patients with ARID1A gene mutations than those without ARID1A gene mutations. Mutation patterns were compared between tissue samples before and after neoadjuvant treatment in seven patients who underwent surgical resection. The allelic fraction of mutations in KRAS codon 12 was lower in the surgically resected samples than in the endoscopic ultrasonography-guided fine needle aspiration biopsy samples of six patients. The number of mutant alleles of the histone lysine methyltransferase gene WHSC1 also decreased after treatment. CONCLUSIONS: These results indicate that tumor tissue from PDA patients is genetically diverse and suggest that ARID1A mutations may be a potential prognostic marker for PDA.

Histone methyltransferase MLL1 accelerates the development of renal fibrosis via promoting the epithelial-mesenchymal transition of HK-2 cells / 中华肾脏病杂志

Objective To investigate the possible effects of histone methyltransferase MLL1 on renal interstitial fibrosis and epithelial-mesenchymal transition(EMT).Methods Forty-two male SD rats were randomly divided into normal group,sham operation group and unilateral ureteral occlusion(UUO)group,and then UUO group was further divided into group 1 d,1 week,2 week,3 week and 4 week after operation.The expression of MLL1,E-cadherin,α-SMA,Vimentin and Col3α1 in UUO rat kidney tissue as well as TGF-β1 stimulated HK-2 cells were detected by real-time PCR and Western blotting.siRNA-MLL1 plasmids was used to inhibit the expression of MLL1 and the protein levels of MLL1,α-SMA,Vimentin,E-cadherin,Col3α1 and H3K4me3 induced by TGF-β1 stimulation were detected by Western blotting.The level of H3K4me3 in promoter region of EMT related genes was observed by chromatin immunoprecipitation(CHIP).Results Compared with normal and sham operated groups,the loss of renal function in UUO group was more obvious with the obstruction time(P ＜ 0.05).The renal fibrosis was most obvious 1 week and 2 weeks after the rats underwent the UUO operation(all P ＜ 0.05),with the highest protein expressions of MLL1,E-cadherin,α-SMA,Vimentin and Col3α1(all P ＜ 0.05).Compared with the control group,3 ng/ml TGF-β1 induced the highest expression of MLL1 and the most obvious EMT in HK-2 cells(all P ＜ 0.05).Moreover,the EMT and the high level of H3K4me3 in HK-2 triggered by TGF-β1 were all inhibited by siRNA-MLL1 plasmids transfection(all P ＜ 0.05).Conclusions MLL1 can enhance the occurrence of EMT induced by TGF-β1 in HK-2 cells by increasing the level of H3K4me3 in the promoter region of α-SMA and Vimentin.

The effect of interference SET8 on regulating the transdifferentiation of vascular smooth muscle cells / 天津医药

Objective To investigate the effect of lysine methyltransferase SET8 on regulating cell transdifferentiation of rat vascular smooth muscle cells(VSMCs)into osteoblast-like cells. Methods VSMCs were obtained from rat thoracic aorta,and then randomly divided into control group(non-transfection),the empty plasmid group(transfect NS-shRNA)and SET8-shRNA group. The expression of SET8, runt-related transcription factor 2 (RUNX2) was detected by RT-PCR and Western blot assay. Alkaline phosphatase (ALP) activity was measured by enzyme linked immunoassay. Results The expression of SET8 in VSMCs was effectively inhibited by SET8-shRNA.RT-PCR and Western blot results showed that the expression of RUNX2 was decreased in cells after SET8-shRNA transfection (P<0.05). ALP activity was significantly reduced after SET8-shRNA transfection(P<0.05).Conclusion Interference with SET8 gene expression could inhibit the differentiation of VSMCs into osteo-like cells.

Histone methyltransferase SMYD3 and tumor / 肿瘤

Histone methyltransferase SMYD3 (SET and MYND domain containing3) plays an important role in tumor epigenetics. Its high expressionin many cancer cells can promote the development and progressionof tumor by regulating several cellular biological functions such asproliferation, apoptosis, invasion and metastasis. Previous studieshave indicated that SMYD3 mainly modulate the methylation of lysineH3K4 to regulate the expression of certain genes. Recent researcheshave shown that SMYD3 can also interact with other histones andcytoplasmic proteins to regulate the expression of tumor-relatedgenes, thereby to promote the development of tumor. This reviewaims to introduce the structure, upstream regulatory pathwayand synergistic molecules of SMYD3, as well as to summarize itsregulation mechanisms, expression and inhibitors in tumor.

Mechanism and effect of hDOT1L on the proliferation inhibition of decitabine in THP-1 cells: a preliminary study / 白血病·淋巴瘤

Objective To analyze the role of hDOT1L signaling pathway on the proliferation of human acute monocytic leukemia cell line THP-1 cell inhibited by demcitabine, and to explore the other effects except DNA demethylation and the mechanism of hDOT1L signaling pathways involved in leukemia. Methods Methyl thiazolyl tetrazolium (MTT) method was used to detect the influence of decitabine on THP-1 cell proliferation, and trypan blue anti-staining was applied to detect the effect of decitabine on THP-1 cell survival rate, reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the expression levels of hDOT1L, HOXA9, HOXA10, MLL-AF10 (MLLT10) mRNA, then the methylation level of H3K79 was detected by Western blotting assay. Results Compared with the control group, decitabine inhibited the proliferation of THP-1 cell in a time and dose-dependent manner. The inhibitory effect of 72 h was the highest, and the highest inhibition rate was 56.18%. In addition to 0.5 μmol/L in decitabine treatment group, the other groups were statistically significant (all P<0.05). There were high expressions of hDOT1L, HOXA9, HOXA10, MLLT10 mRNA and H3K79 hypermethylation in the THP-1 cell, meanwhile decitabine reduced the levels of hDOT1L mRNA after 48 h as well as HOXA9, HOXA10, MLLT10 mRNA, and there was a significant difference compared with the control group (all P < 0.01). Decitabine reduced obviously the methylation of H3K79 in cell after 48 h, especially the expression levels of bis and three methyl protein, and these differences were significant compared with the control group (all P< 0.01). Conclusion Decitabine plays its inhibitory effect on the proliferation of THP-1 cells probably through reducing the expression level of hDOT1L and H3K79 methylation level and decreasing the expression levels of key molecules, HOXA9, MLL-AF10, HOXA10, MLLT10 in the hDOT1L signal pathway.

Inhibition of Nuclear Receptor Binding SET Domain 2/Multiple Myeloma SET Domain by LEM-06 Implication for Epigenetic Cancer Therapies

BACKGROUND: Multiple myeloma SET domain (MMSET)/nuclear receptor binding SET domain 2 (NSD2) is a lysine histone methyltransferase (HMTase) and bona fide oncoprotein found aberrantly expressed in several cancers, suggesting potential role for novel therapeutic strategies. In particular, MMSET/NSD2 is emerging as a target for therapeutic interventions against multiple myeloma, especially t(4;14) myeloma that is associated with a significantly worse prognosis than other biological subgroups. Multiple myeloma is the second most common hematological malignancy in the United States, after non-Hodgkin lymphoma and remains an incurable malignancy. Thus, effective therapeutic strategies are greatly needed. HMTases inhibitors are scarce and no NSDs inhibitors have been isolated. METHODS: We used homology modeling, molecular modeling simulations, virtual ligand screening, computational chemistry software for structure-activity relationship and performed in vitro H3K36 histone lysine methylation inhibitory assay using recombinant human NSD2-SET and human H3.1 histone. RESULTS: Here, we report the discovery of LEM-06, a hit small molecule inhibitor of NSD2, with an IC50 of 0.8 mM against H3K36 methylation in vitro. CONCLUSIONS: We propose LEM-06 as a hit inhibitor that is useful to further optimize for exploring the biology of NSD2. LEM-06 derivatives may pave the way to specific NSD2 inhibitors suitable for therapeutic efforts against malignancies.

Increased lysine N-methylation of a 23-kDa protein during hepatic regeneration

The methylation of a 23-kDa nuclear protein increased after partial hepatectomy and methylation returned to basal levels after the initial stage of regeneration. The methylating enzyme was partially purified from rat liver by ammonium sulfate precipitation, DEAE-anion exchange chromatography and Butyl-Sepharose chromatography. The 23-kDa protein was purified from a nuclear fraction of liver tissue with SP-Sepharose. When the 23-kDa protein was methylated with the partially purified methyltransferase and analyzed on C18 high performance liquid chromatography (HPLC), the methylated acceptor amino acid was monomethyl lysine (MML). Previously, only arginine N-methylation of specific substrate proteins has been reported during liver regeneration. However, in this report, we found that lysine N-methylation increased during early hepatic regeneration, suggesting that lysine N-methylation of the 23-kDa nuclear protein may play a functional role in hepatic regeneration. The methyltransferase did not methylate other proteins such as histones, hnRNPA1, or cytochrome C, suggesting the enzyme is a 23-kDa nuclear protein- specific lysine N-methyltransferase.