Effects of histone acetyltransferase MYST2 on pancreatic cancer / 中华消化杂志

Objective:To investigate the expression at protein level and diagnostic value of histone acetyltransferase MYST2 in pancreatic cancer.Methods:From December 1st, 2017 to June 30th, 2020, at Peking Union Medical College Hospital, a total of 54 cases of pancreatic cancer tissues and corresponding paracancerous pancreatic tissues (>5 cm from the surgical margin) resected and confirmed by pathology were collected. ASPC1 and BXPC3 pancreatic cancer cell lines were knocked down (ASPC1 and BXPC3 knockdown group), CFPAC1 and SW1990 pancreatic cancer cell lines were overexpressed (CFPAC1 and SW1990 overexpression group), the untreated ASPC1, BXPC3, CFPAC1 and SW1990 were set as blank vector control group. The expression at protein level of MYST2 was detected by Western blotting in patients with different degrees of pathological differentiation, human normal pancreatic duct epithelial cell line HPDE, human pancreatic cancer cell lines ASPC1, BXPC3, CFPAC1 and SW1990, knockdown group, overexpression group and blank vector control group. The cell proliferation, migration, invasion and colony formation ability of the knockdown group, overexpression group and blank vector control group were determined by real-time cellular analysis, Transwell migration and invasion test, and plate colony formation assay. MYST2 immunohistochemical scoring was performed on pancreatic cancer tissues and para cancer tissues. Receiver operating characteristic curve was drawn to analyze the value of different MYST2 protein expression levels in the diagnosis of pancreatic cancer. Independent sample t test and variance analysis were used for statistical analysis. Results:Among the pathological slides of 54 cases of pancreatic cancer, 13 cases were highly differentiated, 24 cases were moderately differentiated, 15 cases were poorly differentiated and 2 cases were undifferentiated, the MYST2 expression at protein level in pancreatic cancer cells was 3.12±1.67, 2.87±1.59, 2.12±1.03 and 1.08±0.34, respectively, and the difference was statistically significant ( F=1.241, P<0.05). The MYST2 expression levels of ASPC1, BXPC3, CFPAC1 and SW1990 were all higher than that of normal pancreatic ductal epithelial cell lines HPDE (1.41±0.47, 1.40±0.93, 1.13±0.62 and 1.71±0.46 vs. 0.82±0.25), and the differences were statistically significant( t=1.625, 1.577, 1.319 and 1.832, all P<0.05). The MYST2 expression level of BXPC3 knockdown group was lower than that of BXPC3 blank vector control group (0.39±0.12 vs. 0.75±0.34); that of ASPC1 knockdown group was lower than that of ASPC1 blank vector control group (0.43±0.22 vs. 0.82±0.48); that of CFPAC1 overexpression group was higher than that of CFPAC1 blank vector control group (1.38±0.45 vs. 0.82±0.37); that of SW1990 overexpression group was higher than that of SW1990 blank vector control group (1.34±0.65 vs. 0.51±0.22), and the differences were statistically significant ( t=1.414, 1.378, 1.319 and 1.934, all P<0.05). The cell proliferation of ASPC1 knockdown group was slower than that of ASPC1 blank vector control group, and the proliferation peak at 80 h was lower than that of blank vector control group (1.02±0.77 vs. 4.31±2.45); the cell proliferation of BXPC3 knockdown group was slower than that of BXPC3 blank vector control group, and the proliferation peak at 80 h was lower than that of blank vector control group (0.91±0.24 vs. 2.84±0.53); the proliferation of pancreatic cancer cells in SW1990 overexpression group was faster than that of SW1990 blank vector control group, and the proliferation peak at 80 h was higher than that of blank vector control group (3.10±0.67 vs. 1.04±0.17); the proliferation of pancreatic cancer cells in CFPAC1 overexpression group was faster than that that of CFPAC1 blank vector control group, and the proliferation peak at 80 h was higher than that of blank vector control group (5.45±1.13 vs. 1.01±0.29), and the differences were statistically significant ( t=1.427, 1.316, 1.292 and 1.501, all P<0.05). In the test of migration ability, the number of cells passed through the Transwell chamber of ASPC1 knockdown group was less than that of ASPC1 blank vector control group (34.08±17.62 vs. 118.76±5.31); that of BXPC3 knockdown group was less than that of BXPC3 blank vector control group (18.62±9.64 vs. 57.90±12.67); that of SW1990 overexpression group was more than that of SW1990 blank vector control group (134.84±24.65 vs. 37.82±6.73); that of CFPAC1 overexpression group was more than that of CFPAC1 blank vector control group (65.79±27.46 vs. 11.68±5.13), and the differences were statistically significant ( t=1.475, 1.322, 1.437 and 1.219, all P<0.05). In the test of invasion ability, the number of cells passed through the Transwell chamber of ASPC1 knockdown group was less than that of ASPC1 blank vector control group (9.79±5.75 vs. 45.76±12.71); that of BXPC3 knockdown group was less than that of BXPC3 blank vector control group (23.46±11.13 vs. 84.92±17.65); that of SW1990 overexpression group was more than that of SW1990 blank vector control group (156.42±34.50 vs. 42.13±22.17); that of CFPAC1 overexpression group was more than that of CFPAC1 blank vector control group (112.64±47.82 vs. 39.09±17.23), and the differences were statistically significant ( t=1.324, 1.635, 1.423 and 1.119, all P<0.05). The number of colony formation of the ASPC1 knockdown group was less than that of ASPC1 blank vector control group (13.15±6.42 vs. 86.79±35.17); that of BXPC3 knockdown group was less than that of BXPC3 blank vector control group (14.93±9.30 vs. 52.93±15.76); that of SW1990 overexpression group was more than that of SW1990 blank vector control group (129.10±57.31 vs. 62.42±37.43); that of CFPAC1 overexpression group was more than that of CFPAC1 blank vector control group (157.98±66.45 vs. 74.35±34.69), and the differences were statistically significant ( t=1.148, 1.290, 1.274 and 1.462, all P<0.05). The MYST2 score of pancreatic cancer tissues was higher than that of adjacent paracancerous pancreatic tissues (3.04±2.23 vs. 1.32 ± 0.70), and the difference was statistically significant ( t=3.479, P<0.05). When the total immunohistochemistry score of MYST2 was 3 point, the area under the curve was the largest (0.888, 95% confidence interval 0.827 to 0.948), and the Youden index was 0.56. Conclusion:MYST2 is associated with the proliferation, invasion and migration of pancreatic cancer cells, and promotes the development of pancreatic cancer.

Free fatty acid-induced histone acetyltransferase activity accelerates lipid accumulation in HepG2 cells

BACKGROUND/OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disease triggered by epigenetic alterations, including lysine acetylation at histone or non-histone proteins, affecting the stability or transcription of lipogenic genes. Although various natural dietary compounds have anti-lipogenic effects, their effects on the acetylation status and lipid metabolism in the liver have not been thoroughly investigated. MATERIALS/METHODS: Following oleic-palmitic acid (OPA)-induced lipid accumulation in HepG2 cells, the acetylation status of histone and non-histone proteins, HAT activity, and mRNA expression of representative lipogenic genes, including PPARγ, SREBP-1c, ACLY, and FASN, were evaluated. Furthermore, correlations between lipid accumulation and HAT activity for 22 representative natural food extracts (NExs) were evaluated. RESULTS: Non-histone protein acetylation increased following OPA treatment and the acetylation of histones H3K9, H4K8, and H4K16 was accelerated, accompanied by an increase in HAT activity. OPA-induced increases in the mRNA expression of lipogenic genes were down-regulated by C-646, a p300/CBP-specific inhibitor. Finally, we detected a positive correlation between HAT activity and lipid accumulation (Pearson's correlation coefficient = 0.604) using 22 NExs. CONCLUSIONS: Our results suggest that NExs have novel applications as nutraceutical agents with HAT inhibitor activity for the prevention and treatment of NAFLD.

Effect of Modified Wuzi Yanzong Pill () on Tip60-Mediated Apoptosis in Testis of Male Rats after Microwave Radiation / 中国结合医学杂志

OBJECTIVE@#To investigate the effect of a modified Wuzi Yanzong Pill (, WZYZP) on the male rats' testis after microwave radiation, as well as its potential mechanism.@*METHODS@#Forty-five male rats were randomly assigned to three groups: the control group, the radiation group, and the WZYZP group. The rats in the radiation group and WZYZP group were exposed to microwave radiation for 15 min once, while the rats in the control group were not exposed to any radiation. The rats in the WZYZP group were given a modified of WZYZP by gavage daily for 7 days. Apoptosis in the testis was evaluated using terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay. Histopathological alterations of the testis were observed by haematoxylin-eosin (HE) staining. Tat-interactive protein, 60kD (Tip60) and p53 expressions were determined by Western blotting.@*RESULTS@#The apoptosis index (AI) in the radiation group was higher than that of the WZYZP group and control group on day 1 (D1), day 7 (D7) day 14 (D14) after radiation (P<0.05). The seminiferous tubules were of normal morphology in the control group. In the radiation group, the partial seminiferous tubules were collapsed, basement membranes of the seminiferous epithelia became detached. WZYZP could restore the morphological changes. There was no expression of Tip60 among the three groups on D7 and D14. The expression of p53 was higher in the radiation group than in the control group (P<0.05). WZYZP could down-regulate the rising p53 induced by radiation on D7 and D14 (P<0.05).@*CONCLUSION@#A modified WZYZP may affect germ cells, and its protective effects may partly result from its ability to intervene in Tip60 mediated apoptosis.

Effects of cell-to-cell communication and histone acetyltransferase on the change of osteogenic differentiation ability among single-cell clones from healthy periodontium with heterogeneity of osteogenic differentiation abilities / 中华口腔医学杂志

Objective@#To investigate the effect of cell-to-cell communication amongst single-cell clones from healthy periodontium with different osteogenic differentiation potentials on change of osteogenic differentiation capabilities and the role histone acetyltransferase partaken in this process.@*Methods@#In order to research the change of osteogenic differentiation ability via cell-to-cell communication, indirect co-culture method was used by placing two single-cell clones with different osteogenesis potentials in each of the 6-well plates. Blank control, weak and strong osteogenic groups were set up, corresponding to Transwell chambers with blank, cells of weak osteogenesis ability and cells of strong osteogenesis ability, respectively. Each group was made in triplicate. After co-culture for four days, Transwell chamber was removed. Quantitative real-time PCR (qPCR) and alizarin red staining were employed to detect the change of osteogenic differentiation ability. The acetylation level of H3 was measured by using Western blotting. Histone acetyltransferases were detected by qPCR.@*Results@#Single-cell clones were ensured from mesenchymal stem cells by flow cytometer, the positive expression of CD29, CD90, CD105, CD146 was (99.80±0.02)%, (99.36±0.18)%, (99.41±0.05)% and (95.10±2.11)%, respectively. And CD31 and CD34 expression were (0.29±0.11)% and (0.22±0.13)%, respectively. Alizarin red and oil red O staining confirmed that single-cell clones had the abilities of adipogenesis and osteogenesis. Alkaline phosphatase (ALP) and alizarin red staining indicated that different single-cell clones were heterogeneity in osteogenesis differentiation. Indirect co-culture indicated that the mRNA expression of osteocalcin (OCN) were 14.24±5.60 and 4.78±2.90, respectively and Runt-related transcription factor 2 (RUNX2) were 2.75±1.44 and 1.61±0.44, respectively, in strong and weak osteogenic groups. They were significantly higher compared to the blank group (the mRNA expression of OCN and RUNX2 were 1.00±0.47 and 1.00±0.39, respectively). The expression of OCN and RUNX2 were also higher in strong osteogenic group than that in weak osteogenic group (P<0.05). The mean gray level of the acetylation of H3 in strong osteogenic group (0.76±0.09) and weak osteogenic group (0.54±0.12) were also higher than that in the blank group (0.30±0.04)(P<0.05). qPCR results showed that KAT6A in strong osteogenic group exhibiting higher expression (P<0.05) compared to weak osteogenic group and the blank group, which were corresponding to the changes of acetylation levels.@*Conclusions@#Single-cell clones from healthy periodontium showed heterogeneity in osteogenic differentiation abilities. Single-cell clones with strong osteogenesis abilities had an advantage over others by promoting others' osteogenesis differentiation and this change mediated by cell-to-cell communication might be caused by modulating KAT6A to affect the acetylation level of histone.

Establishment of interfering RNA library of acetyltransferase genes and its infection of HepG2.2.15 cells / 中华传染病杂志

Objective To construct lentivirus vectors carrying 16 short hairpin RNA (shRNA) expression cassettes targeting histone acetyltransferases and provide a powerful research approach to explore the mechanism of epigenetic genes in regulating hepatitis B virus (HBV).Methods Following the rule of short shRNA primer design,eight-pair primers (A ~ H )for each gene,which had stable interfering efficiency,were designed.The annealed primers were connected to the empty lentiviral vectors of shRNA for transformation.In order to confirm the positive clones,clones were analyzed by real-time polymerase chain reaction (RT-PCR ).Then, qualified plasmids were verified by enzyme digestion technology.Four shRNA lentivirus plasmids against the same gene were mixed to build lentivirus respectively.After the virus transfected into 293T cells for 48 and 72 hours,supernatants were collected to infect HepG2.2.15 cells.The percentage of fluorescent cells were observed and assessed by microscope 72 hours after infection.Results One hundred and twenty-eight lentiviral vectors of RNA interference (RNAi)library were constructed against 16 histone acetyltransferases and more than 80% of HepG2.2.15 cells were infected with lentivirus 72 hours after infection.Conclusions Sixteen shRNA lentivirus vectors against histone acetyltransferase are successfully constructed.Thus,a solid foundation for the study of the effect of human histone deacetylase on HBV replication is established.

The biological characteristics of p300/CBP-associated factor and relationship with disease / 国际儿科学杂志

p300/CBP-associated factor (PCAF)is an important histone acetyltransferase in eukaryotic cells. PCAF can acetylizes histone and non-histone. PCAF participates in various biological processes of cells and in the interaction between virus and cells. The imbalance of PCAF would lead to abnormal development of various organs, and related to the development of some tumors.

The Histone Acetyltransferase hMOF is Overexpressed in Non-small Cell Lung Carcinoma

BACKGROUND: One of the histone acetyltransferases (HATs) family of proteins, human MOF (hMOF, MYST1), is involved in histone H4 acetylation, particularly at lysine 16 (H4K16Ac), an epigenetic mark of active genes. Dysregulation of the epigenetic mark influences cellular biology and possibly leads to oncogenesis. We examined the involvement of hMOF and H4K16Ac in primary non-small cell lung cancer (NSCLC). METHODS: Reverse transcription polymerase chain reaction using fresh-frozen lung cancer tissues and lung cancer cell lines and immunohistochemistry for hMOF and H4K16Ac via tissue microarray of 551 formalin-fixed paraffin-embedded NSCLC tissue blocks were conducted. RESULTS: hMOF mRNA was frequently overexpressed in lung cancer tissues, compared with normal lung tissues (10/20, 50%). NSCLC tissues were positive for hMOF in 37.6% (184/489) and H4K16Ac in 24.7% (122/493) of cases. hMOF protein expression was tightly correlated with the H4K16Ac level in tumors (p<0.001). Knockdown of hMOF mRNA with siRNA led to a significant inhibition of growth in the Calu-6 cell line. CONCLUSIONS: hMOF was frequently expressed in NSCLC and was correlated with H4K16Ac. To our knowledge, this is the first study that has focused on the expression status of HATs and hMOF in NSCLC. Our results clearly suggest a potential oncogenic role of the gene and support its utility as a potential therapeutic target.

Spatiotemporal expression patterns of histone acetyltransferase steroid receptor coactivator-1 in developing mouse heart / 第二军医大学学报

Objective: To study the spatiotemporal expression pattern of histone acetyltransferase steroid receptor coactivator-1 (SRC1) in developing mouse heart, so as to explore the relationship of SRC1 with heart development. Methods: The normal mouse hearts were collected at E7.5-E18 and 1 day and 3 months after birth; 9 specimens were chosen for each time point to observe the spatial expression of SRC1 by immunohistochemistry technique, and 6 specimens of each time point were used to examine the temporal expression of SRC1 protein by Western blotting technique and to plot the time-dose curve. Results Immunohistochemistry showed no expression of SRC1 in the heart primordium at E7.5; only very weak SRC1 expression was found in the cardiac tube at E8. 5-E9.5. Weak SRC1 expression was found in the trabeculae after E10.5 and relatively strong and widespread expression was found in other heart regions. Western blotting results demonstrated that SRC1 protein expression at E10.5 was significantly lower than those at E11.5 and E12.5 (P0.05). SRC1 expression reached the peak at E11. 5-E12.5, and there was no significant difference between the two time points (P>0.05). SRC1 expression gradually decreased after Ell. 5-E12.5 till the adulthood, and there were no significant differences in the expression after E13.5 (P>0.05). Conclusion: Widespread distribution of SRCl is present in the developing mouse heart after ES. 5, and the expression is in a dynamical spatiotemporal pattern, suggesting that SRC1 may take part in the overall regulation of the heart development, and it might has a closer relationship with the early induction of the heart septa.

Repression of TNF-alpha-induced IL-8 expression by the glucocorticoid receptor-beta involves inhibition of histone H4 acetylation

Increased expression of a number of proinflammatory genes, including IL-8, is associated with inflammatory conditions such as asthma. Glucocorticoid receptor (GR)beta, one of the GR isoforms, has been suggested to be upregulated in asthma associated with glucocorticoid insensitivity and to work as a dominant negative inhibitor of wild type GRalpha. However, recent data suggest that GRbeta is not a dominant negative inhibitor of GRalpha in the transrepressive process and has its own functional role. We investigated the functional role of GRbeta expression in the suppressive effect of glucocorticoids on tumor necrosis factor (TNF)-alpha-induced IL-8 release in an airway epithelial cell line. GRbeta expression was induced by treatment of epithelial cells with either dexamethasone or TNF-alpha. GRbeta was able to inhibit glucocorticoid-induced transcriptional activation mediated by binding to glucocorticoid response elements (GREs). The suppressive effect of dexamethasone on TNF-alpha-induced IL-8 transcription was not affected by GRbeta overexpression, rather GRbeta had its own weak suppressive activity on TNF-alpha-induced IL-8 expression. Overall histone deacetylase activity and histone acetyltransferase activity were not changed by GRbeta overexpression, but TNF-alpha-induced histone H4 acetylation at the IL-8 promoter was decreased with GRbeta overexpression. This study suggests that GRbeta overexpression does not affect glucocorticoid-induced suppression of IL-8 expression in airway epithelial cells and GRbeta induces its own histone deacetylase activity around IL-8 promoter site.

Repression of TNF-alpha-induced IL-8 expression by the glucocorticoid receptor-beta involves inhibition of histone H4 acetylation

Increased expression of a number of proinflammatory genes, including IL-8, is associated with inflammatory conditions such as asthma. Glucocorticoid receptor (GR)beta, one of the GR isoforms, has been suggested to be upregulated in asthma associated with glucocorticoid insensitivity and to work as a dominant negative inhibitor of wild type GRalpha. However, recent data suggest that GRbeta is not a dominant negative inhibitor of GRalpha in the transrepressive process and has its own functional role. We investigated the functional role of GRbeta expression in the suppressive effect of glucocorticoids on tumor necrosis factor (TNF)-alpha-induced IL-8 release in an airway epithelial cell line. GRbeta expression was induced by treatment of epithelial cells with either dexamethasone or TNF-alpha. GRbeta was able to inhibit glucocorticoid-induced transcriptional activation mediated by binding to glucocorticoid response elements (GREs). The suppressive effect of dexamethasone on TNF-alpha-induced IL-8 transcription was not affected by GRbeta overexpression, rather GRbeta had its own weak suppressive activity on TNF-alpha-induced IL-8 expression. Overall histone deacetylase activity and histone acetyltransferase activity were not changed by GRbeta overexpression, but TNF-alpha-induced histone H4 acetylation at the IL-8 promoter was decreased with GRbeta overexpression. This study suggests that GRbeta overexpression does not affect glucocorticoid-induced suppression of IL-8 expression in airway epithelial cells and GRbeta induces its own histone deacetylase activity around IL-8 promoter site.

A Simple Reliable Fluorescent Assay Method for Histone Acetyltransferase (HAT) Inhibitors / 生物化学与生物物理进展

Histone acetyltransferases (HATs) are involved in the regulation of gene transcription in eukaryotic cells and their inhibitors could be a promising class of drugs due to their ability to modulate transcription and exert antiviral, anti-inflammatory as well as antioxidant effects. Nonradioactive spectrophotometric HAT assay is an alternative method to the widespread radioactive assay but suffers from drawbacks as lack of sensitivity and accuracy. A simple, non-radioactive fluorescent assay that measures the production of CoASH was established by its facile reaction with O-phthalaldehyde and 2-amino-ethanol. This method gives much higher accuracy compared to spectrophotometric assay, and allows screening of various compounds with potential HAT inhibition. The novel assay should be a valuable tool in transcriptional research and especially drug discovery.

Expression of the RERG Gene is Gender-Dependent in Hepatocellular Carcinoma and Regulated by Histone Deacetyltransferases

Ras-related, estrogen-regulated, and growth-inhibitory gene (RERG) is a novel gene that was first reported in breast cancer. However, the functions of RERG are largely unknown in other tumor types. In this study, RERG expression was analyzed in hepatocellular carcinomas of human patients using reverse transcriptase PCR analysis. In addition, the possible regulation of RERG expression by histone deacetyltransferases (HDACs) was studied in several cell lines. Interestingly, the expression of RERG gene was increased in hepatocellular carcinoma (HCC) of male patients (57.9%) but decreased in HCC of females (87.5%) comparison with paired peri-tumoral tissues. Moreover, RERG gene expression was increased in murine hepatoma Hepa1-6 cells, human breast tumor MDA-MB-231 cells, and mouse normal fibroblast NIH3T3 cells after treated by HDAC inhibitor, trichostatin A. Our results suggest that RERG may function in a gender-dependent manner in hepatic tumorigenesis and that the expression of this gene may be regulated by an HDAC-related signaling pathway.

Screening and identification of interaction proteins of human TIP60? by yeast two- hybrid system / 第三军医大学学报

Objective To construct a yeast expression vector containing human TIP60? gene (a spliced form of HIV-1 tat interactive protein,HTATIP,60?103) and screen its interaction proteins by yeast two-hybrid. Methods Human TIP60? gene fragment was amplified by RT-PCR and cloned into pGBKT7 vector. Using TIP60? as bait,the proteins interacting with TIP60? were screened from a human liver cDNA library. The positive clones were analyzed by bioinformatic methods. Results Human TIP60? cDNA was successfully amplified and cloned into the pGBKT7 vector and indentified by double enzyme digestion and DNA sequencing. Using yeast two-hybrid,32 clones were screened from a human liver cDNA library,and 9 positive clones including histone deacetylase 7A (HDAC7A),mouse double minute 2 (DMD2),B-cell CLL/lymphoma 3 (BCL3),endothelin receptor type A (EDNRA),androgen receptor (AR),PHD finger protein 17 (PHF17),Ataxia Telangiectasia Mutated (ATM),CAMP responsive element binding protein 1 (CREB1),orphan steroid receptor BD73 were verified after sequencing. Conclusion The screened interaction proteins with TIP60? by yeast two-hybrid may be involved in the roles of transcriptional regulation of TIP60?.