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Objective:To investigate the role of modification level of lysine trimethylation at position 27 of histone 3 (H3K27me3) on expression of anti-apoptotic protein B lymphocyte tumor-2 gene (BCL2) during arsenic-induced hepatocyte apoptosis.Methods:Rat liver BRL-3A cells were cultured in vitro. According to the arsenic treatment factor, the experiment was divided into two parts, in the first part arsenic was not added, the experiment was divided into normal, transfection reagent, negative transfection, H3K27me3 specific demethylase (JMJD3) small interfering RNA (siRNA) transfection and H3K27me3 methyltransferase (EZH2) siRNA transfection groups. In the second part arsenic was added, the experiment was divided into control, arsenic treatment, arsenic + negative transfection, arsenic + JMJD3siRNA transfection and arsenic + EZH2siRNA transfection groups. When arsenic was not added, the corresponding siRNA and transfection reagent was used to transfect cells at a ratio of 100 pmol : 7.5 μl for 6 h [the normal group was treated with phosphate buffer solution (PBS) of the same volume as transfection reagent], then the medium was changed and the cells were incubated for a total of 48 h. After 24 h of treatment with the above transfection and culture method in arsenic added group, a final concentration of 30 μmol/L sodium arsenite (NaAsO 2) was added and the cells were incubated for 24 h (the control group was treated with PBS with the same volume of NaAsO 2 for 24 h). Real-time cell analysis (RTCA) was used to measure the proliferation of BRL-3A cells in arsenic added group. Apoptosis of BRL-3A cells was analyzed by flow cytometry in arsenic added group. Western blotting was used to detect JMJD3, EZH2, H3K27me3 and BCL2 in no-arsenic and arsenic-added BRL-3A cells. The modification levels of H3K27me3 in BCL2 gene promoter regions were detected by chromatin immunoprecipitation of the cells exposed to arsenic. Results:There were statistically significant differences of the proliferation rates [control, arsenic treatment, arsenic + negative transfection, arsenic + JMJD3siRNA transfection and arsenic + EZH2siRNA transfection groups: (100.00 ± 10.43)%, (12.19 ± 3.37)%, (31.86 ± 1.95)%, (24.58 ± 3.64)%, (11.53 ± 1.11)%] and the apoptosis rates [(1.15 ± 0.04)%, (13.06 ± 1.33)%, (17.39 ± 0.22)%, (23.90 ± 1.66)%, (15.07 ± 0.88)%] between groups ( F = 146.50, 194.30, P < 0.001), correspondingly. The protein expression level of H3K27me3 in JMJD3siRNA transfection group was higher than that of normal, transfection reagent and negative transfection groups, while EZH2siRNA transfection group had an opposite result ( P < 0.05). The protein expression level of BCL2 in JMJD3siRNA transfection group was lower than that of normal, transfection reagent and negative transfection groups, while EZH2siRNA transfection group had an opposite result ( P < 0.05). The protein expression levels of H3K27me3 and BCL2 were not statistically significant differences between normal, transfection reagent and negative transfection groups ( P > 0.05). The protein expression levels of JMJD3, EZH2, H3K27me3 and BCL2 among control, arsenic treatment, arsenic + negative transfection, arsenic + JMJD3siRNA transfection and arsenic + EZH2siRNA transfection groups were compared, and the differences were statistically significant ( F = 26.56, 7.82, 9.81, 31.19, P < 0.05). Compared with control group, the protein expression levels of JMJD3 and EZH2 in arsenic treatment group were significantly reduced ( P < 0.05), and the protein expression level of H3K27me3 was higher ( P < 0.05), meanwhile the protein expression level of BCL2 was lower ( P < 0.05). Compared with arsenic + negative transfection group, the protein expression level of JMJD3 was significantly reduced in arsenic + JMJD3siRNA group, and the protein expression level of EZH2 was significantly reduced in arsenic + EZH2siRNA group ( P < 0.05). In addition, arsenic + JMJD3siRNA increased the level of H3K27me3 modification while reducing the protein expression of BCL2, while arsenic + EZH2siRNA had an opposite result ( P < 0.05). Compared with control group, the enrichment levels of H3K27me3 in BCL2 gene promoter regions (CHIP1 and CHIP2) in arsenic treatment group were significantly higher ( P < 0.05). Conclusion:Arsenic may inhibit the expression of BCL2 by increasing the enrichment level of H3K27me3 in the promoter regions of BCL2 gene, and promoting hepatocyte apoptosis.
RESUMO
Objective:To investigate the changes of microRNA-153 (miR-153) expression and the mechanism of regulating histone H3 lysine 4 (H3K4) methyltransferase (SET7/9) and histone H3K4 methylation (H3K4me1) in the process of arsenic-induced endoplasmic reticulum stress-related hepatocytes apoptosis.Methods:Human normal hepatocytes (L-02 cells) were cultured in vitro and divided into control, arsenic treatment, arsenic + negative transfection, arsenic + miR-153 up-regulation and arsenic+ miR-153 down-regulation groups according to different treatment methods. Arsenic+ negative transfection, arsenic+ miR-153 up-regulation and arsenic+ miR-153 down-regulation groups were transfected with transfection plasmid and transfection reagent according to a certain proportion (3 μg: 8 μl). After 24 h, arsenic treatment, arsenic+ negative transfection, arsenic+ miR-153 up-regulation and arsenic+ miR-153 down-regulation groups were all treated with 100 μmol/L sodium arsenite (NaAsO 2) as the final concentration for 24 h. The control group was treated with phosphate buffer solution (PBS) of the same volume as NaAsO 2 for 24 h. The expression of miR-153 was detected by real-time quantitative polymerase chain reaction (RT-qPCR); cell apoptosis and cell cycle were detected by flow cytometry; real-time cell dynamic analyzer (RTCA) was used to detect cell proliferation; Western blotting was used to detect the expression of endoplasmic reticulum marker proteins glucose regulatory protein 78 (GRP78), SET7/9 and H3K4me1. Results:The expression levels of miR-153 in each group were significantly different ( F = 10.73, P < 0.05). Compared with the control group [(41.10 ± 6.08)%], the expression level of miR-153 in arsenic treatment group [(4.35 ± 0.20)%] was significantly decreased ( P < 0.05); compared with the arsenic+ negative transfection group [(10.00 ± 2.40)%], the expression level of miR-153 in arsenic+ miR-153 up-regulation group [(157.70 ± 42.70)%] was significantly increased ( P < 0.05), and that in arsenic+ miR-153 down-regulation group [(4.20 ± 0.28)%] was significantly decreased ( P < 0.05). There were significant differences in the total cell apoptosis rate and G1 phase cell proportion among the five groups ( F = 29.69, 104.32, P < 0.05). Compared with the control group, the total cell apoptosis rates and G1 phase cell proportions in arsenic treatment, arsenic+ miR-153 up-regulation and arsenic+ miR-153 down-regulation groups were significantly increased ( P < 0.05); compared with the arsenic+ negative transfection group, the total cell apoptosis rate and G1 phase cell proportion in arsenic+ miR-153 up-regulation group were significantly decreased ( P < 0.05), and those in arsenic+ miR-153 down-regulation group were significantly increased ( P < 0.05). The difference of cell proliferation rate in each group was statistically significant ( F = 799.35, P < 0.05). Compared with the control group, the cell proliferation rates in arsenic treatment, arsenic+ miR-153 up-regulation and arsenic+ miR-153 down-regulation groups were significantly decreased ( P < 0.05); compared with the arsenic+ negative transfection group, the cell proliferation rate in arsenic+ miR-153 up-regulation group was significantly increased ( P < 0.05), and that in arsenic+ miR-153 down-regulation group was significantly decreased ( P < 0.05). The protein expression levels of SET7/9, GRP78 and H3K4me1 in each group were significantly different ( F = 78.52, 52.13, 54.32, P < 0.05). Compared with the control group, the protein expression levels of SET7/9, GRP78 and H3K4me1 in arsenic treatment group were significantly increased ( P < 0.05); compared with the arsenic+ negative transfection group, the protein expression levels of SET7/9, GRP78 and H3K4me1 in arsenic+ miR-153 up-regulation group were significantly decreased ( P < 0.05), and those in arsenic + miR-153 down-regulation group were significantly increased ( P < 0.05). Conclusion:miR-153 plays an important role in arsenic-induced endoplasmic reticulum stress-related hepatocytes apoptosis, the expression and regulation are related to the changes of SET7/9 and H3K4me1 levels.
RESUMO
Esophageal variceal bleeding is the most dangerous complications of portal hypertension caused by cirrhosis.It is a common emergency of digestive diseases,with characteristics of a large amount of bleeding,difficulties in rescue and high mortality.Treatment methods include drug therapy,surgical treatment, interventional therapy,combined therapy,endoscopic therapy,etc.Endoscopic therapy has become the main means to treat EVB because of its high success rate of hemostasis,little trauma and rapid hemostasis.With the continuous improvement of endoscopic equipment and technology,endoscopic treatment plays a more prominent role in the rescue process.This article reviews the endoscopic treatment of esophageal variceal bleeding.