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Objective:To investigate the expression of DNA damage repair factor DNA damage-binding protein 1 (DDB1) in hepatocellular carcinoma tissues, and the effect of DDB1 gene silencing on DNA repair and targeted killing in human hepatocellular carcinoma SMMC-7721 cells.Methods:The UALCAN platform was used to perform bioinformatics analysis on the expression of DDB1 in hepatocellular carcinoma tissues (371 cases) and paracancerous tissues (50 cases) in The Cancer Genome Atlas (TCGA) database and the correlation of DDB1 expression with the overall survival of liver cancer patients were analyzed by bioinformatics using the UALCAN platform. SMMC-7721 cells were transfected with small interfering RNA (siRNA) targeting DDB1 and negative control siRNA, which were DDB1 silencing group and negative control group, respectively. X-ray irradiation induced exogenous DNA double strand break (DSB) damage in the two groups of cells. Immunofluorescence staining (γH2AX was used for assessing cellular DSB damage, RPA32s33 and Rad51 were used for assessing homologous recombination repair) and Western blotting (were used to detect the level of RPA32s33 protein) were used to analyze the effect of DDB1 gene silencing on DSB damage repair. Sister chromosome exchange (SCE) experiment was used to analyze the frequency of SCE of homologous recombination of cells in DDB1 silencing group and negative control group. Tetramethylazozolium salt (MTT) method was used to analyze the killing effect of PARP inhibitor olapani (10 μmol/L), cisplatin (1 μg/ml) and olapani combined with cisplatin on SMMC-7721 cells in DDB1 silencing group and negative control group.Results:Bioinformatics analysis showed that the level of DDB1 mRNA in liver cancer tissues was higher than that in paracancerous tissues ( P < 0.001), and the overall survival of patients with high expression of DDB1 was worse than that of patients with low expression of DDB1 ( P = 0.029). When cultured for 4 hours after X-ray irradiation, the number of γH2AX foci in cells of the negative control group had mostly disappeared, and there were still more cells in DDB1 silencing group [(5.1±2.0) per cell vs. (13.4±2.0) per cell, t = -5.08, P = 0.007]. When cultured for 4 hours after X-ray irradiation, the number of RPA32s33 foci in the negative control group [(30.8±5.0) per cell vs. (13.2±1.6) per cell] and the number of Rad51 foci [(19.5±1.8) per cell vs. (8.3±3.3) per cell] were higher than those in the DDB1 silencing group, and the differences between the two groups were statistically significant (both P < 0.01). The frequency of SCE in the negative control group was higher than that in the DDB1 silencing group [(21.2±3.0)% vs. (11.2±1.6)%, t = 5.07, P = 0.007]. MTT assay showed that after olaparib treatment, the survival rate of cells in the DDB1 silencing group was lower than that in the negative control group [(40.3±3.6)% vs. (79.8±1.3)%, t = 17.94, P < 0.001]. When treated with olapani combined with cisplatin, the survival rate of cells in the two groups further decreased, and the survival rate of cells in the DDB1 silencing group was lower than that in the negative control group [(10.2±2.8)% vs. (29.6±3.4)%, t = 7.72, P = 0.002]. When treated with cisplatin alone, there was no significant difference in cell survival between DDB1 silencing group and negative control group [(41.9±5.1)% vs. (49.8±3.3)%, t = 2.71, P = 0.054]. Conclusions:The high expression of DDB1 in hepatocellular carcinoma tissues may be an important factor in the treatment resistance and poor prognosis of hepatocellular carcinoma. Knockdown of DDB1 gene expression can promote the sensitivity of SMMC-7721 cells to PARP inhibitors, and its mechanism may be related to the homologous recombination repair defect of SMMC-7721 cells caused by DDB1 silencing.
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Objective:To investigate the value of antithrombin Ⅲ (AT-Ⅲ) in evaluating patients with decompensated hepatitis B liver cirrhosis and complicated with esophagogastric variceal bleeding (EVB).Methods:From January 1, 2018 to December 31, 2021, clinical data of 193 hospitalized patients with hepatitis B liver cirrhosis diagnosed in the Second Hospital of Shanxi Medical University were retrospectively analyzed, which included coagulation indicator (AT-Ⅲ), liver function indicators (total bilirubin, etc.), abdominal ultrasound results (portal vein diameter, portal vein blood flow velocity), and the occurrence of esophagogastric varices. According to the presence or absence of main complications, 193 patients with hepatitis B liver cirrhosis were divided into compensated group (60 cases) and decompensated group (133 cases). According to the presence or absence of EVB, 133 patients of decompensated group were divided into non-bleeding subgroup (96 cases) and bleeding subgroup (37 cases). The above indicators were compared among compensated group, decompensated group and their subgroups. The independent related factors of decompensated hepatitis B liver cirrhosis and EVB were analyzed. The level of AT-Ⅲ of each group were compared, and the relationship between AT-Ⅲ and Child-Pugh score was analyzed. The diagnostic capability of AT-Ⅲ in decompensated hepatitis B liver cirrhosis and complicated with EVB were analyzed. Mann-Whitney U test, independent sample t test, chi-square test, multiple logistic regression analysis, Pearson correlation analysis and receiver operating characteristic curve (ROC) analysis were used for statistical analysis. Results:The total bilirubin level of the decompensated group was higher than that of the compensated group, the portal vein diameter was larger than that of the compensated group, and the portal vein blood flow velocity was lower than that of the compensated group (31.50 μmol/L (21.90 μmol/L, 48.80 μmol/L) vs. 19.40 μmol/L (15.00 μmol/L, 25.50 μmol/L); (14.31±3.53) mm vs. (12.57±3.83) mm; (13.39±3.49) cm/s vs. (15.08±4.28) cm/s), and the differences were statistically significant ( Z=-5.76, t=-2.78 and 2.40; P<0.001, =0.006 and 0.018). The incidence of esophagogastric varices of the compensated group and the decompensated group was compared (40.0%, 24/60 vs. 87.2%, 116/133), and the difference was statistically significant ( χ2=64.06, P<0.001). The diameter of portal vein of the bleeding subgroup was larger than that of the non-bleeding subgroup, and the portal vein blood flow velocity was lower than that of the non-bleeding subgroup ((15.54±4.23) mm vs. (13.87±3.16) mm; (12.05±3.12) cm/s vs. (13.85±3.51) cm/s), and the differences were statistically significant ( t=-2.15 and 2.23, P=0.034 and 0.028). The AT-Ⅲ levels gradually decreased in the non-bleeding subgroup and bleeding subgroup of the compensated group and decompensated group, which were (79.52±16.02)%, (63.91±19.96)% and (35.92±13.69)%, respectively, the difference was statistically significant ( F=5.71, P=0.018). The AT-Ⅲ level of the compensated group was higher than that of the non-bleeding subgroup and the bleeding subgroup of the decompensated group, and the AT-Ⅲ level of the non-bleeding subgroup of the decompensated group was higher than that of the bleeding subgroup, and the differences were statistically significant ( t=5.11, 13.74 and 7.84, all P<0.001). The results of multivariate logistic regression analysis showed that total bilirubin and AT-Ⅲ were independent related factors of decompensation of hepatitis B liver cirrhosis ( OR (95% confidence interval (95% CI) 1.060 (1.018 to 1.104) and 0.945 (0.922 to 0.970), P=0.005 and <0.001). AT-Ⅲ was an independent related factor of decompensation of hepatitis B liver cirrhosis and complicated with EVB ( OR(95% CI) 0.902 (0.856 to 0.950, P<0.001). AT-Ⅲ was negatively correlated with Child-Pugh score ( r=-0.559, P<0.001). ROC analysis showed that the cut-off values of AT-Ⅲ in the diagnosis of decompensated stage of hepatitis B liver cirrhosis and complicated with EVB were 62.5% and 61.5%, the sensitivity was 88.3% and 89.2%, the specificity was 70.7% and 61.5%, and the area under the curve (95% CI) was 0.815 (0.755 to 0.874, P<0.001) and 0.899 (0.828 to 0.971, P<0.001), respectively. Conclusion:AT-Ⅲ is an important indicator in evaluating the severity of disease progression in patients with hepatitis B liver cirrhosis, and it has a certain clinical value in evaluating the bleeding tendency of patients with decompensated hepatitis B liver cirrhosis and complicated with esophagogastric varices.
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Objective:To investigate the relationship between antithrombin Ⅲ (AT-Ⅲ) levels and CHA2DS2-VASc scores to assess the thromboembolism risk in patients with non-valvular atrial fibrillation (NVAF), and to explore the value of AT-Ⅲ in the risk assessment of thrombosis in these patients.Methods:We enrolled patients diagnosed with NVAF (observation group) and non-atrial fibrillation (control group), hospitalized in Fuwai Hospital of Chinese Academy of Medical Sciences from October 2018 to June 2019, and assessed the two groups for AT-Ⅲ, protein C, protein S, and lipid levels including lipoprotein (a), three acyl glycerin (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C). Based on the CHA2DS2-VASc score, patients with NVAF and a score of less than 2 were assigned to the low-and middle-risk groups; the high-risk group consisted of patients with a score of 2 or more. The diagnostic performance of AT-Ⅲ was evaluated using receiver operating characteristic (ROC) curve analysis, and the risk factors for high CHA2DS2-VASc scores were analyzed using logistic regression.Results:Overall, 206 cases were enrolled in the observation group, including 54 women (26%; aged 59.85±11.06 years). The control group consisted of 76 cases, with 19 women (25%; aged 59.34±9.84 years). The two groups were gender ( χ2=0.043, P=0.836) and age ( t=0.352, P=0.725) matched. In the observation group, AT-Ⅲ activity (98.68%±11.37%) was significantly lower than that in the control group (110.87%±13.91%), demonstrating a statistically significant difference ( t=-6.841, P<0.001). In total, 102 cases (49.5%) were assigned to the high-risk group, with 104 cases (50.5%) in the low-and medium-risk groups. In the high-risk group, the AT-Ⅲ activity (93.67%±9.92%) was significantly lower than that in the low-and middle-risk groups (103.60%±10.56%), with a statistically significant difference observed ( t=6.953, P<0.001). In the high-risk group, protein C ［(94.34±26.61)% vs. (102.63±22.74)%］, TC ［(4.09±1.02) mmol/L vs. (4.69±0.97) mmol/L］, and LDL-C ［(2.18±0.83) mmol/L vs. (2.74±0.88) mmol/L］ levels were lower than those observed in the low-risk group (P<0.05). For NVAF screening, the AT-Ⅲ early warning threshold was 96.5%, and the area under the ROC curve was 0.746 (95% CI: 0.681-0.812, P<0.001). Based on logistic regression analysis, low AT-Ⅲ activity levels were an independent risk factor for high CHA2DS2-VASc scores in NVAF ( OR=7.282，95% CI: 3.098-17.117， P<0.001). Additionally, logistic regression analysis demonstrated that with increasing age ( OR=44.339, 95% CI: 15.207-129.276), lower levels of AT-Ⅲ ( OR=7.282, 95% CI: 3.098-17.117) and TC ( OR=4.349, 95% CI: 1.739-10.875), and higher CHA2DS2-VASc scores were observed for non-valvular AF ( P<0.05). Conclusion:A positive correlation exists between the CHA2DS2-VASc score and old age, low AT-Ⅲ activity, and low TC levels, indicating a high reference value for evaluating thrombosis in NVAF.
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ObjectiveTo investigate the effects and mechanism of rosuvastatin on the expression of tissue factor in cultured human monocyte-macrophages cells which was induced by oxidized low density lipoprotein(ox-LDL).MethodsThe human monocyte-macrophages cells were divided into four groups:control group,ox-LDL group,Poly-inosine monophosphate (Poly Ⅰ) group,rosuvastatin group.The expression of LOX-1mRNA and TF mRNA was assayed by RT-PCR.The ELISA was performed to determine the protein concentration of TF.ResultsCompared with control group,the expression of LOX-1 mRNA and TF mRNA was increased in the ox-LDL group[ (3.25156±0.15772) vs (1±0) ; (2.522451±0.138967) vs (1±0) ],and it was in a concentration-dependent manner (P＜0.01).Compared with the expression of LOX-1 mRNA in the Poly-inosine monophosphate group and rosuvastatin group,TF mRNA were decreased in the ox-LDL group[ (2.95139±0.157253) vs(3.25156±0.15772) ; (2.877343±0.156558) vs(3.25156±0.15772) ; (1.811956±0.169699) vs (2.522451±0.138967) ; (1.687701±0.174647) vs (2.522451±0.138967)],and it was in a concentration-dependent manner(P＜0.05).Compared with control group,the expression of TF in the ox-LDL group was increased [(207.7233±1.154701) vs (184.8467±0.871799) ],and it was in a concentration-dependent manner (P＜0.01).Compared with the Poly-inosine monophosphate group and rosuvastatin group [(197.8733±1.505003) vs (207.7233±1.154701) ;(202.9567±2.722744)vs(207.7233±1.154701) ],the expression of TF in the ox-LDL group were decreased,and it was in a concentration-dependent manner (P＜0.05).ConclusionsLOX-1 may be responsible for the expression of TF in human monocyte-macrophages cells induced by ox-LDL.Rosuvastatin is able to down-regulate the expression of LOX-1mRNA in human monocyte-macrophages cells through oxLDL,and TF mRNA and TF expression can be reduced.
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Objective To investigate the relationship between cytokines and human graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods In 21 patients undergoing allo-HSCT,the plasma concentrations of cytokines[soluble interleukin 2 receptor(sIL-2R), interferon-gama (IFN-γ), transforming growth factor-betal (TGF-β1)] were measured by using sandwich enzyme-linked immunological assay (ELISA) and the gene expressions of three cytokines were analysed by using semi-quantitate reverse transcriptase-polymerase chain reaction(RT-PCR). Results The concentrations and gene expressions of sIL-2R and IFN-γin the patients with GVHD were significantly higher than those without GVHD (P <0.01), and they were higher in the patients with aGVHD than with cGVHD and without GVHD(P <0.05); the levels of TGF-β1 in the patients with GVHD were significantly declined(P <0.01), but in those without aGVHD were obviously increased(P <0.05). After effective treatment, unnormal sIL-2R, IFN-γand TGF-β1 expressions recovered to the levels before transplantation. A multivariate COX analysis showed sIL-2R and TGF-β1 are independent prognostic factors for GVHD (P<0.001). Conclusion Monitoring the changes of sIL-2R, IFN-γand TGF-β1 expression levels (especially sIL-2R and TGF-β1) might provide predictive markers for GVHD after allo-HSCT. The sensitivity between RT-PCR and ELISA for detecting cytokines expressions had no difference.