ABSTRACT
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 determine the effects of Jinlong capsule combined with interventional therapy on the immune functions of primary hepatocellular carcinoma patients. Methods:Sixty randomly selected cases of clinically diagnosed primary hepatocellular carcinoma were divided into the observation group and the control group. Three days after operation, the observation group was given four Jinlong capsules three times a day for 30 days (one treatment). Meanwhile, the control group received interventional therapy after the operation. One to four days following one treatment, peripheral blood specimens were collected from the two groups to determine the cellular immune function indices. Results:The cell numbers (mean) of the peripheral blood components CD3, CD4, NK, SIL-2R, TSGF, and SIL-2R and the CD4/CD8 ratio in the observation group showed no significant difference before and after treatment. In the control group, these indices were significantly different before and after treatment. Conclusion:The Jinlong capsule facilitates the cellu-lar immunity recovery of patients with primary hepatocellular carcinoma after interventional therapy.
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Objective To explore the tumor markers for the diagnosis of primary liver carcinomas (PLC) by detecting the serum protein spectrum differently expressed between PLC patients and healthy controls. Methods We detected the serum protein spectrum in 50 PLC patients and 50 healthy controls using surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) technique and find the significant protein peaks. The serum Alpha-fetoprotein (AFP) levels in all 100 serum samples were also measured by ELISA. Results The protein peaks, which could discriminate healthy individuals from PLC patients, were detected. Four protein molecules (3354.71, 8825.80, 4345.08, 13 715.01) had a significant difference between PLC patients and the normal controls (P <10-5), indicating that these protein molecules might be a potential marker for PLC. The specificity and sensitivity of SELDI-TOF-MS were 94% and 90% respectively. Sixteen PLC patients were AFP positive and the sensitivity was 54%(27/50). Conclusion With a high specificity and sensitivity, the detection of serum protein spectrum can be performed easily and quickly by SELDI-TOF-MS technique, which provides a serological way in identifying PLC and most likely to benefit from AFP strategies.