Objective To investigate the anticancer effects and detailed mechanisms of Saikosaponin D (SSD) in human hepatoma HepG2 cells. Methods Cell proliferation and apoptosis were tested by MTT assay and Annexin-V/PI assay respectively. The expressions of CCAAT enhancer binding protein β(C/EBPβ) and p53 were detected by RT-PCR and Western blotting. Results SSD inhibited cell proliferation in a dose-dependent manner and induced apoptosis at the concentration of 5.0mg/L. SSD significantly increased the mRNA and protein levels of C/EBPβ and p53 in a dose-dependent manner. Conclusion SSD exerts its anticancer effect by inhibiting cell proliferation and inducing apoptosis partly through C/EBPβ-p53 signal pathway in HepG2 cells.

Objective: To investigate the cell death-inducing effect of saikosaponin-d (SSD) on human hepatoma Hep3B cells and its potential mechanism. Methods: Hepatoma Hep3B cells were divided into five groups: blank control group, DMSO (vehicle) group, and three SSD treatment groups treated with various doses of SSD (5, 10, and 15 μmol/L). MTT assay was used to evaluate cell viability. Flow cytometer was employed to quantitatively detect the percentage of dead cells after Annexin V-FITC/PI double staining. Apoptosis was detected morphologically after Hoechst 33258 staining. The activity of caspase-3 apoptotic protease was determined by spectrophotometry. Cell morphologic changes were observed with an inverted microscope. Western blotting and Real-time PCR were employed to evaluate the expression levels of C/EBP homology (CHOP) protein and mRNA, respectively. Results: MTT assay showed that SSD inhibited the viability of human hepatoma Hep3B cells in concentration- and time-dependent manners. Sinomenine hydrochloride induced the death of Hep3B cells in a concentration-dependent manner indicated by flow cytometry. After staining with Hoechst 33258, the nuclei of SSD-treated cells showed nucleosomal agglutination, nucleosomal shrinkage and fragmentation under the fluorescence microscope, which are the characteristics of apoptotic cells. SSD significantly activated the key apoptotic executor caspase-3. The occurrence of paraptosis, characterized by extensive cytoplasmic vacuoles, was observed in SSD-treated cells under an inverted microscope. The pretreatment of a pancaspase inhibitor Z-VAD-FMK completely inhibited caspase-3 activity triggered by SSD, but only partially suppressed cell death and could not reduce the cytoplasmic vacuolation in SSD-treated cells. The protein and mRNA expressions of CHOP, a stress-inducible molecule, were upregulated by SSD, which could not be inhibited by Z-VAD-FMK. Conclusion: SSD can simultaneously induce caspase-dependent apoptosis and caspase-independent paraptosis in human hepatoma Hep3B cells. The upregulated expression of CHOP may be the mechanism involved in SSD-induced paraptosis.

Objective To investigate the expression of DNA methyltransferase 3b (DNMT3B) in hepatocellular carcinoma (HCC) and its effect and mechanism on the proliferation,invasion and migration of HCC cells.Methods The expression of DNMT3B gene was detected by qRT-PCR in 46 cases of HCC tissues and corresponding adjacent tissues;the results and clinical pathological parameters were analyzed.SiRNA targeting DNMT3B was transfected into MHCC97-H cells by RNA interference (RNAi) technique.The mRNA and protein expression levels of related genes were detected by qRT-PCR and Western blot.The cell proliferation was measured by MTT assay,and the invasion and migration abilities were measured by Transwell assay.Results In 46 HCC patients,the expression of DNMT3B (73.91％) was significantly higher in HCC than in adjacent normal tissue.The high expression of DNMT3B gene was associated with histological type and tumor size of HCC (all P＜0.05).Inhibition of DNMT3B gene expression decreased proliferation,invasion and migration of MHCC97-H cells.Interference with DNMT3B gene increased the expressions of tumor suppressor genes RASSFA1,APC and MTSS1 at mRNA and protein levels.Conclusion DNMT3B is associated with the progression of HCC.It may inhibit the proliferation,invasion and migration of HCC cells by regulating the methylation of downstream tumor suppressor gene.

ABSTRACT:Objective To observe the influence of saikosaponin-d (SSd)on the proliferation and the function of autophagy of human hepatocellular carcinoma (HCC)cell line SMMC-7721 to explore the possible mechanisms. Methods SMMC-7721 was cultured invitro and then treated with SSd of various concentrations (5.0,7.5,10.0, 12.5,15.0 and 17.5 mg/L)for 24,48 and 72 h.We used MTT to detect cell proliferation,selected the optimal concentration and time,and detected the expressions of BECN1 at mRNA and protein levels by PCR and Western blot.Results The inhibition rate of the proliferation of SMMC-7721 cell line increased with the increase of the concentration of SSd,and the highest inhibition rate (60%)appeared when the concentration reached 12.5 mg/L. The expression of BECN1 in the group with SSd was obviously higher than that in the control group (P<0.05). 3-MA decreased not only the expressions of BECN1 at mRNA and protein levels but also the expression of BECN1 when used in conjunction with SSd.Conclusion The inhibiting function of SSd on SMMC-7721 presents a dependency between drug concentration and function time,basically in line with the drug dose-effect relationship. SSd induces the occurrence of autophagic cell death through up-regulating the expression of BECN1 ,thus inhibiting the proliferation of SMMC-7 7 2 1 .

Objective:To explore the expression of polypyrimidine tract-binding protein 1 (PTBP1) in gastric cancer (GC) tissues and GC cell lines, and the role of PTBP1 in the proliferation and metastasis of GC cells.Methods:From January to June in 2019 at The First Affiliated Hospital of Xi′an Jiaotong University, the cancer tissues and corresponding para-cancer tissues of GC patients underwent surgical resection were collected. The Kaplan-Meier Plotter database was used to analyze the survival of GC patients. The expression of PTBP1 was down-regulated by transfecting small interfering RNA (siRNA) in human GC cell lines SGC7901 and AGS with relatively high expression of PTBP1. The cells were divided into blank control group, negative control group, and PTBP1 knockdown group. The expression of PTBP1 at mRNA and protein level were detected by real-time fluorescence quantification polymerase chain reaction (RT-qPCR) and Western blotting. At 24, 48, 72 and 96-hour after transfection, the effect of PTBP1 on the proliferation of GC cells was observed by 3-(4, 5 dimethylthiazol)-2, 5 diphenyltetrazolium bromide (MTT) experiment. The changes of invasion and migration of GC cells after down-regulation of PTBP1 were detected by transwell assay. The expression changes of epithelial-mesenchymal transition (EMT) markers E-cadherin, N-cadherin and vimentin after down-regulation of PTBP1 in GC cells were determined by Western blotting. Indenpendent samples t test, analysis of variance and rank sum test were used for statistical analysis. Results:The Kaplan-Meier Plotter prognostic analysis showed that the overall survival of GC patients with high PTBP1 expression was shorter than that of GC patients with low PTBP1 expression (9.2 months, 6.2 months to 17.2 months vs. 19.0 months, 14.5 months to 28.4 months), and the difference was statistically significant ( Z=5.31, P<0.05). The results of RT-qPCR showed that in GC cell lines SGC7901 and AGS, the expression of PTBP1 at mRNA level of PTBP1 knockdown group was lower than that of blank control group and negative control group (SGC7901: 0.78±0.11 vs.3.10±0.19 and 2.99±0.23; AGS: 0.80±0.09 vs. 3.55±0.24 and 3.50±0.18), and the differences were statistically significant ( tSGC7901=10.57 and 8.08, tAGS=10.91 and 13.42; all P<0.01). The results of Western blotting indicated that in GC cell lines SGC7901 and AGS, the expression of PTBP1 at protein level of PTBP1 knockdown group was lower than those of blank control group and negative control group (SGC7901: 0.38±0.04 vs. 1.42±0.05 and 1.35±0.09; AGS: 0.17±0.02 vs. 1.52±0.08 and 1.38±0.45), and the differences were statistically significant ( tSGC7901=15.94 and 10.57, tAGS=16.60 and 20.80; all P<0.01). The results of MTT showed that at 48, 72 and 96-hour after transfection the absorbance values of PTBP1 knockdown group decreased by 0.25±0.01, 0.38±0.02, and 0.84±0.04 as compared with those of negative control group, and the decrease was the most significant at 96-hour after transfection, and the differences were statistically significant ( t=10.21、14.32, both P<0.01). The results of transwell experiment demonstrated that the number of invasion and migration cells of PTBP1 knockdown group were both less than that of the blank control group and the negative control group (SGC7901: 42.00±5.91 vs. 116.40±10.23 and 114.40±10.43; 39.60±6.77 vs. 125.80±11.51 and 122.40±5.90; AGS: 40.20±7.25 vs. 115.60±14.63 and 117.40±9.12; 36.00±5.20 vs. 122.40±12.10 and 125.40±12.74), and the differences were statistically significant ( tSGC7901=14.07, 13.50, 14.43 and 20.62; tAGS=10.27, 14.75, 14.68 and 16.76; all P<0.01). The results of Western blotting showed that the expression of E-cadherin of PTBP1 knockdown group was higher than that of the blank control group and the negative control group (SGC7901: 1.42±0.05 vs. 0.53±0.05 and 0.57±0.03; AGS: 1.34±0.04 vs. 0.54±0.03 and 0.61±0.01), however the expression levels of N-cadherin and vimentin were both lower than those of the blank control group and the negative control group (SGC7901: 0.50±0.03 vs. 1.64±0.05 and 1.46±0.07; 0.32±0.07 vs. 1.42±0.07 and 1.33±0.07; AGS: 0.37±0.06 vs. 1.47±0.04 and 1.36±0.04; 0.41±0.04 vs. 1.53±0.06 and 1.37±0.04), and the differences were statistically significant ( tSGC7901=11.63, 13.19, 18.83, 11.68, 11.43 and 10.43; tAGS= 15.02, 16.23, 14.67, 12.97, 14.45 and 17.18; all P<0.01). Conclusions:The expression levels of PTBP1 increase in GC tissues and cells, which may be involved in regulating the proliferation, metastasis and EMT of GC cells.

Objective To detect the expression profile of transcription factor C/EBPβ in human immortalized normal hepatic cell lines and hepatocellular carcinoma cell lines so as to determine the correlation between C/EBP3 with cell death mediated by endoplasmic reticulum stress in hepatocellular cells.Methods We cultured the human immortalized normal hepatic cells lines HHL5 and HL7702 and hepatocellular carcinoma cell lines SMMC7721;Bel7402,HepG2 and Hep3B.Hep3B cells were used as the cell model in tunicamycin-induced endoplasmic reticulum stress.Cellular morphology was observed under an inverted optical microscope.MTT assay was used to assess the inhibition of cell growth.To detect cell apoptosis,the cells were dyed with Hoechst 33258 and observed using a fluorescence microscope.RToPCR and Western blotting were used to detect the expression of at mRNA and protein levels,respectively.Results We found that normally the mRNA and protein isoform of C/EBPβ,C/EBPβ-1,were both expressed in all of the four hepatocellular cell lines and the two immortalized normal hepatic cell lines,while C/EBPβ protein isoform C/EBPβ-3 was only expressed in the two immortalized normal hepatic cell lines.Tunicamycin increased the expressions of both mRNA and protein of C/EBPβ in Hep3B cells and the increase of protein isoform C/EBPβ-3 was the most remarkable.In Hep3B cells,cell death was induced by tunicamycin through endoplasmic reticulum stress activity.Apoptosis as well as paraptosis was observed in tunicamycin-induced cell death.Conclusion C/EBPβ-3,one of the protein isoforms of C/EBPβ,is only expressed in normal hepatic cell lines,but not in hepatocellular cell lines.C/EBPβ is involved in cell death mediated by endoplasmic reticulum stress activity in hepatocellular carcinoma cells.

OBJECTIVE: To compare the efficacy and safety of combined radiofrequency ablation (RFA) and transcatheter arterial chemoembolization (TACE) with RFA alone for hepatocellular carcinomas (HCC). MATERIALS AND METHODS: Randomized controlled trial (RCT) studies that compared the clinical or oncologic outcomes of combination therapy of TACE and RFA versus RFA for the treatment of HCC were identified through literature searches of electronic databases (Pubmed, Embase, Cochrane Library, China Biology Medicine disc, China National Knowledge Infrastructure, and Google Scholar). Hazard ratios (HRs) or odds ratios (ORs) with their corresponding 95% confidence interval (CI) were combined as the effective value to assess the summary effects. The strength of evidence was rated by the Grading of Recommendations Assessment, Development, and Evaluation system. RESULTS: Six RCTs with 534 patients were eligible for inclusion in this meta-analysis. The meta-analysis showed that the combination of TACE and RFA is associated with a significantly longer overall survival (HR = 0.62, 95% CI: 0.49-0.78, p < 0.001) and recurrence-free survival (HR = 0.55, 95% CI: 0.40-0.76, p < 0.001) in contrast with RFA monotherapy. The seemingly higher incidence of major complications in the combination group compared with RFA group did not reach statistical significance (OR = 1.17, 95% CI: 0.39-3.55, p = 0.78). CONCLUSION: In patients with HCC, the combination of TACE and RFA is associated with significantly higher overall survival and recurrence-free survival, as compared with RFA monotherapy, without significant difference in major complications.
Carcinoma, Hepatocellular/*surgery ; Catheter Ablation/adverse effects/*methods ; Chemoembolization, Therapeutic/adverse effects/*methods ; China ; Combined Modality Therapy ; Disease-Free Survival ; Humans ; Liver Neoplasms/*surgery ; Odds Ratio ; Proportional Hazards Models ; Treatment Outcome

Objective:To evaluate the feasibility and effectiveness of magnetic anchor-guided endoscopic submucosal dissection (MAG-ESD).Methods:A total of 36 patients with gastrointestinal tumors at different sites who underwent MAG-ESD in the First Affiliated Hospital of Xi'an Jiaotong University from March 2020 to October 2022 were enrolled. The anchor success rate, en bloc resection rate, the anchor time, the procedure time, and the complication incidence were observed and analyzed.Results:Among the 36 patients, there were 9 lesions in stomach, 2 in duodenum, 6 in cecum and 19 in colorectum. Thirty-five (97.2%) patients successfully underwent magnetic anchor, and en bloc resection of lesions were completed. No adverse events such as bleeding or perforation occurred. The anchor time and procedure time was 4.0 (2.0-9.5) min and 36 (16-82) min, respectively.Conclusion:MAG-ESD is feasible and effective for gastrointestinal tumors at different sites, with a high anchor success rate and en bloc resection rate, and shorter operation time, especially for difficult submucosal dissection.

Objective To assess the clinical diagnostic performance of liver stiffness measurement (LSM) and aspartate transaminase (AST)-to-platelet (PLT) ratio index (APRI) for liver fibrosis in chronic hepatitis B (CHB) patients with alanine aminotransferase (ALT) less than or equal to five times of the upper limit of normal (≤5×upper limit of normal [ULN]).Methods FibroScan,blood routine and liver function test were conducted at the day or one day before liver biopsy in 383 CHB patients with ALT≤5 × ULN.The Scheuer scoring system was used for liver histologic assessment.APRI was calculated.Based on the results of liver pathology,the areas under receiver operating characteristic curve (AUC) of LSM and APRI for diagnosis of liver fibrosis stage were compared.Results The median LSM were 5.10 kPa for S0 fibrosis stage,5.20 kPa for S1,6.60 kPa for S2,10.10 kPa for S3,and 18.80 kPa for S4.The median APRI values were 0.36,0.38,0.63,0.61 and 1.27,respectively.The AUC of LSM were 0.817 for ≥S2,0.891 for ≥S3 and 0.913 for ≥S4.And the AUC of APRI were 0.717 for ≥S2,0.711 for ≥S3 and 0.746 for ≥S4.The cut-offs of LSM values were 6.8 kPa for ≥S2,8.7 kPa for ≥S3,and 10.9 kPa for ≥S4.Conclusion LSM can accurately assess the degree of liver fibrosis in CHB patients with ALT ≤5 × ULN,which is superior to APRI in clinical utility.

Objective To investigate the difference of hepatic microvessel density, neovasculariza-tion of regenerating liver tissue after ablation of two ways of irreversible electroporation and radiofrequency ablation in rats. Methods 90 male Sprague-Dawley rats were randomly divided into 3 groups, including the control group ( n =30), the irreversible electroporation group ( n =30 ) and the radiofrequency ablation group (n=30). 3,7 and 10 days were executed after the operation and draw material, expression of vascu-lar endothelial growth factor(VEGF) and CD34 in tissue was studied by immunohistochemistry, and the mi-crovascular density of tissue and VEGF positive cells were measured. Results The microvascular density of 3, 7 and 10 days in the control group was 50. 3 ± 12. 5, 54. 6 ± 11. 9 and 58. 2 ± 14. 7, the microvascular density of the radiofrequency ablation group was 18. 4 ± 4. 7, 17. 3 ± 5. 1 and 18. 1 ± 5. 9, respectively. The microvascular density of the irreversible electroporation group was 42. 8 ± 10. 4, 45. 6 ± 10. 2 and 49. 2 ± 13. 8, respectively. The positive cells of VEGF in control group was 50, 56 and 57 at 3, 7 and 10 days, and 32, 30 and 33 at 3, 7 and 10 days in radiofrequency ablation group, 44, 43 and 45 at 3, 7 and 10 days in irreversible electroporation group; expression of VEGF and CD34 in 3, 7, 10 d and the microvascular density of ablation area in radiofrequency ablation group was significantly lower than those in control group after irreversible electroporation and radiofrequency ablation. No significant differences were found between irreversible electroporation group and control group. Conclusion The irreversible electroporation can effectively protect the microvessels in the ablation area, ensure the tissue’s blood supply after the ablation, and provide a guarantee for the repair and regeneration of the tissue.