DNA N6-methyladenine involvement and regulation of hepatocellular carcinoma development.

DNA N6-methyladenine (6 mA) is a new type of DNA methylation identified in various eukaryotic cells. However, its alteration and genomic distribution features in hepatocellular carcinoma (HCC) remain elusive. In this study, we found that N6AMT1 overexpression increased HCC cell viability, suppressed apoptosis, and enhanced migration and invasion, whereas ALKBH1 overexpression induced the opposite effects. Further, 23,779 gain-of-6 mA regions and 11,240 loss-of-6 mA regions were differentially identified in HCC tissues. The differential gain and loss of 6 mA regions were considerably enriched in intergenic regions. Moreover, 7% of the differential 6 mA modifications were associated with tumors, with 60 associated with oncogenes and 57 with tumor suppressor genes (TSGs), and 17 were common to oncogenes and TSGs. The candidate genes affected by 6 mA were filtered by gene ontology (GO) and RNA-seq. Using quantitative polymerase chain reaction (qPCR), BCL2 and PARTICL were found to be correlated with DNA 6 mA in certain HCC processes.

Exosome-mediated miRNA delivery promotes liver cancer EMT and metastasis.

The deregulation of exosomal microRNAs (miRNAs) plays an important role in the progression of hepatocarcinogenesis. In this study, we highlight exosomes as mediators involved in modulating miRNA profiles in liver cancer cells after induction of the epithelial-mesenchymal transition (EMT) and metastasis. Initially, we induced EMT in a hepatocellular carcinoma cell (HCC) line (Hep3B) by stimulation with transforming growth factor-ß (TGF-ß) and confirmed by western blot detection of EMT markers such as vimentin and E-cadherin. Exosomes were then isolated from the cells and identified by nanoparticle tracking analysis (NTA). The isolated exosomal particles from unstimulated Hep3B cells (Hep3B exo) or TGF-ß-stimulated EMT Hep3B cells (EMT-Hep3B exo) contained higher levels of exosome marker proteins, CD63 and TSG101. After incubation with EMT-Hep3B exo, Hep3B cell proliferation increased. EMT-Hep3B exo promoted the migration and invasion of Hep3B and 7721 cells. High-throughput sequencing of miRNAs and mRNA within the exosomes showed 119 upregulated and 186 downregulated miRNAs and 156 upregulated and 166 downregulated mRNA sequences in the EMT-Hep3B exo compared with the control Hep3B exo. The most differentially expressed miRNAs and target mRNA sequences were validated by RT-qPCR. Based on the known miRNA targets for specific mRNA sequences, we hypothesized that GADD45A was regulated by miR-374a-5p. Inhibition of miR-374a-5p in Hep3B cells resulted in exosomes that inhibited the proliferation, migration, and invasion of HCC cells. These results enhance our understanding of metastatic progression of liver cancer and provide a foundation for the future development of potential biomarkers for diagnosis and prognosis of hepatic cancer.

Stent placement with iodine-125 seeds strand effectively extends the duration of stent patency and survival in patients with unresectable malignant obstructive jaundice.

Background: This study aimed to compare the treatment outcomes and safety between stent placement with or without Iodine-125 (125I) seeds strand for patients with unresectable malignant obstructive jaundice (MOJ).Methods: A total of 84 patients with unresectable MOJ treated in our hospital were retrospectively included and divided into the stent group (n = 54) undergoing biliary stent placement and the stent + seeds group (n = 30) receiving stent placement with 125I seeds strand. The therapeutic outcome, postoperative complications, duration of patient survival and stent patency were compared between groups. Kaplan-Meier survival analysis was performed to compare the duration of patient survival and stent patency between groups. Cox-regression analysis was performed to investigate predictive factors for disease-free survival and overall survival.Results: The stent + seeds group had significantly longer duration of patency (231.57 ± 256.54 vs. 110.37 ± 120.52) and overall survival (310.57 ± 330.54 vs. 173.15 ± 219.40) than the stent group (both p < .05). In addition, Kaplan-Meier survival analysis confirmed that the stent + seeds group had longer duration of patency (log-rank test, p = .001) and higher overall survival rate (log-rank test, p = .020) than the stent group. Furthermore, Cox-regression analysis demonstrated that treatment methods was an independent factor associated with disease-free survival (HR: 0.36, 95% CI: 0.19-0.70; p = .003) and overall survival (HR: 1.01, 95% CI: 1.00-1.01; p < .001).Conclusion: The stent placement with 125I seeds strand can significantly improve the primary patency rate and overall survival time in MOJ patients.

In Vivo Bioluminescence Imaging of Transplanted Mesenchymal Stromal Cells and Their Rejection Mediated by Intrahepatic NK Cells.

PURPOSE: Mesenchymal stromal cells (MSCs) hold promise in the treatment of liver disease. However, short survival time of MSCs after intrahepatic transplantation limits their value; therefore, understanding the basis of MSCs survival and rejection may increase their utility. This study was aimed at determining the role of intrahepatic natural killer (NK) cells on MSCs survival and their retention in the liver shortly after transplant. PROCEDURES: Human MSCs were labeled with the Luc2-mKate2 dual-fusion reporter gene (MSCs-R), and the residence time and survival of MSCs-R xenografts after intrahepatic transplantation were evaluated by in vivo bioluminescence imaging (BLI). Coculture of MSCs and NK cells was performed to assess cytotoxicity. To evaluate the role of NK cells in rejection of the xenografted cells, the fates of transplanted MSCs-R were then assessed in vivo by BLI after activation of intrahepatic NK cells. RESULTS: We observed a linear correlation between luciferase activity from live MSCs-R and cell number in vitro (R 2 = 0.9956). In vivo, we observed a gradual decline in bioluminescent signals from transplanted MSCs-R over a region corresponding to the liver in both the control group and the NK-activated group. However, the survival time and retention of intrahepatic MSCs-R decreased more rapidly in the NK-activated group of mice compared to the control group. This indicated that activated NK cells accelerate the elimination of transplanted MSCs. Also, we found that the number of hepatic NK cells and the expression of NK activation markers significantly increased after intrahepatic delivery of MSCs. This suggested that resident NK cells, in a resting state, were activated by intrahepatic transplantation of human MSCs. Taken together, the data suggests that activated hepatic NK cells mediate, in part, rejection of the MSCs xenografts. Cytotoxicity assays showed that activated NK cells may inhibit the proliferation of MSCs and, to a certain extent, induce MSCs death. CONCLUSION: Human MSCs could be followed dynamically in vivo by BLI, and the role of murine hepatic NK cells, especially activated NK cells, could be inferred from the loss of signals from MSCs. This finding may have practical clinical implications in MSCs transplantation in treating liver disease.

The inhibitory effect of MSCs expressing TRAIL as a cellular delivery vehicle in combination with cisplatin on hepatocellular carcinoma.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been demonstrated to induce cell apoptosis in many types of tumors, while many hepatocellular carcinoma (HCC) cells display high resistance to TRAIL. Another outstanding limitation of TRAIL is the short half-life in vivo. Stem cell-based therapies provide a promising approach for the treatment of many types of tumors because of the ability of tropism. Therefore, as a new therapeutic strategy, the combination of chemotherapeutic agents and TRAIL gene modified MSCs (TRAIL-MSCs) would improve the therapeutic efficacy of HCC in vivo. This is the first time to show the potential of combination of chemotherapeutic agents and MSCs as a gene vector in the therapy of HCC.

Different methods of detaching adherent cells significantly affect the detection of TRAIL receptors.

AIMS AND BACKGROUND: As a powerful technique allowing analysis of large numbers of cells, fluorescence-activated cell sorting (FACS) is used more and more widely. For FACS analysis, adherent cells are usually detached by trypsinization, followed by centrifugation and resuspension. However, trypsinization can cut off some receptors from the cell surface like fine scissors, which will affect the accuracy of FACS results. Though non-enzymatic methods such as citric saline buffer have been used to determine cell surface receptors, how much of the receptors is cut off by trypsinization has been rarely studied. This work aimed to investigate whether different methods of detaching adherent cells could affect the detection of cell surface receptors. METHODS: Human hepatocellular carcinoma cell lines (HepG2, Huh7 and Hep3B) were detached enzymatically with trypsin-EDTA solution or non-enzymatically with citric saline buffer, and then the receptors of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were detected by FACS analysis. Cell viability, cell cycle and apoptosis (sub-G1 fraction detected by FACS) of the trypsin-EDTA group and citric saline buffer group were also studied. RESULTS: Different methods of detaching adherent cells could significantly affect the detection of TRAIL receptors. Compared to the conventional trypsin-EDTA group, the non-enzymatic group showed a 3.42-fold increase in the mean fluorescence intensity index of DcR HepG2 and a 1.25-fold increase in DR Huh 7 (P <0.05). However, the viability, cell cycle and apoptosis of these cells were not affected. CONCLUSIONS: Citric saline buffer might be recommended as the first choice to detach adherent cells for FACS analysis of cell surface receptors.

[Labeling of liver cancer cell for fluorescence imaging study by far-red fluorescence protein reporter gene mKate2].

OBJECTIVE: To create far-red fluorescence protein reporter gene mKate2 lentivirus, label human liver cancer cell line HepG2 with lentivirus and explore the feasibility of in vitro fluorescence imaging of labeled tumor cells so as to provide experimental rationales for in vivo fluorescence tumor imaging. METHODS: mKate2 gene was amplified from pmKate2-N plasmid. Then the fragment was inserted into the lentivirus expression vector pLenti6.3/V5-DEST. The expression plasmids pLenti6.3-mKate2 and the packaging plasmids were cotransfected into 293T cells. The biological titer of lentivirus was determined. HepG2 cells were infected with mKate2 lentivirus at a MOI (virus multiplicity of infection) of 6 for 96 hours. The infection efficiency was assayed through fluorescence microscope and fluorescent-activated cell scanning (FACS). And 2 × 10(6) mKate2-HepG2 cells were collected for fluorescence imaging through an optical imaging system. And the optimal imaging parameters were determined. RESULTS: DNA sequencing analysis confirmed that mKate2 gene sequence was correct and there was no mutation or deletion. The biological titer of produced mKate2 lentivirus was 1.6 × 10(6) TU/ml. At 96 hours after mKate2 lentivirus infection, fluorescence microscope showed that mKate2 was expressed in a large percentage of cells. FACS assay showed that the mKate2 positive rate was 93.8% ± 0.4%. Excitation light 530 ± 15 nm and emission light 710 ± 28 nm were the optimal imaging parameters for mKate2-HepG2 cells. CONCLUSION: Lentivirus can mediate efficiently the mKate2 reporter gene labeling of human liver cancer cell line HepG2. The mKate2-labeled HepG2 cells can be detected through in vitro fluorescence imaging. Further tracing studies of in vivo tumor fluorescence imaging are technically feasible.

Cisplatin sensitizes human hepatocellular carcinoma cells, but not hepatocytes and mesenchymal stem cells, to TRAIL within a therapeutic window partially depending on the upregulation of DR5.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines and has been shown to induce cell apoptosis in many types of tumors, but not in normal cells. This tumor-selective property has made TRAIL a promising approach for the development of cancer therapy. However, hepatocellular carcinoma (HCC) cells display a striking resistance to TRAIL. Although some chemotherapeutic agents can overcome this resistance, safety issues remain a concern because the combination of these agents and TRAIL has been reported to induce toxicity in normal hepatocytes. In this study, we examined whether cisplatin could reverse TRAIL resistance in HCC cells with different p53 status and evaluated the toxicity of combination TRAIL and cisplatin to normal hepatocytes and mesenchymal stem cells (MSCs). We observed that cisplatin could efficiently sensitize HCC cells, but not hepatocytes and MSCs to TRAIL-induced apoptosis within a wide therapeutic window. The apoptosis of HCC cells only partially depended on the upregulation of DR5 and the status of p53. In addition, we provide favorable evidence supporting the feasibility of the combination of chemotherapy and MSCs transduced with TRAIL.

[Enhanced green fluorescence protein reporter gene labeling of mesenchymal stem cells mediated by lentivirus].

OBJECTIVE: To explore the effect of enhanced green fluorescence protein (EGFP) labeling mediated by lentivirus on the biophysical properties of mesenchymal stem cells (MSC), and whether the EGFP gene expression is permanent and stable. METHODS: MSC were infected with EGFP lentivirus at different virus multiplicity of infection (MOI). EGFP positive rate was measured with fluorescent-activated cell scanning (FACS) analysis, and EGFP expression in MSC was investigated under a fluorescence microscope. Cell viability, proliferation, apoptosis and cell cycle were detected with trypan blue stain, MTT colorimetric assay, Hoechst stain and FACS analysis respectively. To evaluate the stability of EGFP expression, EGFP lentivirus infected MSC were harvested after cultured continuously in vitro for 2, 4, 8 or 16 weeks, and EGFP positive rate and fluorescence strength were detected with FACS analysis. RESULTS: After infected with EGFP lentivirus (MOI = 20) for 96 h, EGFP positive rate of MSC was 97.39% +/- 0.68%. Cell viability, proliferation, apoptosis and cell cycle of MSC infected with EGFP lentivirus were unaffected, as compared with control MSC (P > 0.05). When cultured in vitro continuously for 2, 4, 8 or 16 weeks, EGFP positive rates of EGFP-MSC were 97.50% +/- 0.54%, 97.32% +/- 0.51%, 97.39% +/- 0.11%, and 97.48% +/- 0.13% respectively, while EGFP fluorescence strength were 440 +/- 13, 445 +/- 12, 458 +/- 13 and 456 +/- 16 respectively. Both EGFP positive rate and fluorescence strength kept in a stable level. CONCLUSION: EGFP lentivirus can efficiently label MSC and has no significant effect on the biophysical properties of MSC. EGFP gene expression in MSC is permanent and stable. EGFP-MSC can be used for further cell tracing research.

[Transportal variceal sclerotherapy with n-butyl-2-cyanoacrylate for gastric fundal varices].

OBJECTIVE: To evaluate the technique, safety and clinical efficacy of transportal variceal sclerotherapy with n-butyl-2-cyanoacrylate (NBCA) for gastric fundal varices. METHODS: Twenty-one patients with gastric fundal varices confirmed by endoscopy were enrolled in this study. The causes of the gastric varices were cirrhosis caused by hepatitis virus B or C (n = 16) and hepatocellular carcinoma with portal venous obstruction (n = 5). Percutaneous transhepatic or transplenic portography were performed on all 21 patients. The gastric varices were treated with NBCA-lipiodol mixture injected via a microcatheter introduced into the varices. For 8 patients who had large gastrorenal shunts (GRS), a balloon-occluded catheter was introduced into the GRS via the right femoral and left renal veins before injecting the NBCA-lipiodol. During the NBCA-lipiodol injection, the balloon was inflated to block the flow of GRS. Follow-up evaluations included findings of the laboratory liver function tests, upper intestinal endoscopies, and the occurrences of rebleeding. RESULTS: In 20 patients (95.2%), the gastric varices were successfully obliterated with 2-8 ml of NBCA-lipiodol. In one patient with a large GRS, sclerotherapy was not successfully performed because a balloon-occluded catheter was not available during the procedure. In five patients, small amounts of NBCA-lipiodol entered into the distal pulmonary artery branches. Two of them suffered from transient irritable coughs; no patient developed severe pulmonary embolism. Embolization of portal venous branches occurred in two patients, which were not treated specifically. In comparison with the findings before the treatments, the serum alanine aminotransferase levels decreased at both 3 and 6 months after treatments (P less than 0.05); serum albumin levels increased at 6 months (P less than 0.05); the prothrombin times decreased at 6 months (P less than 0.05); but no significant changes were seen in the serum bilirubin levels. Fifteen patients were followed-up endoscopically for 3 months after the treatment. Gastric varices were completely resolved in 10 patients (66.7%) and were markedly smaller in 4 patients (26.6%). Worsening of the esophageal varices occurred in 3 patients (20%). All the patients were followed-up from 1 to 30 months [(16.7+/-8.8) months]. Rebleeding was observed in 4 patients, and the cumulative rebleeding rate at 1 year was 9.52%. CONCLUSION: Transportal variceal sclerotherapy with NBCA is a safe and effective method for treating gastric varices. Microcatheter technique and occlusion of the large gastrorenal shunt with a balloon-occluded catheter are necessary to ensure obliteration of gastric varices and prevent pulmonary embolism.

[Radiofrequency ablation with or without transcather arterial chemoembolization for management of hepatocellular carcinoma].

OBJECTIVE: To evaluate the efficacy and complications of radiofrequency ablation (RFA) with or without transcatheter arterial chemoembolization (TACE) for management of hepatocellular carcinoma (HCC). METHODS: A retrospective analysis was conducted for 62 small HCC cases undergoing RFA with or without TACE, and in each case, the tumors were not more than 3 with a diameter below 5 cm. Nineteen cases were managed with RFA alone (RFA group) while the other 27 underwent RFA combined with TACE (TACE+RFA group). Percutaneous RFA (RITA 1500) procedure was performed under CT guidance 1-3 weeks after TACE in TACE+RFA group. RESULTS: The complete tumor necrosis rate was 77.8% (21/27) in TACE+RFA group, significantly higher than that in RFA group [57.9% (11/19), P<0.01], and the former group had a significantly lower local recurrence rate than the latter [22.2% (6/27) vs 42.1% (8/19), P<0.01]. Postoperative fever, local pain and temporary hepatic function abnormality were the common complications that were relieved after proper interventions, and mortality did not occur in these cases. CONCLUSION: The combination of TACE and RFA significantly increases the complete tumor necrosis rate and decreases the recurrence rate of small HCC. CT-guided percutaneous RFA can be a safe and effective therapy for small HCC.

[The role of multi-detector row CT in the diagnosis and hemodynamic studies of gastric varices in portal hypertension].

OBJECTIVE: To evaluate the value of multi-detector row CT (MDCT) in the diagnosis and hemodynamic studies of gastric varices (GV) in portal hypertension by comparison with endoscopy and DSA direct portography. METHODS: Thirty-six consecutive cirrhotic patients with GV confirmed by endoscopy underwent tri-phase contrast-enhanced CT scans and CT portography (CTP) within 2 weeks after endoscopy examination. Three independent experienced radiologists, who were blinded to the patients' clinical data, analyzed the CT images, including the size and location of GV as well as afferent and efferent veins of GV, separately. Interobserver agreement among the 3 radiologists with regard to the diagnosis of submucosal and perigastric GV was determined by Kappa (k) values. The findings of endoscopy were used as standards. RESULTS: Sub mucosal GV was diagnosed in 34 of the 36 patients (94.4%) and perigastric GV in all 36 patients (100%) by the observation of the 3 radiologists. MDCT showed an excellent interobserver reliability with regard to the diagnosis of submucosal GV (kappa = 0.85) and perigastric GV (kappa = 1.0). Agreement between MDCT and endoscopy with regard to the opacification of variceal size and location were 86.1% and 88.9% respectively. The sensitivity, specificity, accuracy, and positive predictive value of CTP in the opacification of afferent and efferent veins of GV were all more than 80%. The frequencies of participation of posterior gastric vein and short gastric vein in blood supply to gastric fundal varices in the isolated gastric varices and gastroesophageal varices type 2 (GEV2) were 94.1% and 70.6% respectively, both significantly higher than those in the gastroesophageal varices type 1 (GEV1, 52.6% and 31.6%, respectively, both P < 0.05). The main blood drainage route of GEV1 was via azygous system into the super vena cava (100%), whereas in the gastric fundal varices the main blood drainage route was via the gastrorenal shunts into the inferior vena cava (82.4%). CONCLUSION: MDCT can be used as an important tool for detecting submucosal and perigastric GV, and can clearly reveal the size, location, and hemodynamics of GV.

Transjugular intrahepatic portosystemic shunt for palliative treatment of portal hypertension secondary to portal vein tumor thrombosis.

AIM: To evaluate the palliative therapeutic effects of transjugular intrahepatic portosystemic shunt (TIPS) in portal vein tumor thrombosis (PVTT) complicated by portal hypertension. METHODS: We performed TIPS for 14 patients with PVTT due to hepatocellular carcinoma (HCC). Of the 14 patients, 8 patients had complete occlusion of the main portal vein, 6 patients had incomplete thrombosis, and 5 patients had portal vein cavernous transformation. Clinical characteristics and average survival time of 14 patients were analysed. Portal vein pressure, ascites, diarrhoea, and variceal bleeding and circumference of abdomen were assessed before and after TIPS. RESULTS: TIPS was successful in 10 cases, and the successful rate was about 71%. The mean portal vein pressure was reduced from 37.2 mmHg to 18.2 mmHg. After TIPS, the ascites decreased, hemorrhage stopped and the clinical symptoms disappeared in the 10 cases. The average survival time was 132.3 d. The procedure failed in 4 cases because of cavernous transformation in portal vein and severe cirrhosis. CONCLUSION: TIPS is an effective palliative treatment to control hemorrhage and ascites due to HCC complicated by PVTT.

Comparison of long-term effects between intra-arterially delivered ethanol and Gelfoam for the treatment of severe arterioportal shunt in patients with hepatocellular carcinoma.

AIM: To evaluate long-term effect of ethanol embolization for the treatment of hepatocellular carcinoma (HCC) with severe hepatic arterioportal shunt (APS), compared with Gelfoam embolization. METHODS: Sixty-four patients (ethanol group) and 33 patients (Gelfoam group) with HCC and APS were respectively treated with ethanol and Gelfoam for APS before the routine interventional treatment for the tumor. Frequency of recanalization of shunt, complete occlusion of the shunt, side effects, complications, and survival rates were analyzed between the two groups. RESULTS: The occlusion rate of APS after initial treatment in ethanol group was 70.3%(45/64), and recanalization rate of 1 month after embolization was 17.8%(8/45), and complete occlusion rate was 82.8%(53/64). Those in Gelfoam group were 63.6%(21/33), 85.7%(18/21), and 18.2%(6/33). There were significant differences in recanalization rate and complete occlusion rate between the two groups (P<0.05). The survival rates in ethanol group were 78% at 6 months, 49% at 12 months, 25% at 24 months, whereas those in Gelfoam group were 58% at 6 months, 23% at 12 months, 15% at 24 months. The ethanol group showed significantly better survival than Gelfoam group (P<0.05). In the ethanol group, there was a significant prolongation of survival in patients with monofocal HCC (P<0.05) and Child class A (P<0.05). There were no significant differences in survival rate in the Gelfoam group with regard to the number of tumor and Child class (P>0.05). The incidence rate of abdominal pain during procedure in ethanol group was 82.8%. There was no significant difference in postembolization syndromes between two groups. Procedure-related hepatic failure did not occur in ethanol group. CONCLUSION: Ethanol embolization for patients with HCC and severe APS is efficacious and safe, and may contribute to prolongation of the life span versus Gelfoam embolization.

Superselective uterine arterial embolization with pingyangmycin-lipiodol emulsion for management of symptomatic uterine leiomyoma.

BACKGROUND: Uterine arterial embolization (UAE) is a safe and effective therapy for symptomatic uterine leiomyoma. This study was to assess the effectiveness and the feasibility of pingyangmycin-lipiodol emulsion (PLE) for the management of symptomatic uterine leiomyoma. METHODS: One hundred consecutive patients (aged 21 - 53 years, with 38 in average) with symptomatic uterine leiomyoma underwent superselective UAE with PLE. Clinical symptoms of the patients (including menorrhagia, bulk-related symptoms, and postprocedure-related abdominal pain) and the changes in uterine volume and tumor size after the embolization were analyzed. The patients were followed up for 8 - 21 months (mean, 15 months). RESULTS: Ninety-nine patients (99%, 99/100) were interviewed in their first menses circle after embolization, showing improvements in their abnormal bleeding and bulk-related symptoms to some extent. Imagiological results during follow-up showed a mean of 48% reduction in uterine volume at 6 months and a mean of 75% reduction in tumor size at 9 months. Eighty-three percent of the patients reported complete resolution of postprocedure pain within 7 days. CONCLUSIONS: PLE is effective in the management of uterine leiomyoma, having superiority in alleviating postprocedure-related pain.