RAD54L promotes progression of hepatocellular carcinoma via the homologous recombination repair pathway.

Hepatocellular carcinoma (HCC) is a malignant tumor with high incidence worldwide. The underlying mechanisms remain poorly understood. The DNA metabolic process of homologous recombination repair (HRR) has been linked to a high probability of tumorigenesis and drug resistance. This study aimed to determine the role of HRR in HCC and identify critical HRR-related genes that affect tumorigenesis and prognosis. A total of 613 tumor and 252 para-carcinoma tissue samples were collected from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) to obtain differentially expressed genes (DEGs). HRR-related genes were assessed using gene enrichment and pathway analyses. Survival analysis was performed using the Kaplan-Meier method in the Gene Expression Profiling Interactive Analysis portal. The levels of RAD54L in the HRR pathway were detected by RT-qPCR and western blotting in para-carcinoma and HCC tissues and in L02 normal human liver cells and Huh7 HCC cells. Immunohistochemistry (IHC) was performed on the clinical specimens to determine the connection between gene expression and clinical features. Bioinformatics analysis revealed that the HRR pathway was enriched in HCC tissues. Upregulation of HRR pathway DEGs in HCC tissues was positively correlated with tumor pathological staging and negatively associated with patient overall survival. RAD54B, RAD54L, and EME1 genes in the HRR pathway were screened as markers for predicting HCC prognosis. RT-qPCR identified RAD54L as the most significantly expressed of the three genes. Western blotting and IHC quantitative analyses further demonstrated that RAD54L protein levels were higher in HCC tissues. IHC analysis of 39 pairs of HCC and para-carcinoma tissue samples also revealed an association between RAD54L and Edmondson-Steiner grade and the proliferation-related gene Ki67. The collective findings positively correlate RAD54L in the HRR signaling pathway with HCC staging and implicate RAD54L as a marker to predict HCC progression.

COTE1 Facilitates Intrahepatic Cholangiocarcinoma Progression via Beclin1-Dependent Autophagy Inhibition.

COTE1 was recently described as an oncogene in hepatocellular carcinoma and gastric cancer. However, the roles of COTE1 in intrahepatic cholangiocarcinoma (ICC) are little known. Our study is aimed at clarifying novel functions of COTE1 in ICC progression, including proliferation, invasion, and autophagy. By using quantitative real-time PCR, immunohistochemistry staining, and western blotting, we found that COTE1 expression was frequently upregulated in ICC tissues, compared to paracarcinoma tissues. High COTE1 expression was significantly correlated with aggressive clinical features and predicted poor prognosis of ICC patients. Functional experiments revealed that ectopic COTE1 expression promoted ICC cell proliferation, colony formation, cellular invasion, migration, and in vivo tumorigenicity; in contrast, COTE1 knockdown resulted in the opposite effects. At molecular mechanism in vitro and vivo, our study revealed that COTE1 overexpression suppressed autophagy via Beclin1 transcription inhibition; conversely, COTE1 silencing facilitated autophagy through promoting Beclin1 expression. Furthermore, the suppression of COTE1 knockdown on cellular growth and invasion was rescued/aggravated by Beclin1 inhibition/accumulation. Our data, for the first time, illustrate that COTE1 is an oncogene in ICC pathogenesis, and the ectopic COTE1 expression promotes ICC proliferation and invasion via Beclin1-dependent autophagy inhibition.

Anti-Liver Fibrosis Role of miRNA-96-5p via Targeting FN1 and Inhibiting ECM-Receptor Interaction Pathway.

The aberrant expression of mRNAs participates in the pathogenesis of hepatic fibrosis. However, the precise mechanisms regulated by microRNAs (miRNAs) remain unclear. This study aims to investigate the functions about differentially expressed mRNAs (DEMs) in liver fibrosis and their regulatory mechanisms. The DEMs datasets about hepatic stellate cells (HSCs) obtained from hepatic fibrosis mice versus HSCs obtained from normal mice were downloaded from the GEO database (GSE120281). According to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the GSE120281 datasets, ECM-receptor interaction was the most significant enrichment pathway that was correlated with hepatic fibrosis, and the fibronectin 1 (FN1) gene was upregulated most significantly in the signaling pathway. Downregulation of the expression of the FN1 gene by transfecting with FN1-siRNA alleviated the activity of HSCs. Four different bioinformatics web-based tools were used to predict that microRNA-96-5p (miR-96-5p) would directly target FN1, and a luciferase assay further confirmed this. Moreover, miR-96-5p was declined in activated HSCs and FN1, whereas laminin γ1 (LAMC1), collagen 1α1 (COL1A1) in the ECM-receptor interaction pathway, and the fibrosis marker α-smooth muscle actin (α-SMA) could be reduced by upregulation of the miRNA. Additionally, miR-96-5p expression was low in CCl4-induced liver fibrosis mice. Increased miR-96-5p expression alleviated liver fibrosis, improved liver function, and inhibited the expression of α-SMA, FN1, COL1A1, and LAMC1. In conclusion, this study indicated that upregulation of miR-96-5p could reduce HSC activation and relieve hepatic fibrosis by restraining the FN1/ECM-receptor interaction pathway.

PTD-hFOXP3 protein acts as an immune regulator to convert human CD4(+)CD25(-) T cells to regulatory T-like cells.

Regulatory T cells (Tregs) are critical for maintaining self-tolerance and homeostasis, and have potential application in clinical disease therapy, such as autoimmune diseases and transplant rejection, but their numbers are limited. FOXP3 is a key transcription factor controlling Tregs development and function. Although transfection of CD4(+)CD25(-) lymphocytes with the FOXP3 gene can convert them to Treg-like cells, there is the risk of insertional mutagenesis and thus an alternative to genetic intervention is sought. The protein transduction domain (PTD) from the HIV transactivator of transcription is a useful tool to deliver protein to the cytoplasm and nucleus. In this study, we generated a fusion protein linking the human FOXP3 to PTD (PTD-hFOXP3), and explored its function in T cells. The results showed that the PTD rapidly and effectively delivered the hFOXP3 protein into cells where it localized not only in the cytoplasm, but also to the nucleus. PTD-hFOXP3-transduced Jurkat cells (human T lymphoma cell line) and CD4(+)CD25(-) T cells failed to proliferate and produce IL-2 and IFN-γ, but produced large amounts of the cytokines IL-4, IL-10, and TGF-ß, in response to TCR stimulation in vitro. PTD-hFOXP3-transduced CD4(+)CD25(-) T cells also expressed high levels of CTLA-4 and low levels of CD25 after stimulation. Most importantly, PTD-hFOXP3-transduced T cells inhibited the proliferation of activated CD4(+)CD25(-) T cells. Furthermore, chromatin immunoprecipitation assays demonstrated that PTD-hFOXP3 can bind with the IL-2 gene promoter and repress the expression of IL-2. These results indicate that PTD-hFOXP3 has the capability to convert conventional T cells to Treg-like cells.

Mesenchymal stem cells improve liver fibrosis and protect hepatocytes by promoting microRNA-148a-5p-mediated inhibition of Notch signaling pathway.

BACKGROUND: Mesenchymal stem cells (MSCs) are considered to be a potential therapeutic tool for liver fibrosis. Inhibiting the activation of hepatic stellate cells (HSCs) and protecting hepatocytes are important mechanisms for the anti-fibrotic effect of MSCs. However, how MSCs inhibit liver fibrosis by regulating the expression of microRNAs (miRNAs) has not been fully clarified. METHODS: Transforming growth factor-ß1 (TGF-ß1)-activated HSCs LX-2 were single cultured or co-cultured with human umbilical cord mesenchymal stem cells (HUC-MSCs). High-throughput sequencing was used to evaluate the differentially expressed microRNAs (DEMs) between the two groups. Quantitative real-time PCR (qRT-PCR), Western blot, and transfection experiments were used to investigate and screen the most significantly up-regulated DEM. Bioinformatics analysis was used to predict the target mRNAs and the potential functions of the DEM. The possible mechanism of HUC-MSCs against liver fibrosis was analyzed by co-culture experiment of HUC-MSCs with LX-2 cells, and HUC-MSCs treatment of Bile duct ligation (BDL)-induced liver fibrosis in mice. Finally, the mechanism of the DEM regulating liver fibrosis was confirmed in human liver fibrosis specimens. RESULTS: MicroRNA-148a-5p (miR-148a-5p) was the most significantly up-regulated DEM in activated LX-2 cells co-cultured with HUC-MSCs compared with LX-2 cells single cultured. Up-regulation of the expression of miR-148a-5p in activated LX-2 cells could significantly inhibit the expression of hepatic fibrosis markers α-SMA and Col1α1. Notch2 was one target gene of miR-148a-5p. Co-cultured with HUC-MSCs could inhibit the activation of LX-2 cells by inhibiting the expression of the Notch2 and the Notch signaling pathway. In addition, HUC-MSCs treatment could up-regulate the expression of miR-148a-5p in liver tissue and hepatocytes, promote the proliferation and avoid the apoptosis of hepatocytes, and reduce the degree of fibrosis by inhibiting expression of the Notch2 and the Notch signaling pathway in BDL-induced liver fibrosis mice. Moreover, miR-148a-5p was down-regulated and Notch2 was up-regulated in fibrotic human liver tissues compared with the normal livers. CONCLUSIONS: HUC-MSCs treatment could inhibit HSCs activation, protect hepatocytes, and alleviate BDL-induced liver fibrosis in mice by up-regulating the expression of miR-148-5p and inhibiting the Notch signaling pathway. The down-regulation of miR-148-5p and up-regulation of Notch2 could be used as biomarkers to monitor the progression of liver fibrosis.

Human Umbilical Cord Mesenchymal Stem Cells Ameliorate Hepatic Stellate Cell Activation and Liver Fibrosis by Upregulating MicroRNA-455-3p through Suppression of p21-Activated Kinase-2.

Mesenchymal stem cells (MSCs) were shown to have potential therapeutic effects for treatment of liver fibrosis, and dysregulated expression of microRNAs (miRNAs) played a pivotal role in the pathogenesis of liver fibrosis by regulating their downstream target genes. However, the mechanism by which MSCs affect the progression of liver fibrosis by regulating miRNA expression remains unclear. Here, we investigated whether human umbilical cord MSCs (HUC-MSCs) attenuated hepatic fibrosis by regulating miR-455-3p and its target gene. Significantly upregulated miRNA (miR-455-3p) was screened out by GEO datasets analysis and coculture HUC-MSCs with hepatic stellate cell (HSC) LX-2 cells. p21-activated kinase-2 (PAK2) was forecasted to be the target gene of miR-455-3p by bioinformatics analyses and confirmed by luciferase reporter assay. HUC-MSCs were transplanted into mice with carbon tetrachloride- (CCl4-) induced liver fibrosis, the result showed that HUC-MSC transplantation significantly ameliorated the severity of CCl4-induced liver fibrosis, attenuated collagen deposition, improved liver function by reducing the expression of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, upregulated miR-455-3p, and suppressed PAK2 expression of liver tissue in mice. Taken together, our study suggests that HUC-MSCs inhibit the activation of HSCs and mouse CCl4-induced liver fibrosis by upregulation of miR-455-3p through targeting PAK2.

NFATc1 is a tumor suppressor in hepatocellular carcinoma and induces tumor cell apoptosis by activating the FasL-mediated extrinsic signaling pathway.

Nuclear factor of activated T cells (NFAT) is a family of transcription factors that have important functions in many tumors. However, the expression level and functional role of NFAT in hepatocellular carcinoma (HCC) remain unclear. In this study, we showed that NFATc1 expression was decreased in both HCC tissues and cell lines. Low expression of NFATc1 was correlated with larger tumor size, advanced tumor-node-metastasis (TNM) stage, high serum AFP level, and liver cirrhosis. Furthermore, patients with low NFATc1 expression exhibited poor prognosis. Ectopic expression of NFATc1 in HCC cells inhibited proliferation and colony formation, leading to G1 arrest and induction of apoptosis. In addition, we demonstrated that NFATc1 increased Fas ligand (FasL) expression by directly binding to its promoter and activated the extrinsic apoptotic pathway. NFATc1 and FasL expression patterns and their prognostic value for patients with HCC were also evaluated in TCGA Liver Hepatocellular Carcinoma database. Knock-down of FasL expression by siRNA in HCC cell lines abolished NFATc1's antiproliferative and pro-apoptotic effects. In conclusion, NFATc1 is frequently inactivated in HCC and functions as a tumor suppressor in liver carcinogenesis. Ectopic expression of NFATc1 in HCC cells induces apoptosis by activating the FasL-mediated extrinsic signaling pathway.

[Adult-to-adult living donor liver transplantation for decompensated end-stage liver diseases].

OBJECTIVE: To summarize our clinical experience in adult-to-adult living donor liver transplantation (ALDLT). METHODS: Clinical data of 12 patients with ALDLT performed in our center from September 2000 to June 2005 were analyzed, retrospectively. RESULTS: Left lobe (segments II, III, IV, including the middle hepatic veins) transplantation was performed in 3 patients and right lobe (segments V, VI, VII, VIII, with or without the middle hepatic veins) transplantation was performed in 9 patients. Donors: There were no operative deaths. The median operative time was 6.20+/-1.40 hours and their blood loss ranged from 300 ml to 1200 ml. Postoperative complications included biliary fistula (1 donor) and wound fat liquefaction (1 donor). During a 6-12 months follow-up, no long-term complications were found. Recipients: The operating time ranged from 5 to 11 hours and their blood loss ranged from 800 to 7000 ml. Modified outflow reconstruction, microvascular reconstruction of the hepatic artery and duct-to-duct biliary reconstruction were done during the recipient operations. The median cold ischemia time was 1.90+/-0.50 hours. The median anhepatic phase of recipients was 1.63+/-0.43 hours. Graft/recipient weight ratio (GRWR) was (1.20+/-0.26)%. One recipient presented a postoperative complication of biliary fistula and another recipient died 1 month after the operation from serious infection. The other 11 recipients had long-term survivals. CONCLUSION: ALDLT is an effective treatment for decompensated end-stage liver disease patients and is relatively safe for the donors.

[Adult to adult living donor liver transplantation for decompensated end stage liver disease].

OBJECTIVE: To summarize the clinical experience and some principal surgical techniques of adult to adult living donor liver transplantation (ALDLT). METHODS: The clinical data of 9 patients receiving ALDLT from September 2000 to September 2004 in liver transplantation center in the First Affiliated Hospital of Nanjing Medical University were analyzed retrospectively. The left lobe (segments II, III, IV, including the middle hepatic veins) was transplanted in 3 patients, and the right lobe (segments V, VI, VII, VIII, not including the middle hepatic veins) was transplanted in 6 patients. RESULTS: There was no operative death in donors. The median operative time was (6.2+/-1.4) hours. The blood loss ranged from 300 to 1,200 ml. Postoperative complications included biliary fistula (1 donor) and wound fat liquefaction (1 donor). They were followed up for 6-12 months, and no long term complications were found. In recipients, the operating time ranged from 5 to 11 hours. The blood loss ranged from 800 to 7,000 ml. Modified outflow reconstruction method, microvascular reconstruction of the hepatic artery and duct to duct biliary reconstruction were performed in recipients. The median cold ischemic time of the grafts was (1.9+/-0.5) hours. The mean non hepatic stage of recipients was (98+/-26) minutes. Graft/recipient weight ratio (GRWR) was (1.20+/-0.26)%. One recipient presented postoperative complication of biliary fistula. One recipient died of serious infection 1 month postoperatively. The other 8 recipients enjoyed longterm survival. CONCLUSION: The procedure of ALDLT is an effective method in the treatment of decompensated end stage liver disease, and it is relatively safe for the donor. Reconstruction of vessels is the key surgical technique in the operative procedure.

Effects of propofol versus sevoflurane on cerebral oxygenation and cognitive outcome in patients with impaired cerebral oxygenation.

BACKGROUND: Postoperative neurocognitive dysfunction induced by anesthetics, particularly in elderly patients with impaired oxygenation, is a common complication of surgery and is eliciting increased interest in clinical practice. To investigate the effects of anesthetics on neurocognition, we compared the effects of propofol versus sevoflurane on cerebral oxygenation and cognitive outcome in patients with impaired cerebral oxygenation undergoing general anesthesia. METHODS: Sixty-three patients with impaired cerebral oxygenation (jugular venous bulb oxygen saturation [SjvO2] <50%) or cerebral blood flow/cerebral metabolic rate of oxygen ([CBF/CMRO2] ≤15%) undergoing elective abdominal surgery were randomly allocated into propofol group (group P) or sevoflurane group (group S). The clinical parameters and jugular venous bulb blood gas analysis were monitored throughout the surgical procedure. Cognitive function was assessed with the mini-mental state examination and Montreal Cognitive Assessment at day 1 and day 7 following surgery. S100ß protein in plasma was measured using enzyme-linked immunosorbent assay. RESULTS: The SjvO2 increased during anesthesia induction and surgery when compared to baseline but had no significant difference between group P and group S. When compared to baseline, the CBF/CMRO2 was increased only at the end of surgery and extubation in group P; however, the CBF/CMRO2 in group S was increased during anesthesia induction at 1 hour, 2 hours, end of surgery, and extubation. Furthermore, the CBF/CMRO2 in group S was significantly higher than that in group P during anesthesia induction at 1 hour, 2 hours, and end of surgery. S100ß protein did not significantly change at extubation and 1 day after surgery in both groups when compared to baseline. There was no significant difference in mini-mental state examination and Montreal Cognitive Assessment scores between group P and group S at all time points. CONCLUSION: Sevoflurane showed similar effects in postoperative neurocognitive function as propofol but could improve cerebral oxygenation in patients with impaired cerebral oxygenation.

[Expressions of aquaporin-1 on peritonea in hepatic cirrhotic rats with ascites].

OBJECTIVE: To investigate the changes of aquaporin (AQP-1) expressions on peritonea in liver cirrhotic rats with ascites, and to study the correlation between AQP-1 expressions and ascites form. METHODS: 32 healthy Sprague-Dawley (SD) rats were divided into two groups randomly, 20 rats were used to produce liver cirrhotic models induced with phenobarbitol sodium and CCl(4). The distribution and protein expressions of AQP-1 on the rats' peritonea were measured with immunohistochemistry assay, and the expressions of APQ-1 mRNA were tested with relative GAPDH quantitative RT-PCR. RESULTS: (1) The expressions of AQP-1 were mainly on the endothelial cells of capillary vessels and venules on the rats' peritonea, and also on mesothelial cells. (2) There was no statistically significant difference between the expressions of AQP-1 protein and mRNA in the two groups on the early stage of liver cirrhosis. (3) Downregulations of the expressions of AQP-1 protein and mRNA were observed in B group on the advanced cirrhotic stage. CONCLUSION: Expressions of AQP-1 were downregulated on the peritonea of rats with decompensated liver cirrhosis, which may play a role in the formation of ascites. The changes of AQP-1 Expressions on peritoneal mesothelial cells which were fewer than those on the endothelial cells may be few relations to ascites form.

Inhibitory effects of docetaxel on expression of VEGF, bFGF and MMPs of LS174T cell.

AIM: To study the effects of non-cytotoxic concentrations of docetaxel on some important angiogenic factors of LS174T Cells. METHODS: The non-cytotoxic concentration of docetaxel and the activity of gelatinase were determined with MTT and gelatin zymography respectively, the expression of VEGF(vascular endothelial growth factor), bFGF (basic fibroblast growth factor), MMP (matrix metalloproteinase) 2 and MMP 9 was investigated with RT-PCR and Western blot. RESULTS: The maximum non-cytotoxic concentration of docetaxel on LS174T Cells was 1.0 ng/ml. Compared with the solvent control group, 0.1, 0.5, 1.0 ng/ml of docetaxel could downregulate the expression of VEGF, bFGF, MMP 2 and MMP 9 and suppress the activity of gelatinase. CONCLUSION: Our study suggests that the non-cytotoxic concentrations of docetaxel have strong antiangiogenic activity on LS174T Cells, which suggests docetaxel may be a promising antiangiogenic agent.

[Effect of PTD-mFoxp3 fusion protein on graft-versus-host disease after allogeneic bone marrow transplantation].

This study was aimed to investigate the effect of PTD-mFoxp3 fusion protein on graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation. The 10-weeks-old C57BL/6 mice as recipients were randomly divided into three groups (A,B and C), 10 mice were in each group. The mice on day of transplantation as on day 0 received total body irradiation (TBI) 6.0 Gy, then the bone marrow cells (BMC) from BALB/c mice were injected through tail vein within 4-6 hours. At 2 days before transplantation and 0, 1, 3, 5, 7, 9 and 13 days after transplantation, mice in group A were injected with saline, mice in group B were injected with mFoxp3 protein and mice in group C were injected with PTD-mFoxp3 fusion protein. Symptoms of GVHD, survival time and histopathological changes were observed. The establishment of mixed chimerism was determined by flow cytometry in day 60, and IL-2 and IFN-γ expression profiles in the recipient peripheral blood were assessed by ELISA. The results showed that the mean survival time of recipients in group A,B and C was (32.95 ± 5.48) , (38.00 ± 5.45) and (55.30 ± 3.15) respectively. Graft rejection was observed in the liver and small intestine specimens of group A and group B. The serum levels of IL-2 and IFN-γ significantly decreased in the recipients of group C, as compared with the other groups. The flow cytometry analysis revealed that the survival recipient mice developed high chimerism levels, the percentages of donor cells in group A,B and C were (79.46 ± 1.80) %, (79.13 ± 2.23) % and (85.92 ± 2.82) % respectively. It is concluded that PTD-mFoxp3 fusion protein can reduce the incidence and mortality of GVHD after allogeneic bone marrow transplantation.

[Cost-effectiveness of Helicobacter pylori screening to prevent gastric cancer: Markov decision analysis].

OBJECTIVE: Using Markov model Monte Carlo simulation to conduct a cost-effectiveness analysis of screening Helicobacter pylori (H. pylori) infection to prevent gastric cancer. METHODS: The Markov model was developed based on the natural course from H. pylori infection to gastric cancer. Two strategies were compared: (1) screening for H. pylori and treatment for those with positive tests, and (2) without screening and treatment. Data used for model simulation including transition probability, efficacy of test and treatment were collected from related research publications. Markov model Monte Carlo simulation combined with bootstrap method was used to perform base-case analysis and estimate the confidence interval of cost-effectiveness ratios. The probability sensitivity analysis was used to estimate the cost-effectiveness in multiple uncertainty factors. RESULTS: Assuming H. pylori eradication will prevent 50% of attribute gastric cancer, the screening strategies would prevent 16.6% cases of gastric cancer. Cost-effectiveness were 10,405 Yuan (95% CI: 4,238 - 27,727 Yuan) per GC prevented, 64 Yuan (95% CI: 31 - 97 Yuan) per QALY saved and 1,374 Yuan (95% CI: 352 - 86,624 Yuan) per life year saved. CONCLUSION: Screening and treatment for H. pylori infection in population was potentially effective in the prevention of gastric cancer, and screening in high incidence area of gastric cancer would be more effective and economic.