Xitai Sun

Identification and Clinical Significance of Mobilized Endothelial Progenitor Cells in Tumor Vasculogenesis of Hepatocellular Carcinoma

Purpose: To investigate the distribution, frequency, and clinical significance of mobilized endothelial progenitor cells (EPC) in hepatocellular carcinoma (HCC). Experimental Design: In healthy controls and patients with HCC, the frequency of circulating EPCs was determined by colony-forming assays, fluorescence-activated cell sorting, and real-time PCR. One hundred sixty-five–amino acid form of vascular endothelial growth factor and platelet-derived growth factor-BB in plasma and tissue were quantified by ELISA. The distribution and frequency of EPCs were evaluated by immunofluorescence, immunohistochemistry, and real-time PCR in normal liver (n = 8), and tumor tissue (TT), adjacent nonmalignant liver tissue (AT), and tumor-free tissue 5 cm from the tumor edge (TF) from 64 patients with HCC. Clinicopathologic data for these patients were evaluated. Results: Compared with values for healthy controls, colony-forming unit scores were higher in the peripheral blood of patients with HCC. Plasma 165-amino acid form of vascular endothelial growth factor and platelet-derived growth factor-BB correlated with the expression level of the AC133 gene, which was also higher in the peripheral blood of patients with HCC. Immunohistochemical analysis showed that EPCs were incorporated into the microvessels in cirrhotic and tumor tissue. Compared with normal liver (9.00), increased AC133+ microvessel density (microvessels/0.74 mm2) was found in TT (53.56), AT (84.76), and TF (48.33). The levels of AC133 gene expression and AC133-microvessel density in AT, which were the highest among four groups, correlated with clinicopathologic variables (the absence of tumor capsule, venous invasion, proliferating cell nuclear antigen intensity, and early recurrence). Conclusions: Mobilized EPCs participate in tumor vasculogenesis of HCC. AC133 gene or antigen in peripheral blood and liver tissue could be used as a biomarker for predicting the progression of HCC.

Extrahepatic bile duct regeneration in pigs using collagen scaffolds loaded with human collagen-binding bFGF

Extrahepatic bile duct defects and their complications are benign lesions but with malignant outcomes. Extrahepatic bile duct regeneration at the injury site could be important for the repair. In our previous work, a human basic fibroblast growth factor (bFGF) fused with a collagen-binding domain (CBD) was produced to activate the collagen membrane to obtain targeted tissue regeneration. This collagen/growth factor functional biomaterial could promote the regeneration of skin, bladder and full-thickness abdominal wall by accelerating vascularization and cellularization of autologous tissues. We speculate that the functional biomaterial could also provide the repairing effect on extrahepatic bile duct injuries. Using a pig extrahepatic bile duct injury model, we found that the collagen/CBD-bFGF composite biomaterial could significantly promote the extrahepatic bile duct regeneration at the injury site without causing structure deformation or hepatic dysfunction during both short- and long-time observations.

Efficacy and safety of liver transplantation in patients with cholangiocarcinoma: A systematic review and meta‐analysis

The aim of our study was to evaluate the efficacy and safety of liver transplantation in patients with cholangiocarcinoma. According to the requirements of Cochrane systematic review, a thorough literature search was performed in PubMed/Medline, Embase and Cochrane electronic databases between 1995 and 2009 in terms of the key words “liver transplantation” and “cholangiocarcinoma,” “cholangiocellular carcinoma” or “bile duct cancer,” with restricted articles for the English language. Data were processed for a meta‐analysis by Stata 10 software. Altogether 14 clinical trials containing 605 transplanted patients of bile duct cancers were finally enrolled in our study. The overall 1‐, 3‐ and 5‐year pooled survival rates were 0.73 [95% confidence interval (CI) = 0.65–0.80], 0.42 (95% CI = 0.33–0.51) and 0.39 (95% CI = 0.28–0.51), respectively. Of note, preoperative adjuvant therapies [orthotopic liver transplantation (OLT)‐PAT group] rendered the transplanted individuals with comparably favorable outcomes with 1‐, 3‐ and 5‐year pooled survival rates of 0.83 (95% CI = 0.57–0.98), 0.57 (95% CI = 0.18–0.92) and 0.65 (95% CI = 0.40–0.87). In addition, the overall pooled incidence of complications was 0.62 (95% CI = 0.44–0.78), among which that of OLT‐PAT group (0.58; 95% CI = 0.20–0.92) was relatively acceptable compared to those of liver transplantation alone (0.61; 95% CI = 0.33–0.85) and liver transplantation with extended bile duct resection (0.78; 95% CI = 0.55–0.94). In comparison to curative resection of cholangiocarcinoma with the 5‐year survival rate reported from 20 to 40%, the role of liver transplantation alone is so limited. In the future, attention will be focused on liver transplantation following neoadjuvant radiochemotherapy, which requires a well‐designed, prospective randomized controlled study.

USP39 promotes the growth of human hepatocellular carcinoma in vitro and in vivo

Ubiquitin specific protease 39 (USP39) plays an important role in mRNA splicing. In the present study, we investigated the role of USP39 in regulating the growth of hepatocellular carcinoma (HCC). We detected USP39 expression in more than 100 HCC clinical samples. The USP39 expression was significantly higher in the tumor tissues compared to the adjacent normal tissues, and was strongly associated with the pathological grade of HCC. USP39 knockdown inhibited cell proliferation and colony formation in vitro in the HepG2 cells, while upregulation of USP39 promoted tumor cell growth. FCM assay showed that USP39 knockdown led to G2/M arrest and induced apoptosis in the HepG2 cells. USP39 knockdown by shRNA inhibited xenograft tumor growth in nude mice. Moreover, USP39 knockdown led to the upregulation of p-Cdc2 and downregulation of p-Cdc25c and p-myt1, while the expression of total Cdc2, Cdc25c and myt1 was not changed in the USP39-knockdown cells. We also found that p-Cdc2 was decreased in the USP39-overexpressing cells and was upregulated in the xenografted tumors derived from the HepG2/KD cells from nude mice. Meanwhile, the expression levels of FoxM1 and its target genes PLK1 and cyclin B1 were decreased in the USP39-knockdown cells. These results suggest that USP39 may contribute to FoxM1 splicing in HCC tumor cells. Our data indicate that USP39 knockdown inhibited the growth of HCC both in vitro and in vivo through G2/M arrest, which was partly achieved via the inhibition of FoxM1 splicing.

In vitro biocompatibility of polypyrrole/PLGA conductive nanofiber scaffold with cultured rat hepatocytes

To intruduce conductive biomaterial into liver tissue engineering, a conductive nanofiber scaffold, polypyrrole/poly(lactic-co-glycolic)acid(PLGA), was designed and prepared via electro-spinning and oxidative polymerization. Effects of the scaffold on hepatocyte adhesion, viability and function were then investigated. SEM revealed pseudopodium formation and abundant extracellular matrix on the surface of PLGA membrane and polypyrrole/PLGA membrane. The adhesion rate, cellular activity, urea synthesis and albumin secretion of the hepatocytes cultured on polypyrrole/PLGA group were similar to those on the PLGA group, but were significantly higher than those on the control group. There were no significant differences in concentrations of LDH and TNF-α among three groups. These results suggested the potential application of this conductive nanofiber scaffold as a suitable substratum for hepatocyte culturing in liver tissue engineering.

Repair of Extrahepatic Bile Duct Defect Using a Collagen Patch in a Swine Model

Extrahepatic bile duct (EBD) injury can happen during surgery. To repair a defect of the EBD and prevent postoperative biliary complications, a collagen membrane was designed. The collagen material was porous, biocompatible, and degradable and could maintain its shape in bile soaking for about 4 weeks. The goal was to induce rapid bile duct tissue regeneration. Twenty Chinese experimental hybrid pigs were used in this study and divided into a patch group and a control group. A spindle-shaped defect (20 mm × 6 mm) was made in the anterior wall of the lower EBD in the swine model, and then the defect was reconstructed using a collagen patch with a drainage tube and wrapped with greater omentum. Ultrasound was performed at 2, 4, 8, and 12 weeks postoperatively. Liver function tests and white blood cell count (WBC) were measured. Hematoxylin-eosin staining, cytokeratin 7 immunohistochemical staining, and Van Giesons staining of EBD were used. The diameter and thickness of the EBD at the graft site were measured. There was no significant difference in liver function tests or WBC in the patch group compared with the control group. No evidence of leakage or stricture was observed, but some pigs developed biliary sludge or stone at 4 and 8 weeks. The drainage tube was lost within 12 weeks. The neo-EBD could withstand normal biliary pressure 2 weeks after surgery. Histological study showed the accessory glands and epithelial cells gradually regenerated at graft sites from 4 weeks, with increasing vessel infiltration and decreasing inflammation. The collagen fibers became regular with full coverage of epithelial cells. The statistical analysis of diameter and thickness showed no stricture formation at the graft site, but the EBD wall was slightly thicker than in the normal bile duct due to collagen fiber deposition. The structure of the neo-EBD was similar to that of the normal EBD. The collagen membrane patch associated with a drainage tube and wrapped with greater omentum effectively induced the regeneration of the EBD defect within 12 weeks.

USP39 promotes colorectal cancer growth and metastasis through the Wnt/β-catenin pathway

In the present study, we first examined the expression of USP39 protein using tissue array containing 90 colorectal cancer (CRC) tissues and 9 clinical samples, and observed that it has significantly higher expression in cancer tissues as compared to the corresponding adjacent normal tissues. Also, we tested USP39 expression level in four CRC cancer cell lines and identified that it indeed had higher expression in all these CRC cell lines. In addition, its knockdown inhibited not only the cell growth of SW480 and HT29 cells, but also the cell migration and invasion. Further analysis of its molecular mechanism suggested that the expression of four crucial proteins of Wnt/β-catenin pathway, including β-catenin, TCF4, MMP2 and MMP9 was reduced as a result of USP39 knockdown. Taken together, all these findings demonstrated that USP39 protein plays an important role in the growth and metastasis of colorectal cancer mainly through Wnt/β-catenin pathway.

Geranylgeranyl diphosphate synthase (GGPPS) regulates non-alcoholic fatty liver disease (NAFLD)-fibrosis progression by determining hepatic glucose/fatty acid preference under high-fat diet conditions: GGPPS regulates NAFLD-fibrosis by glycolysis

Patients with obesity have a high prevalence of non‐alcoholic fatty liver disease (NAFLD) and, in parallel, increased susceptibility to fibrosis/cirrhosis/hepatocellular carcinoma (HCC). Herein, we report that a high‐fat diet (HFD) can augment glycolysis and then accelerate NAFLD–fibrosis progression by downregulating the expression of geranylgeranyl diphosphate synthase (GGPPS), which is a critical enzyme in the mevalonate pathway. Long‐term HFD overloading decreases GGPPS expression in mice, which shifts the fuel preference from fatty acids towards glucose. Liver‐specific Ggpps deficiency drives the Warburg effect by impairing mitochondrial function, and then induces hepatic inflammation, thus exacerbating fibrosis. Ggpps deficiency also enhances the hyperfarnesylation of liver kinase B1, and promotes metabolic reprogramming by regulating 5′‐AMP‐activated protein kinase activity. Clinical data further imply that GGPPS expression can predict the stage of NAFLD and recurrence of NAFLD‐associated HCC. We conclude that the level of GGPPS is a susceptibility factor for NAFLD–fibrosis progression, and requires more stringent surveillance to ensure early prediction and precision of treatment of NAFLD‐related HCC. Copyright

Hepatic expression of Yin Yang 1 (YY1) is associated with the non-alcoholic fatty liver disease (NAFLD) progression in patients undergoing bariatric surgery

BackgroundThis study is to investigate the association between the hepatic expression of Yin Yang 1 (YY1) and the progression of non-alcoholic fatty liver disease (NAFLD) in patients undergoing bariatric surgery.MethodsObese patients undergoing bariatric surgery were included. Liver tissues were subjected to the quantitative real-time PCR, Western blot analysis, and immunohistochemical assay, to determine the expression levels of YY1.ResultsTotally 88 patients were included. According to the NAFLD activity score (NAS), these patients were divided into the control (n = 12), steatosis (n = 20), non-defining NASH (n = 38), and NASH (n = 18) groups. Significant differences in the serum glucose, insulin, ALT, AST, and HOMA-IR levels were observed among these different NAFLD groups. Hepatic YY1 expression had correlation with serum glucose, insulin, HOMA-IR, ALT, AST, triglycerides, HDL, and GGT. Immunohistochemical analysis showed that, compared with the control group, the expression levels of YY1 were significantly higher in the non-defining NASH and NASH groups. In addition, multivariate regression model showed that the serum ALT and YY1 levels were strongly associated with the NAFLD activity.ConclusionsSeveral factors are associated with NAFLD progression, including the expression of YY1. Our findings contribute to understanding of the pathogenesis of NAFLD.Trial registrationNCT03296605, registered on September 28, 2017.

USP39 regulates the growth of SMMC‑7721 cells via FoxM1

The present study investigated ubiquitin specific peptidase 39 (USP39) gene knockdown on SMMC-7721 cells in vitro and in vivo, and the role of USP39 in regulating the growth of hepatocellular carcinoma (HCC). Two small interfering RNAs (siRNA) were constructed, which targeted the USP39 gene and control sequences were synthesized and inserted into a pGCSIL-GFP lentiviral vector. The full length of USP39 cDNA was amplified by polymerase chain reaction (PCR) and cloned into pEGFP-N2, and the recombinant plasmids were transfected into cells. Knockdown efficiency and upregulation of USP39 was determined by reverse transcription-quantitative PCR and western blotting. The impact of USP39 on the growth of SMMC-7721 cells in vitro was examined using an MTT assay, colony formation, flow cytometry (FCM) and immunohistochemical staining. The impact of USP39 on the growth of SMMC-7721 cells in vivo was examined by assessing tumorigenicity in nude mice. Western blotting was performed to examine the mechanism of USP39 regulation on SMMC-7721 cell growth. Recombinant vectors containing specific and scrambled USP39 siRNA sequences were constructed and transfected into SMMC-7721 cells. USP39 knockdown inhibited cell proliferation and colony formation in SMMC-7721 cells, while upregulation of USP39 promoted the growth of tumor cells. FCM indicated that USP39 knockdown led to G2/M arrest and induced apoptosis in SMMC-7721 cells. USP39 knockdown inhibited xenograft tumor growth in nude mice and led to the downregulation of the transcription factor Forkhead Box M1 (FoxM1). Gene expression of FoxM1 targets, including polo-like kinase 1, cyclin B1 and centromere protein A also decreased following USP39 knockdown. The results suggest that knockdown of USP39 inhibits the growth of HCC in vitro and in vivo, potentially through the induction of G2/M arrest by regulating the pre-mRNA splicing of FoxM1.