Xuyong Wei

NDRG1 as a biomarker for metastasis, recurrence and of poor prognosis in hepatocellular carcinoma

N-myc downstream-regulated gene 1 (NDRG1) has been reported to be a multifunctional protein associated with carcinogenesis, however, the cellular function of NDRG1 remains elusive in human cancers. Here, our proteomics profile analysis of HCC tissues with different metastatic capabilities revealed that NDRG1 was correlated with metastasis and recurrence in HCC patients after liver transplantation (LT). Immunohistochemical staining of 143 HCC patients after LT showed that NDRG1-positive expression had poor prognosis, either for shorter disease-free survival or overall survival (P < 0.001), compared with NDRG1-negative expression. Multivariate analysis confirmed NDRG1 as an independent prognostic value (P < 0.001). In addition, in vitro experiments HCC cells with small interfering RNA against NDRG1 significantly suppressed its proliferation, colony formation, invasion and migration ability. Microarray analysis revealed that NDRG1 modulated the expression of genes associated with transmembrane transporter activity, chemoattractant activity, immune response, cell adhesion and cell proliferation process. Taken together, these results suggested that NDRG1 was an important molecule in controlling HCC metastasis and thus suggested as a novel biomarker for predicting HCC recurrence after LT.

Oxymatrine induces human pancreatic cancer PANC-1 cells apoptosis via regulating expression of Bcl-2 and IAP families, and releasing of cytochrome c

BackgroundOxymatrine, an isolated extract from traditional Chinese herb Sophora Flavescens Ait, has been traditionally used for therapy of anti-hepatitis B virus, anti-inflammation and anti-anaphylaxis. The present study was to investigate the anti-cancer effect of oxymatrine on human pancreatic cancer PANC-1 cells, and its possible molecular mechanism.MethodsThe effect of oxymatrine on the viability and apoptosis was examined by methyl thiazolyl tetrazolium and flow cytometry analysis. The expression of Bax, Bcl-2, Bcl-x (L/S), Bid, Bad, HIAP-1, HIAP-2, XIAP, NAIP, Livin and Survivin genes was accessed by RT-PCR. The levels of cytochrome c and caspase 3 protein were assessed by Western blotting.ResultsOxymatrine inhibited cell viability and induced apoptosis of PANC-1 cells in a time- and dose-dependent manner. This was accompanied by down-regulated expression of Livin and Survivin genes while the Bax/Bcl-2 ratio was upregulated. Furthermore, oxymatrine treatment led to the release of cytochrome c and activation of caspase-3 proteins.ConclusionOxymatrine can induce apoptotic cell death of human pancreatic cancer, which might be attributed to the regulation of Bcl-2 and IAP families, release of mitochondrial cytochrome c and activation of caspase-3.

The impact of miR-34a on protein output in hepatocellular carcinoma HepG2 cells

MicroRNAs are small non‐coding RNA molecules that play essential roles in biological processes ranging from cell cycle to cell migration and invasion. Accumulating evidence suggests that miR‐34a, as a key mediator of p53 tumor suppression, is aberrantly expressed in human cancers. In the present study, we aimed to explore the precise biological role of miR‐34a and the global protein changes in HCC cell line HepG2 cells transiently transfected with miR‐34a. Transfection of miR‐34a into HepG2 cells caused suppression of cell proliferation, inhibition of cell migration and invasion. It also induced an accumulation of HepG2 cells in G1 phase. Among 116 protein spots with differential expression separated by 2‐DE method, 34 proteins were successfully identified by MALDI‐TOF/TOF analysis. Of these, 15 downregulated proteins may be downstream targets of miR‐34a. Bioinformatics analysis produced a protein–protein interaction network, which revealed that the p53 signaling pathway and cell cycle pathway were two major hubs containing most of the proteins regulated by miR‐34a. Cytoskeletal proteins such as LMNA, GFAP, MACF1, ALDH2, and LOC100129335 are potential targets of miR‐34a. In conclusion, abrogation of miR‐34a function could cause downstream molecules to switch on or off, leading to HCC development.

Structure-Based Rational Design of Prodrugs To Enable Their Combination with Polymeric Nanoparticle Delivery Platforms for Enhanced Antitumor Efficacy

Drug-loaded nanoparticles (NPs) are of particular interest for efficient cancer therapy due to their improved drug delivery and therapeutic index in various types of cancer. However, the encapsulation of many chemotherapeutics into delivery NPs is often hampered by their unfavorable physicochemical properties. Here, we employed a drug reform strategy to construct a small library of SN-38 (7-ethyl-10-hydroxycamptothecin)-derived prodrugs, in which the phenolate group was modified with a variety of hydrophobic moieties. This esterification fine-tuned the polarity of the SN-38 molecule and enhanced the lipophilicity of the formed prodrugs, thereby inducing their self-assembly into biodegradable poly(ethylene glycol)-block-poly(d,l-lactic acid) (PEG-PLA) nanoparticulate structures. Our strategy combining the rational engineering of prodrugs with the pre-eminent features of conventionally used polymeric materials should open new avenues for designing more potent drug delivery systems as a therapeutic modality.

Liver transplantation for hepatocellular carcinoma beyond the Milan criteria

Objective Liver transplantation is an optimal radical therapy for selected patients with hepatocellular carcinoma. The stringent organ allocation system driven by the Milan criteria has been challenged by alternative sets of expanded criteria. Careful analysis is needed to prove that the Milan criteria can be expanded safely and effectively. Design This study collectively reviewed 6012 patients of hepatocellular carcinoma from the China Liver Transplant Registry. Expanded criteria were evaluated to characterise an optimised expansion with acceptable outcomes beyond the Milan criteria. Results Compared with the Milan criteria, Valencia, University of California, San Francisco, University Clinic of Navarra and Hangzhou criteria provided an expansion of 12.4%, 16.3%, 19.6%, and 51.5%, respectively. The post-transplant survivals of patients fulfilling the expanded criteria were comparable to that of the Milan criteria. The analysis of net reclassification improvement and area under the receiver operating characteristic curves showed an excellent efficiency in recurrence prediction for the expanded criteria compared with the Milan criteria. In patients exceeding Milan but fulfilling the Hangzhou criteria (N=1352), α-fetoprotein (AFP) >100 ng/mL and tumour burden>8 cm were the only two independent prognostic factors (p<0.001). Accordingly, the Hangzhou criteria were stratified as type A (tumour burden ≤8 cm, or tumour burden >8 cm but AFP≤100 ng/mL) and type B (tumour burden >8 cm but AFP between 100 and 400 ng/mL). Type A showed significantly higher 5-year tumour-free survival rates compared with type B (p<0.001). Conclusions The Milan criteria can be expanded safely and effectively. The prognostic stratification system based on the Hangzhou criteria serves as a hierarchy of transplant candidates for hepatocellular carcinoma.

Association between donor and recipient TCF7L2 gene polymorphisms and the risk of new-onset diabetes mellitus after liver transplantation in a Han Chinese population

BACKGROUNDS & AIMS New-onset diabetes mellitus (NODM) is a frequent and serious complication arising after liver transplantation (LT). Transcription factor 7-like 2 (TCF7L2) polymorphisms have been reported to strongly associate with type 2 diabetes. In addition, the donor liver plays a vital role in regulating blood glucose levels. In this study, we aim at evaluating the association between donor and recipient TCF7L2 gene polymorphisms with NODM after LT. METHODS A total of 125 patients undergoing primary LT, without a history of diabetes were included. Four single nucleotide polymorphisms (rs290487, rs7903146, rs11196205, and rs12255372), closely associated with type 2 diabetes in the Eastern Asia population, were genotyped and analyzed. RESULTS Both donor and recipient rs290487 polymorphisms (CC vs. TT genotype) were found to be significantly associated with NODM. In multivariate analysis, donor rs290487 genetic variation (OR = 2.172 per each C allele, p = 0.015), blood tacrolimus levels at 1 month post-LT >10 ng/ml (OR = 3.264, p = 0.017), and recipient age >55 years (OR = 2.638, p = 0.043) were identified as independent risk factors of NODM. Furthermore, donor rs290487 CC genotype could predict a high probability (>40%) of the onset of NODM. Predictive model containing donor rs290487 polymorphism showed a significantly higher prognostic ability on NODM than the model with only clinical parameters (p = 0.031). CONCLUSIONS Donor TCF7L2 rs290487 polymorphism is associated with an increased risk of NODM after LT and has a potential clinical value for the prediction of NODM.

ZIP4, a Novel Determinant of Tumor Invasion in Hepatocellular Carcinoma, Contributes to Tumor Recurrence after Liver Transplantation

Background and purpose: Recently, evidence that Zinc transporter ZRT/IRT-like protein 4 (ZIP4) is involved in invasiveness and apoptosis has emerged in pancreatic cancer and prostate cancer. Our aim was to assess the role of ZIP4 in invasiveness, migration and apoptosis of hepatocellular carcinoma (HCC). The prognostic value of ZIP4 in HCC after liver transplantation was evaluated. Methods: The role of ZIP4 in HCC was investigated by overexpressing ZIP4 in BEL7402 and HepG2 cells and inhibiting ZIP4 in HuH-7 and HepG2 cells, using overexpression and shRNA plasmids in vitro studies. Immunohistochemical analysis was used to evaluate ZIP4 expression in HCC tissues from 60 patients undergoing liver transplantation, 36 cirrhotic tissue samples, and 6 normal tissue samples. Prognostic significance was assessed using the Kaplan-Meier method and the log-rank test. Results: Specific suppression of ZIP4 reduced cell migration and invasiveness, whereas ZIP4 overexpression caused increases in cell migration and invasiveness. Furthermore, overexpression of ZIP4 resulted in increased expression of pro-metastatic genes (MMP-2, MMP-9) and decreased expression of pro-apoptotic genes (caspase-3, caspase-9, Bax). In contrast, suppression of ZIP4 resulted in an opposite effect. ZIP4 was more highly expressed in tumor tissues than non-tumor tissues (P < 0.0001). ZIP4 expression was significantly associated with tumor recurrence (P = 0.002), tumor node metastasis stage (P = 0.044), Child-Turcotte-Pugh score (P = 0.042), and tumor size (P = 0.022). Univariate analysis showed that ZIP4 expression was significantly associated with overall survival (P = 0.020) and tumor-free survival (P = 0.049). Multivariate analysis revealed that ZIP4 was an independent predictor of overall survival (P = 0.037) after liver transplantation. Conclusions: ZIP4 could promote migration, invasiveness, and suppress apoptosis in hepatocellular carcinoma, and represent a novel predictor of poor prognosis and therapeutic target for patients with HCC who undergo liver transplantation.

Evaluation of hepatitis B viral replication and proteomic analysis of HepG2.2.15 cell line after knockdown of HBx

BACKGROUND Hepatitis B virus (HBV) is one of the major pathogens of human liver disease. Studies have shown that HBV X protein (HBx) plays an important role in promoting viral gene expression and replication. In this study we performed a global proteomic profiling to identify the downstream functional proteins of HBx, thereby detecting the mechanisms of action of HBx on virion replication. METHODS HBx in the HepG2.2.15 cell line was knocked down by the transfection of small interfering RNA (siRNA). The replication level of HBV was evaluated by microparticle enzyme immunoassay analysis of HBsAg and HBeAg in the culture supernatant, and real-time quantitative PCR analysis of HBV DNA. Two-dimensional electrophoresis combined with MALDI-TOF/TOF was performed to analyze the changes in protein expression profile after treatment with HBx siRNA. RESULTS Knockdown of HBx disturbed HBV replication in vitro. HBx target siRNA significantly inhibited the expression of HBsAg, HBeAg and the replication of HBV DNA. Twelve significantly changed proteins (7 upregulated and 5 downregulated) were successfully identified by MALDI-TOF/TOF using proteomics differential expression analysis after the knockdown of HBx. Among these identified proteins, HSP70 was validated by Western blotting. CONCLUSION The results of the study indicated the positive effect of HBx on HBV replication, and a group of downstream target proteins of HBx may be responsible for this effect.

Donor miR-196a-2 polymorphism is associated with hepatocellular carcinoma recurrence after liver transplantation in a Han Chinese population.

Recurrence of hepatocellular carcinoma (HCC) is one of the leading causes of death after liver transplantation (LT). We aim to evaluate the association of donor and recipient single nucleotide polymorphisms (SNPs) with the risk of HCC recurrence after LT. A total of 155 adult patients who underwent primary LT for HCC were enrolled. Ten SNPs associated with HCC susceptibility were genotyped. Patients who received donor livers with the rs11614913 homozygous CC variant presented significantly higher recurrence rates of HCC (41.7 vs. 15.3%, p = 0.009) and lower cumulative tumor‐free survival (p = 0.005) than those who received TT wild‐type donor livers. The donor rs11614913 genetic variant was an independent risk factor for HCC recurrence (odds ratio = 2 per each C allele, p < 0.05) and could significantly improve the predictive abilities of clinical models (Milan, UCSF and Hangzhou criteria). Donor livers homozygous for rs11614913 CC were associated with a higher miR‐196a expression than TT (p = 0.002). In a lentiviral infection of mouse liver and orthotopic mouse model of HCC, the liver miR‐196a overexpression group showed a significantly larger tumor size than the control group (p = 0.001). There is a close association between the tumor size and expression of miR‐196a in the liver (r = 0.693, p = 0.001). In conclusion, the donor miR‐196a‐2 rs11614913 polymorphism is associated with HCC recurrence after LT and improves the predictive value of clinical models. The overexpression of miR‐196a in the liver might provide a tumor‐favorable environment for the development of HCC.

Characterization of genome-wide TFCP2 targets in hepatocellular carcinoma: implication of targets FN1 and TJP1 in metastasis

BackgroundTranscription factor CP2 (TFCP2) is overexpressed in hepatocellular carcinoma(HCC) and correlated with the progression of the disease. Here we report the use of an integrated systems biology approach to identify genome-wide scale map of TFCP2 targets as well as the molecular function and pathways regulated by TFCP2 in HCC.MethodsWe combined Chromatin immunoprecipitation (ChIP) on chip along with gene expression microarrays to study global transcriptional regulation of TFCP2 in HCC. The biological functions, molecular pathways, and networks associated with TFCP2 were identified using computational approaches. Validation of selected target gene expression and direct binding of TFCP2 to promoters were performed by ChIP -PCR and promoter reporter.ResultsTFCP2 fostered a highly aggressive and metastatic phenotype in different HCC cells. Transcriptome analysis showed that alteration of TFCP2 in HCC cells led to change of genes in biological functions involved in cancer, cellular growth and proliferation, angiogenesis, cell movement and attachment. Pathways related to cell movement and cancer progression were also enriched. A quest for TFCP2-regulated factors contributing to metastasis, by integration of transcriptome and ChIP on chip assay, identified fibronectin 1 (FN1) and tight junction protein 1 (TJP1) as targets of TFCP2, and as key mediators of HCC metastasis. Promoter reporter identified the TFCP2-responsive region, and located the motifs of TFCP2-binding sites in the FN1 promoter, which then was confirmed by ChIP-PCR. We further showed that FN1 inhibition blocks the TFCP2-induced increase in HCC cell aggression, and that overexpression of TFCP2 can rescue the effects of FN1 inhibition. Knock down of TJP1 could also rescue, at least in part, the aggressive effect of TFCP2 knockdown in HCC cells.ConclusionsThe identification of global targets, molecular pathways and networks associated with TFCP2, together with the discovery of the effect of TFCP2 on FN1 and TJP1 that are involved in metastasis, adds to our understanding of the mechanisms that determine a highly aggressive and metastatic phenotype in hepatocarcinogenesis.