Zhikun Liu

MicroRNA-30a-3p inhibits tumor proliferation, invasiveness and metastasis and is downregulated in hepatocellular carcinoma.

BACKGROUND MicroRNAs (miRNAs) are small non-coding RNAs that regulate physiological and pathological processes by suppressing target gene expression. Altered expression of miR-30a-3p has been demonstrated in several cancers. However, little about how miR-30a-3p functions in these cancers has been reported, and the role of miR-30a-3p in hepatocellular carcinoma (HCC) is unknown. The purpose of this study was to identify the role and underlying molecular mechanism of action of miR-30a-3p in HCC. METHODS A total of 110 HCC patients, primarily treated by surgical removal of tumors, were involved in the study. HCC cell line Bel-7402 was selected to characterize the function of miR-30a-3p in vitro. RESULTS Our results showed that in 83.6% of the 110 HCC patients, expression of miR-30a-3p was significantly downregulated (P < 0.0001) in tumors compared to adjacent normal tissues. In a clinicopathological correlation analysis, downregulation of miR-30a-3p correlated with a significantly higher incidence of portal vein tumor thrombus (PVTT, P = 0.009). Moreover, miR-30a-3p markedly inhibited the invasion and migration of Bel-7402 HCC cells in vitro. Furthermore, miR-30a-3p overexpression had an inhibitory effect on cell proliferation, induced apoptosis and increased arrest of cells in the S phase. We further demonstrated that miR-30a-3p regulates HCC cell function by a mechanism involving reduced vimentin and MMP3 expression and restoration of E-cadherin expression. CONCLUSIONS our data suggest that miR-30a-3p is downregulated in HCC and acts as a tumor suppressor in vitro. Regulation of vimentin, E-cadherin and MMP3 by miR-30a-3p suggests a useful therapeutic strategy for tumors with reduced miR-30a-3p expression.

Artificial Liver Support System Combined with Liver Transplantation in the Treatment of Patients with Acute-on-Chronic Liver Failure

Background The search for a strategy to provide temporary liver support and salvage the patients with acute-on-chronic liver failure (ACLF) remains an important issue. This study was designed to evaluate the experience in artificial liver support system (ALSS) combined with liver transplantation (LT) in the treatment of ACLF. Methodology/Principal Findings One hundred and seventy one patients with HBV related ACLF undergoing LT between January 2001 and December 2009 were included. Of the 171 patients, 115 received 247 sessions of plasma exchange-centered ALSS treatment prior to LT (ALSS-LT group) and the other 56 received emergency LT (LT group). The MELD score were 31±6 and 30±7 in ALSS-LT group and LT group. ALSS treatment resulted in improvement of liver function and better tolerance to LT. The average level of serum total bilirubin before LT was lower than that before the first time of ALSS treatment. The median waiting time for a donor liver was 12 days (2–226 days) from the first run of ALSS treatment to LT. Compared to LT group, the beneficial influences of ALSS on intraoperative blood loss and endotracheal intubation time were also observed in ALSS-LT group. The 1-year and 5-year survival rates in the ALSS-LT group and LT group were 79.2% and 83%, 69.7% and 78.6%. Conclusions/Significance Plasma exchange-centered ALSS is beneficial in salvaging patients with ACLF when a donor liver is not available. The consequential LT is the fundamental treatment modality to rescue these patients and lead to a similar survival rate as those patients receiving emergency transplantation.

MicroRNA-452 promotes stem-like cells of hepatocellular carcinoma by inhibiting Sox7 involving Wnt/β-catenin signaling pathway

The decrease of microRNA-452 (miR-452) in gliomas promoted stem-like features and tumorigenesis. However, the role of miR-452, especially in regulating cancer stem cells (CSCs) in hepatocellular carcinoma (HCC) remains ambiguous. We enriched stem-like HCC cells by serial passages of hepatospheres with chemotherapeutic agents. Stem-like characteristics including the capabilities of chemo-resistance, stemness-related gene expression profiling, self-renewal, tumorigenicity and metastasis formation were detected. MiR-452 was markedly increased in the chemo-resistant hepatospheres and human HCC tissues. and the overexpression of miR-452 in HCC patients predicted poor overall survival. MiR-452 significantly promoted stem-like characteristics in vitro and in vivo. Further, Sox7 was identified as the direct target of miR-452, which could physically bind with β-catenin and TCF4 in the nucleus and then inhibit the activity of Wnt/β-catenin signaling pathway. Finally, the combined chemotherapy of doxorubicin and all-trans retinoic acid (ATRA) showed dramatically efficiency in suppressing HCC metastasis. These data suggested that miR-452 promoted stem-like traits of HCC, which might be a potential therapeutic target for HCC. The combination of doxorubicin and ATRA might be a promising therapy in HCC management.

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.

Role of overexpression of MACC1 and/or FAK in predicting prognosis of hepatocellular carcinoma after liver transplantation

Background: Metastasis-associated in colon cancer-1 (MACC1) acts as a promoter of tumor metastasis; however, the predictive value of MACC1 for hepatocellular carcinoma (HCC) after liver transplantation (LT) remains unclear. Methods: We examined the expression of MACC1 and its target genes MET and FAK by quantitative PCR in 160 patients with HCC that was undergone LT. Results: The patients with MACC1high or FAKhigh in HCCs showed a significantly shorter overall survival and higher cumulative recurrence rates after liver transplantation (LT), compared with MACC1low or FAKlow group. Multivariate analysis indicated that MACC1 alone or combination of MACC1/FAK was an independent prognostic factor for overall survival and cumulative recurrence. Conclusions: MACC1 or combination of MACC1/FAK could serve as a novel biomarker in predicting the prognosis of HCC after LT.

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.

Global proteomic profiling in multistep hepatocarcinogenesis and identification of PARP1 as a novel molecular marker in hepatocellular carcinoma

The more accurate biomarkers have long been desired for hepatocellular carcinoma (HCC). Here, we characterized global large-scale proteomics of multistep hepatocarcinogenesis in an attempt to identify novel biomarkers for HCC. Quantitative data of 37874 sequences and 3017 proteins during hepatocarcinogenesis were obtained in cohort 1 of 75 samples (5 pooled groups: normal livers, hepatitis livers, cirrhotic livers, peritumoral livers, and HCC tissues) by iTRAQ 2D LC-MS/MS. The diagnostic performance of the top six most upregulated proteins in HCC group and HSP70 as reference were subsequently validated in cohort 2 of 114 samples (hepatocarcinogenesis from normal livers to HCC) using immunohistochemistry. Of seven candidate protein markers, PARP1, GS and NDRG1 showed the optimal diagnostic performance for HCC. PARP1, as a novel marker, showed comparable diagnostic performance to that of classic markers GS and NDRG1 in HCC (AUCs = 0.872, 0.856 and 0.792, respectively). A significant higher AUC of 0.945 was achieved when three markers combined. For diagnosis of HCC, the sensitivity and specificity were 88.2% and 81.0% when at least two of the markers were positive. Similar diagnostic values of PARP1, GS and NDRG1 were confirmed by immunohistochemistry in cohort 3 of 180 HCC patients. Further analysis indicated that PARP1 and NDRG1 were associated with some clinicopathological features, and the independent prognostic factors for HCC patients. Overall, global large-scale proteomics on spectrum of multistep hepatocarcinogenesis are obtained. PARP1 is a novel promising diagnostic/prognostic marker for HCC, and the three-marker panel (PARP1, GS and NDRG1) with excellent diagnostic performance for HCC was established.

Annexin A2 is not a good biomarker for hepatocellular carcinoma in cirrhosis.

In China, hepatocellular carcinoma (HCC) usually develops following a long history of chronic hepatitis B infection or cirrhosis. To evaluate the diagnostic role of annexin A2 (ANXA2), a possible tumor marker, in patients with hepatitis B virus (HBV)-related HCC, particularly those with a history of cirrhosis, the present study prospectively enrolled 87 patients with HBV-related HCC (with cirrhosis), 39 patients with HBV-related cirrhosis and 27 healthy controls. The expression levels of serum and tissue ANXA2 were determined using an enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining, respectively. The serum levels of ANXA2 were significantly elevated in the patients with HCC (median, 567.2 μg/ml; P=0.003) and cirrhosis (median, 414.8 μg/ml; P=0.011) compared with the healthy controls (median, 241.9 μg/ml). However, no significant differences were observed in the serum ANXA2 levels between the patients with HCC and those with cirrhosis. The immunohistochemical staining analysis showed that the healthy controls did not show positive staining, while the number of cases immunoreactive for ANXA2 steadily increased from the liver cirrhosis tissues (20/39, 51.3%) to the non-cancer (53/87, 60.9%) and cancer tissues (68/87, 78.2%). The cancer tissues exhibited a significantly higher ANXA2-positive rate compared with the non-cancer (P=0.013) and liver cirrhosis tissues (P=0.002). Furthermore, marked ANXA2 staining was more prevalent in the cancer tissues (16/87, 18.4%) than the non-cancer (4/87, 4.6%; P=0.004) and liver cirrhosis (1/39, 2.6%; P=0.034) tissues. The sensitivity, specificity and diagnostic accuracy of tissue ANXA2 for HCC in cirrhosis were 78.2, 42.1 and 56.8%, respectively. The ANXA2 expression levels in the serum and cancer tissues were not associated with tumor-free survival or patient survival following liver transplantation. Serum or tissue ANXA2 is not a good diagnostic marker for HCC in HBV-related cirrhosis and is not associated with prognosis.

LEPREL1 Expression in Human Hepatocellular Carcinoma and Its Suppressor Role on Cell Proliferation.

Background. Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies worldwide. It is characterized by its high invasive and metastatic potential. Leprecan-like 1 (LEPREL1) has been demonstrated to be downregulated in the HCC tissues in previous proteomics studies. The present study is aimed at a new understanding of LEPREL1 function in HCC. Methods. Quantitative RT-PCR, immunohistochemical analysis, and western blot analysis were used to evaluate the expression of LEPREL1 between the paired HCC tumor and nontumorous tissues. The biology function of LEPREL1 was investigated by Cell Counting Kit-8 (CCK8) assay and colony formation assay in HepG2 and Bel-7402 cells. Results. The levels of LEPREL1 mRNA and protein were significantly lower in the HCC tissues as compared to those of the nontumorous tissues. Reduced LEPREL1 expression was not associated with conventional clinical parameters of HCC. Overexpression of LEPREL1 in HepG2 and Bel-7402 cells inhibited cell proliferation (P < 0.01) and colony formation (P < 0.05). LEPREL1 suppressed tumor cell proliferation through regulation of the cell cycle by downregulation of cyclins. Conclusions. Clinical parameters analysis suggested that LEPREL1 was an independent factor in the development of HCC. The biology function experiments showed that LEPREL1 might serve as a potential tumor suppressor gene by inhibiting the HCC cell proliferation.

The prognostic relevance of primary tumor location in patients undergoing resection for pancreatic ductal adenocarcinoma

Different clinical presentations and prognoses have been implied between pancreatic head and body/tail cancers. We aimed to identify the prognostic relevance of primary tumor location in patients undergoing resection for pancreatic ductal adenocarcinoma (PDAC). Thirty-two pairs of patients with strictly matched early stage (II) pancreatic head and body/tail cancers were enrolled. The molecular feature of the two subtypes of PDAC was assessed on the level of miRNA expression. Out of the 64 patients, 34 (53.1%) had tumor recurrence after radical resection during the follow-up period (2.3 ± 0.8 years). Both overall and tumor-free survival were significantly higher in the patients with pancreatic body/tail cancer compared with those with pancreatic head cancer. Patient age and tumor location were the independent prognostic factors for tumor recurrence. A remarkably lower expression of miR-501-3p and higher expression of miR-375 were found and were further verified in pancreatic body/tail cancer tissues compared with pancreatic head cancer tissues. The low expression of miR-501-3p was significantly associated with a low risk of tumor recurrence. Both, subcutaneous and orthotopic PDAC mouse models presented highly invasive tumor phenotypes upon up-regulated miR-501-3p expression. An in vitro study showed that miR-501-3p promoted the invasiveness of PDAC cells possibly via suppressing E-cadherin. In summary, at resectable early stage, pancreatic body/tail cancer presents a less malignant phenotype associated with deregulation of miR-501-3p compared with pancreatic head cancer.