Weijia Liao

Preoperative Neutrophil-to-Lymphocyte Ratio as a New Prognostic Marker in Hepatocellular Carcinoma after Curative Resection

BACKGROUND: Preoperative peripheral blood neutrophil-to-lymphocyte ratio (NLR) has been proposed to predict prognosis of hepatocellular carcinoma (HCC). However, the cutoff value of NLR in several studies is not consistent. This study aims to investigate the correlation of preoperative NLR with clinicopathologic features and the prognosis in patients who have undergone resection for HCC. METHODS: Clinical data of 256 patients with HCC who underwent radical hepatectomy were retrospectively analyzed. The patients were divided into the low-NLR group (NLR ≤ 2.31) and the high-NLR group (NLR > 2.31). A univariate analysis was performed to assess clinicopathologic characteristics that influenced disease-free survival (DFS) and overall survival (OS) in patients. The significant variables were further analyzed by a multivariate analysis using Cox regression. The Kaplan-Meier method was used to assess the DFS and OS rate. RESULTS: The value of NLR was associated with tumor size, clinical tumor-node-metastasis (TNM) stage, portal vein tumor thrombus (PVTT), distant metastasis, and aspartate aminotransferase (AST) in HCC. NLR > 2.31, size of tumor > 5 cm, number of multiple tumors, III-IV of TNM stage, PVTT, distant metastasis, and AST > 40 U/l were predictors of poorer DFS and OS. NLR > 2.31, size of tumor > 5 cm, III-IV of TNM stage, and AST > 40 U/l were independent predictors of DFS and OS. CONCLUSION: Preoperative NLR > 2.31 was an adverse predictor of DFS and OS in HCC after hepatectomy. This study suggested that NLR might be a novel prognostic biomarker in HCC after curative resection.

Silencing of DLGAP5 by siRNA Significantly Inhibits the Proliferation and Invasion of Hepatocellular Carcinoma Cells

Background The dysregulation of oncogenes and tumor suppressor genes plays an important role in many cancers, including hepatocellular carcinoma (HCC), which is one of the most common cancers in the world. In a previous microarray experiment, we found that DLGAP5 is overexpressed in HCCs. However, whether the up-regulation of DLGAP5 contributes to hepatocarcinogenesis remains unclear. Methodology/Principal Findings In this study, we showed that DLGAP5 was significantly up-regulated in 76.4% (168 of 220) of the analyzed HCC specimens when compared with adjacent liver tissue. DLGAP5 overexpression was evident in 25% (22 of 88) of the HCC specimens without AFP expression, suggesting that DLGAP5 may be a novel biomarker for HCC pathogenesis. The silencing of DLGAP5 gene expression by RNA interference significantly suppressed cell growth, migration and colony formation in vitro. The expression level of DLGAP5 was also found to be related to the methylation level of its promoter in the HCC specimens. Conclusions/Significance Taken together, these data suggest that the expression of DLGAP5 is regulated by methylation and that the up-regulation of DLGAP5 contributes to HCC tumorigenesis by promoting cell proliferation.

TTK activates Akt and promotes proliferation and migration of hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is one of the most malignant cancers with poor clinical outcome. The protein kinase human monopolar spindle 1 (hMps1/TTK) gene expression is significantly increased in HCCs. However, its contributions to hepatocarcinogenesis remain unclear. In this study, we found that TTK was overexpressed in 77.63% (118/152) HCC specimens. Elevated TTK expression positively correlated with large tumor size and presence of the portal vein tumor thrombus (PVTT). Demethylation in its promoter increased TTK expression in HCC. In vitro assays revealed that TTK not only promoted cell proliferation and anchorage-independent growth, but also cell migration. Subsequent investigations revealed that TTK activated Akt/mTOR pathway in a p53 dependent manner. We also found that TTK specific kinase inhibitor AZ3146 could decrease HCC cell growth. In conclusion, TTK contributes to HCC tumorigenesis via promoting cell proliferation and migration. It may serve as a novel biomarker and a potential target in HCC cancer therapy.

Aldo-keto Reductase Family 1 Member B 10 Mediates Liver Cancer Cell Proliferation through Sphingosine-1-Phosphate.

AKR1B10 is involved in hepatocarcinogenesis via modulation of fatty acid and lipid synthesis. AKR1B10 inhibition results in apoptosis of tumor cells whose lipids, especially phospholipids, were decreased by over 50%, suggesting involvement of phospholipids like sphingosine-1-phosphate (S1P) in AKR1B10’s oncogenic function. Using a co-culture system, we found that co-culture of QSG-7701 (human hepatocyte) with HepG2 (hepatoma cell line) increases QSG-7701’s proliferation, in which AKR1B10-S1P signaling plays a pivotal role. Consistent with previous findings, AKR1B10 mRNA and protein levels were higher in primary hepatocellular carcinoma (PHC) tissues than in peri-tumor tissues. Interestingly, the level of S1P was also higher in PHC tissues than in peri-tumor tissues. After analyzing the correlation between AKR1B10 mRNA expression in PHC tissues and the clinical data, we found that AKR1B10 mRNA expression was associated with serum alpha-fetoprotein (AFP), tumor-node-metastasis (TNM) stage, and lymph node metastasis, but not with other clinicopathologic variables. A higher AKR1B10 mRNA expression level is related to a shorter DFS (disease free survival) and OS (overall survival), serving as an independent predictor of DFS and OS in PHC patients with surgical resection.

RNA over-editing of BLCAP contributes to hepatocarcinogenesis identified by whole-genome and transcriptome sequencing

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, although the treatment of this disease has changed little in recent decades because most of the genetic events that initiate this disease remain unknown. To better understand HCC pathogenesis at the molecular level and to uncover novel tumor-initiating events, we integrated RNA-seq and DNA-seq data derived from two pairs of HCC tissues. We found that BLCAP is novel editing gene in HCC and has over-editing expression in 40.1% HCCs compared to adjacent liver tissues. We then used RNA interference and gene transfection to assess the roles of BLCAP RNA editing in tumor proliferation. Our results showed that compared to the wild-type BLCAP gene, the RNA-edited BLCAP gene may stably promote cell proliferation (including cell growth, colony formation in vitro, and tumorigenicity in vivo) by enhancing the phosphorylation of AKT, mTOR, and MDM2 and inhibiting the phosphorylation of TP53. Our current results suggest that the RNA over-editing of BLCAP gene may serve as a novel potential driver in advanced HCC through activating AKT/mTOR signal pathway.

High level of intercellular adhesion molecule-1 affects prognosis of patients with hepatocellular carcinoma.

AIM To determine the cut-off value of intercellular adhesion molecule-1 (ICAM-1) and assess the correlation of ICAM-1 with clinicopathological features and the prognosis of hepatocellular carcinoma (HCC) patients who underwent surgical resection. METHODS We prospectively collected clinicopathological data from 236 HCC patients who had undergone successful hepatectomy. Receiver operating characteristic curve analysis was performed to determine the optimal cut-off value of ICAM-1. Enzyme-linked immunosorbent assay was used to measure the concentration of ICAM-1 in 236 serum samples isolated from HCC patients and the stratified analysis was used to compare the serum level of ICAM-1 in different HCC subgroups. Immunohistochemistry was performed to test the expression level of the ICAM-1 protein in 76 cases of HCC tissues and their adjacent normal liver tissues (ANLT). The survival probability of HCC patients was estimated using Kaplan-Meier plots and differences between the groups were obtained using the log-rank test. Furthermore, independent indicators of the prognosis were acquired using a stepwise Cox proportional hazard model to analyze a series of predictors that were associated with disease-free survival (DFS) and overall survival (OS) in HCC patients. RESULTS Our findings suggested that ICAM-1 promotes HCC metastasis and high serum ICAM-1 is significantly associated with alpha-fetoprotein (AFP) (P = 0.022), clinical tumor-node-metastasis stage (P < 0.001), portal vein tumor thrombus (P = 0.005), distant metastasis (P = 0.016) and recurrence (P = 0.034). We further detected the ICAM-1 protein in HCC specimens and found that 56 of 76 (73.7%) HCC tissues had ICAM-1 positive staining while only 23 of 76 (30.3%) ANLT were positively stained (P < 0.0001). Survival analysis indicated that HCC patients with increased ICAM-1 concentrations had significantly shorter DFS and OS after resection. A multivariate analysis showed that ICAM-1 > 684 ng/mL was an independent factor for DFS (HR = 1.643; 95%CI: 1.125-2.401; P = 0.010) and OS (HR = 1.692; 95%CI: 1.152-2.486; P = 0.007). CONCLUSION ICAM-1 may be a promising serological biomarker for HCC diagnosis and an independent predictor of DFS and OS after surgical resection and may provide a useful reference for the prediction of intra- and extrahepatic metastasis.

DEP Domain Containing 1 is a Novel Diagnostic Marker and Prognostic Predictor for Hepatocellular Carcinoma

BACKGROUND This study was conducted to determine DEPDC1 expression in hepatocelluar carcinomas (HCCs) and to reveal its potential role in diagnosis and prognosis of affected patients. MATERIALS AND METHODS DEPDC1 expression at the mRNA level was detected by quantitative real-time PCR (qRT-PCR) in 205 cases of HCC and paired adjacent normal liver tissues, and by semi-quantitative RT-PCR in 20 cases. Survival curves were obtained by using Kaplan-Meier method and Log-rank test. Independent predictors associated with regard to disease free survival (DFS) and overall survival (OS) were identified using the Cox proportional hazard model. RESULTS High DEPDC1 mRNA levels were detected in 144 out of 205 cases (70.24%) of HCC, significantly associated with clinicopathological parameters, including tumor size (≥4cm), alpha-fetoprotein (≥100ng/ml), B-C of BCLC stage and recurrence. Kaplan-Meier survival analysis revealed that HCC patients with high DEPDC1 expression had poor OS and DFS. Multivariate analysis demonstrated that high DEPDC1 expression was an independent predictor of OS (HR=1.651; 95% 95%CI, 1.041- 2.617; p=0.033) and DFS (HR=1.583; 95%CI, 1.01- 2.483; p=0.045). CONCLUSIONS Our results indicate DEPDC1 might be a novel diagnostic marker and an independent prognostic predictor for HCC patients.

A novel prognostic biomarker SPC24 up-regulated in hepatocellular carcinoma

SPC24 is an important component of the nuclear division cycle 80 (Ndc80) kinetochore complex, which plays an essential role in the coupling of kinetochore to spindle microtubules (MTs) and the accurate segregation of chromosomes during mitosis. However, the functional role of SPC24 in hepatocellular carcinoma (HCC) remains unknown. Here, we detected the expression of SPC24 in HCC and analyzed its association with clinicopathologic features and prognosis of HCC patients. The expression of SPC24 mRNA was investigated in 212 cases of paired HCC and adjacent liver tissues by quantitative real-time PCR (qRT-PCR) and in the tissues of 20 HCC patients by semi-quantitative RT-PCR. Additionally, the expression of SPC24 protein was detected in 69 cases of HCC by immunohistochemistry (IHC) or in 2 cases of HCC tissues by Western-blotting. Furthermore, small interfering RNA (siRNA)-mediated silencing of SPC24 was employed in SMMC7721 and HepG2 human HCC cells to investigate cell proliferation, invasion and apoptosis. Survival curves were plotted using the Kaplan-Meier method, and differences in survival probability were obtained using the log-rank test. Independent predictors associated with disease-free survival (DFS) and overall survival (OS) were analyzed using the Cox proportional-hazards regression model. In this study, we showed that SPC24 was noticeably increased in HCC tissues compared to normal adjacent noncancerous tissues, at both mRNA and protein levels. High expression of SPC24 was significantly correlated with alpha-fetoprotein (AFP) (p = 0.044), median size (p = 0.030), tumor number (p = 0.019), and Barcelona-Clinic Liver Cancer (BCLC) stage (p = 0.015). Kaplan-Meier analysis showed that the DFS and OS of high SPC24 expression group was significantly shorter than that of low SPC24 expression group (p < 0.001; p = 0.001; respectively). The prognostic impact of SPC24 was further confirmed by stratified survival analysis. Importantly, multivariate analysis identified SPC24 upregualtion (p = 0.001), PVTT (p = 0.007), size of tumor > 5 cm (p < 0.001) as independent risk factors of DFS after resection, and SPC24 upregualtion (p < 0.001), PVTT (p = 0.029), size of tumor > 5 cm (p = 0.002), recurrence (p < 0.001) as independent prognostic factors for the OS of HCC patients. Additionally, siRNA-mediated silencing of SPC24 dramatically suppressed cell growth, adhesion, invasion and increased apoptosis in HCC cells. In conclusion, these results showed for the first time that SPC24 expression was significantly up-regulated in HCC, which may act as a novel prognostic biomarker for patients suffering from this deadly disease. Additionally, silence of SPC24 inhibiting HCC cell growth indicated that SPC24 may be a promising molecular target for HCC therapy.

Association between miR-199a rs74723057 and MET rs1621 polymorphisms and the risk of hepatocellular carcinoma

MicroRNAs (miRNAs) can regulate gene expression at post-transcriptional levels, thereby influence cancer risk. The aim of the current study is to investigate association between miR-199a rs74723057 and MET rs1621 and HCC risk in 1032 HCC patients and 1060 cancer-free controls. These two SNPs were genotyped by using the Agena MassARRAY genotyping system. Odds ratio (OR) and 95% confidence interval (95%CI) were calculated to assess the strength of the associations. We found that compared with the wild-type AA genotype of MET rs1621, the variant GG genotype was associated with a decreased risk for HCC (OR = 0.24, 95% CI = 0.06–0.96, P = 0.043). No association between miR-199a rs74723057 and HCC risk was observed. In addition, an interaction effect on HCC risk between the selected two SNPs was found. Among those who carried the CG/GG genotypes of miR-199a rs74723057, those who carried the GG genotype of MET rs1621 had a reduced risk of HCC, when compared with those who carried the AG/AA genotypes of MET rs1621 (OR = 0.15, 95% CI = 0.03~0.73, P for interaction = 0.018). Our results suggest that MET rs1621 polymorphism, alone and combined with miR-199a rs74723057, may influence susceptibility to HCC. Further large-scale association studies and functional studies are needed to validate our findings.

Up-regulation of BRCA1-associated RING Domain 1 Promotes Hepatocellular Carcinoma Progression by Targeting Akt Signaling

The present study was designed to investigate the potential clinical, pathological, prognostic value, role and mechanism of BRCA1-associated RING Domain 1 (BARD1) in Hepatocellular carcinoma (HCC). Quantitative real-time PCR and immunohistochemistry were performed to evaluate the expression of BARD1 mRNA and protein. The expression of BARD1 in the HCC tissue samples was markedly higher than that in the adjacent noncancerous liver tissues. Elevated BARD1 expression was positively correlated with tumor-node-metastasis stage, Barcelona-Clinic Liver Cancer stage, hepatitis B surface antigen, large tumor size, serum alpha-fetoprotein levels, and serum aspartate aminotransferase levels. Univariate and multivariate analyses revealed the BARD1 was an independent predictor for decreased progression-free survival and overall survival in HCC. In vitro experiments demonstrated that knocking down BARD1 significantly inhibited the proliferation, invasion and migration of HCC cells. Moreover, silencing BARD1 inhibit the signaling pathway via decreased the levels of Akt, mTOR, and MMP-9 and inhibited the phosphorylation of Akt (Ser473) and mTOR (Ser2248). Collectively, our findings suggest that BARD1 may be a novel diagnostic and prognostic biomarker of HCC, and up-regulation of BARD1 can contribute to HCC progression by targeting Akt signaling.