Proteomic identification of RhoA as a potential biomarker for proliferation and metastasis in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world, and there is an urgent need to discover novel factors that can act as biomarkers for prognostic assessment and therapeutic targets of HCC. In this study, highly purified plasma membrane proteins from clinical tissue samples were obtained using a strategy combining sucrose density gradient centrifugation and subsequent phase partition. Using a two-dimensional gel electrophoresis and MALDI-Q-TOF MS/MS-based proteomics approach, we identified 13 plasma membrane-associated proteins that were differentially expressed in HCC and normal liver tissues. Of those, RhoA was one of the most significantly upregulated proteins in HCC, and its overexpression was confirmed using Western blotting. Immunohistochemistry suggested a link between RhoA expression and poor differentiation and clinicopathologic stage. Suppression of RhoA expression in HepG2 and Hep3B cells by RNA interference led to significant inhibition of cell growth, induction of apoptosis, and a decrease in migration. Our data suggest that RhoA may serve as a potential biomarker and an attractive therapeutic target for HCC.     

Chaperonin containing TCP1, subunit 8 (CCT8) is upregulated in hepatocellular carcinoma and promotes HCC proliferation

The development of molecular pathogenesis of hepatocellular carcinoma (HCC) is complex and involves alterations in the expression and conformation of assorted oncoproteins and tumor suppressors. Chaperonin containing TCP1 (CCT) is a cytolic molecular chaperone complex that is required for the correct folding of numerous proteins. In this study, we investigated a possible involvement of CCT subunit 8 (CCT8) in HCC development. Immunohistochemical analysis was performed in 102 human HCC samples. High CCT8 expression was detected in clinical HCC samples compared with adjacent noncancerous tissues. The univariate and multivariate survival analyses were also performed to determine their prognostic significance. Western blot confirmed the high expression of CCT8 in HCC compared with adjacent normal tissue. Moreover, the biological significance of the aberrant expression of CCT8 was investigated in HCC cell lines. Expression of CCT8 was correlated directly with the histologic grades and tumor size of HCC and high expression of CCT8 was associated with a poor prognosis. CCT8 depletion by siRNA inhibited cell proliferation and blocked S‐phase entry in HuH7 cells. These results suggested that CCT8 might be an oncogene and participate in HCC cell proliferation. These findings provide a potential therapeutic strategy for the treatment of HCC.     

Prognostic significance of long non-coding RNA PCAT-1 expression in human hepatocellular carcinoma

Background: Long non-coding RNAs (lncRNAs) play widespread roles in gene regulation and cellular processes. However, the functional roles of lncRNAs in hepatocellular carcinoma (HCC) are not yet well elucidated. The aim of the present study was to measure the levels of lncRNA PCAT-1 expression in HCC and evaluate its clinical significance in the development and progression of HCC. Methods: We examined the expression of PCAT-1 in 117 HCC tissues and adjacent non-tumor tissues using quantitative real-time-PCR and analyzed its correlation with the clinical parameters. Results: Our data showed that PCAT-1 expression in HCC tissues was significantly increased compared with adjacent non-tumor tissues (P<0.05). Up-regulated expression of PCAT-1 was significantly associated with TNM stage and metastasis (P<0.05), but not other clinical parameters. Moreover, Kaplan-Meier survival analysis showed that a high expression level of PCAT-1 resulted in a significantly poor overall survival of HCC patients. The multivariate Cox regression analysis demonstrated that PCAT-1 expression level was an independent prognostic factor for the overall survival rate of HCC patients. Conclusions: Our data suggested that the increased expression of PCAT-1 was associated with advanced clinical parameters and poor overall survival of HCC patients, indicating that PCAT-1 up-regulation may serve as a novel biomarker of poor prognosis in HCC patients.     

High expression of long non-coding RNA ANRIL is associated with poor prognosis in hepatocellular carcinoma

Introduction: long non-coding RNA ANRIL (lncRNA ANRIL) has been demonstrated to play a crucial role in cancer progression. However, its effects in hepatocellular carcinoma (HCC) have not been explored. The aim of this study was to investigate the clinical significance of lncRNA NRIL in human HCC. Methods: In this study, we determined for the first time the expression of lncRNA ANRIL in human HCC by quantitative Real-time-PCR analysis. Kaplan-Meier curves and multivariate Cox proportional models were used to study the impact on clinical outcome. Small interfering RNA (siRNA) was used to silence lncRNA ANRIL and to explore the effects of reduced lncRNA ANRIL expression on cell growth and metastasis. Results: lncRNA ANRIL expression in HCC tissues was significantly higher than in the adjacent non-tumor tissues (P < 0.05). The expression of lncRNA ANRIL was remarkably associated with the histologic grade and TNM stage of HCC patients (P < 0.05). In addition, HCC patients with higher lncRNA ANRIL expression had significantly poorer overall survival (P < 0.05). Multivariate analysis suggested that high lncRNA ANRIL expression was an independent predictor of poor prognosis (P < 0.05). Moreover, in vitro assays revealed that the decreased expression of lncRNA ANRIL could suppress the cell proliferation, migration and invasion HCC cells. Conclusions: Our results suggest that lncRNA ANRIL may serve as an efficient clinical biomarker and a therapeutic target for HCC patients.     

组织因子在肝细胞癌的发生及侵袭转移中的研究

目的：检测组织因子（TF）在肝细胞癌患者中的表达并探讨其意义。方法:采用酶联免疫法检测肝细胞癌患者50例及对照组30例的血浆TF水平，采用RT-PCR法检测其中27例肝癌、癌旁、正常肝组织TF mRNA表达。结果:①肝细胞癌患者血浆TF显著高于对照组（P<0.05）。血浆TF在分化高低、肿瘤大小及是否合并肝硬化组间表达有显著差异（P<0.05），在有淋巴转移、肝外脏器转移及门脉癌栓组高于无转移及癌栓组(P<0.05)，而在不同癌灶数目及包膜情况组间无显著差异（P＞0.05）。②TF mRNA在肝癌组织中阳性率及相对表达强度分别为62.96%（17/27）、0.567±0.268，均显著高于癌旁组织及正常组织。阳性患者相对表达强度在肿瘤大小及部分侵袭转移指标中表达有显著差异（P<0.05） 。结论：TF在肝癌患者血浆及组织中升高且与部分侵袭转移指标相关，提示其TF可能促进了肝癌的发生与侵袭转移。     

MicroRNA-194 acts as a prognostic marker and inhibits proliferation in hepatocellular carcinoma by targeting MAP4K4

MicroRNAs (miRNAs) play a crucial role in cancer development and progression of hepatocellular carcinoma (HCC). In this study, we aimed to analyze the role of microRNA-194 (miR-194) in HCC. We found that miR-194 expression was significantly reduced in HCC and its expression was an independent poor prognostic factor for HCC patient overall and disease-free survival rate. A significant correlation was observed between miR-194 reduction and unfavourable variables including tumor size (P = 0.0315), histologic grade (P = 0.0038), TNM stage (P = 0.0083), intrahepatic metastasis (P = 0.0184). Overexpression of miR-194 in HCC cell lines HepG2 and Hep3B inhibited cell proliferation by blocking G1-S transition and inducing apoptosis. Mitogen-activated protein kinase 4 (MAP4K4), a potential target gene of miR-194, was inversely correlated with miR-194 expression in HCC tissues and cell lines. Further studies demonstrated that miR-194 regulated the progression of HCC through directly inhibiting the expression of MAP4K4 and the restoration of MAP4K4 expression reversed the inhibitory effects of miR-194 on HCC cell proliferation. Together, our findings indicate that miR-194 may serve as a valuable prognostic marker and promising interventional therapeutic target for HCC.     

Co-expression of XIAP and cyclin D1 complex correlates with a poor prognosis in patients with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. Despite improved diagnosis and treatment, the prognosis for HCC patients remains poor. The goal of this study was to identify key regulatory proteins and signaling pathways important for cell apoptosis and proliferation as biomarkers for prognostication and targeted therapy. Protein Pathway Array was applied to screen 38 signaling proteins and phosphoproteins in 12 paired HCC tumors and surrounding benign tissues and found that 20 of them, including XIAP, CDK4, CDK6, and Cyclin D1, were overexpressed in HCC tissues. Immunostaining results of XIAP, CDK4, and Cyclin D1 in an additional 59 HCC tissues showed that the expression of XIAP correlated with the expression of CDK4/Cyclin D1, and that the increased expression of these proteins correlated with poor overall survival in these patients. Further studies using the HCC Huh7 cell line transfected with XIAP siRNA or expression vector demonstrated that XIAP regulated the expression of CDK4, CDK6, and Cyclin D1 via NF-êB and PTEN pathways. Finally, inhibition of XIAP using embelin, a XIAP-specific small molecule, leads to an increased apoptosis and decreased cell proliferation via arrest at G1 phase. Taken together, XIAP is a central modulator regulating cell apoptosis and cell cycle progression. Therefore, XIAP together with cell cycle regulatory proteins can be used as prognostic markers and therapeutic targets.     

Profilin 1, negatively regulated by microRNA-19a-3p,serves as a tumor suppressor in human hepatocellular carcinoma

Profilin 1 (PFN1) is a critical actin-regulatory protein; however, its functional role in hepatocellular carcinoma (HCC) progression remains to be further elucidated. In the present study, we observed that the expression levels of PFN1 were significantly decreased in HCC tissues and cell lines. Low PFN1 expression was significantly correlated with aggressive clinicopathological characteristics and poor prognosis of HCC patients. Further in vitro experiments demonstrated that overexpression of PFN1 remarkably inhibited the proliferation, migration, invasion and EMT of HCC cells. Moreover, we also found that PFN1 was a direct target gene of miR-19a-3p, and in HCC tissues, and there was a significantly inverse correlation between PFN1 mRNA and miR-19a-3p expression. Collectively, our results showed that PFN1 functions as a tumor suppressor in HCC, and might serve as a diagnostic and therapeutic target for HCC patients.     

High expression of N-acetyltransferase 10: a novel independent prognostic marker of worse outcome in patients with hepatocellular carcinoma

N-acetyltransferase 10 (NAT10) is a nucleolar protein involved in histone acetylation, telomerase activity regulation, DNA damage response and cytokinesis. The expression of NAT10 was found to be enhanced in several types of tumors, suggesting its correlation with tumor development. However, the specific role of NAT10 in hepatocellular carcinoma (HCC) is still unclear. The aim of this study was to investigate the expression of NAT10 in HCC patients and to assess the relationship of NAT10 expression with clinicopathological characteristics and tumor prognosis. We selected 17 pairs of HCC samples and adjacent non-neoplastic tissue for mRNA expression analysis. We also performed immunohistochemistry in 186 HCC samples to evaluate the NAT10 protein expression. Cox regression and Kaplan-Meier analysis was used to study the diagnostic and prognostic value of NAT10. The results showed that NAT10 expression was mainly localized in the nuclei/nucleoli and was significantly higher in HCC tissues than peritumoral tissues (P < 0.01). High NAT10 expression was positively correlated with histological differentiation (P < 0.01) and TNM classification (P < 0.01). Cox regression univariate and multivariable analysis revealed that expression of NAT10 in HCC was an independent prognostic factor for patient survival time. Our data suggested that NAT10 might be a promising prognostic marker and potential therapeutic target in HCC.     

Clinicopathologic significance of the expression of Snail in hepatocellular carcinoma

Background/Aims

E-cadherin is involved in intercellular binding and cellular polarity formation. Snail is a key regulator of the epithelial-mesenchymal transition and is closely associated with tumor invasiveness due to its ability to suppress E-cadherin expression. We investigated the expressions of E-cadherin and Snail in hepatocellular carcinoma (HCC) tissue to determine the clinical significance of these proteins in HCC.

Methods

Immunohistochemistry was used to examine the expressions of E-cadherin and Snail in resected tissues from 59 patients diagnosed with HCC. We also evaluated the relationship between the expressions of these two molecules in HCC tissue and clinicopathologic factors in the patients.

Results

Immunohistochemistry showed that Snail was stained in 20.3% of the HCC tissues and 3.4% of noncancerous tissues. Snail was not stained in the area of E-cadherin expression. The expression of Snail in the HCC tissue was associated with poorly differentiated HCC (P=0.028). The expression of Snail without E-cadherin staining in HCC tissue was significantly associated with postoperative HCC recurrence (P=0.013).

Conclusions

The expression of Snail in HCC tissue was associated with decreased expression of E-cadherin and poorly differentiated HCC. The expression of Snail without E-cadherin staining in HCC was associated with postoperative recurrence.     

BICD1 functions as a prognostic biomarker and promotes hepatocellular carcinoma progression

Hepatocellular carcinoma (HCC) is the most predominant type of primary liver cancer and has a high degree of malignancy as well as mortality rate. Many drivers are involved in the development and progression of HCC. A recent study has reported that high BICD cargo adaptor 1 (BICD1) expression indicates poor prognosis of glioblastoma. But, the expression and biological function of BICD1 in HCC remain unclear. In current study, we found that the expression of BICD1 was markedly up-regulated in HCC tissues compared to adjacent nontumor tissues. GEPIA dataset and GSE datasets were consistently indicated the elevated expression of BICD1 in HCC. Furthermore, BICD1 was highly expressed in HCC cell lines including Hep3B, Huh7, MHCC97H and HCCLM3 as compared with that in LO2 cells. High BICD1 expression was positively correlated with malignant clinical features, such as tumor size ≥ 5 cm, venous infiltration and advanced tumor stages. HCC patients highly expressing BICD1 showed a significant shorter overall survival compared to BICD1 low-expression cases. Moreover, TCGA-LIHC data further demonstrated that the up-regulated BICD1 expression predicted poor prognosis of HCC patients. Next, we revealed that BICD1 knockdown prominently suppressed the proliferation, migration and invasion of HCCLM3 cells. Conversely, ectopic expression of BICD1 remarkably facilitated these malignant behaviors of Hep3B cells. Interestingly, BICD1 knockdown abolished hypoxia-induced HCC cell proliferation, migration and invasion. In conclusion, we provide the first evidence to support that BICD1 functions as a predictor for the prognosis of HCC and may serve as a promising therapeutic target for further HCC treatment.     

Cytohesin-3 is upregulated in hepatocellular carcinoma and contributes to tumor growth and vascular invasion

Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality, and is characterized by high potential for metastasis and recurrence. The outcome of it is still poor due to lacking of targeted therapeutic strategies. There is an urgent need to find new therapeutic targets for interventions against HCC metastasis and recurrence. In the present study, we found cytohesin-3, a member of the cytohesin family, was upregulated in HCC tissues, and its expression was negatively correlated with the overall survival and relapse-free survival of HCC patients. Further clinicopathological correlation analysis revealed that cytohesin-3 expression was related with tumor size and vascular invasion. And in vitro studies revealed that knock-down of cytohesin-3 suppressed HCC cells proliferation and migration. These results suggest that cytohesin-3 may act as a novel prognostic factor of HCC, and it might also be useful to exploit targeted therapeutic drugs against HCC growth and metastasis.     

Identification of Gem as a new candidate prognostic marker in hepatocellular carcinoma

GTP binding protein overexpressed in skeletal muscle (Gem) is a Ras-related protein whose expression is induced in several cell types upon activation by extracellular stimuli. To investigate the potential roles of Gem in hepatocellular carcinoma (HCC), expression of Gem was examined in human HCC samples. Western blot analysis showed that compared with primary human hepatocytes and adjacent noncancerous tissue, significant down-regulation of Gem was found in HCC cells and tumor tissues. In addition, immunohistochemical analysis of Gem expression was investigated in 108 specimens of HCC tissues. Clinicopathological data were collected to analyze the association with Gem expression. Expression of Gem was significantly negatively correlated with histological grade (P = 0.001), tumor size (P = 0.020), and vascular invasion (P = 0.005), and Gem was also negatively correlated with proliferation marker Ki-67 (P < 0.01). More importantly, the Kaplan–Meier survival curves analysis revealed that low expression of Gem was associated with poor prognosis in HCC patients. Univariate analysis showed that Gem expression was associated with poor prognosis (P = 0.006). Multivariate analysis indicated that Gem expression was an independent prognostic marker for HCC (P = 0.007). Finally, serum starvation and release experiments showed that Gem expression was negatively related with cell proliferation. In the conclusion, our results suggested that down regulation of Gem expression was involved in the pathogenesis of hepatocellular carcinoma, and it might be a favorable independent prognostic parameter for HCC.     

Overexpression of TONSL might be an independent unfavorable prognostic indicator in hepatocellular carcinoma

Background

TONSL has been suggested to function as an oncogene in lung, esophageal and cervical cancer. This study was aimed to identify the expression of TONSL and its role in hepatocellular carcinoma (HCC).

Comparative study of Hsp27, GSK3β, Wnt1 and PRDX3 in Hirschsprung's disease

Hirschsprung's disease (HSCR) is a developmental disorder of the enteric nervous system characterized by aganglionosis in distal gut. In this study, we used two‐dimensional gel electrophoresis (2‐DE) technology coupled with matrix assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) analysis to identify differentially expressed proteins in the aganglionic (stenotic) and ganglionic (normal) colon segment tissues from patients with HSCR. We identified 15 proteins with different expression levels between the stenotic and the normal colon segment tissues from patients with HSCR. Nine proteins were upregulated and six proteins downregulated in the stenotic colon segment tissues compared to the normal colon segment tissues. Based on the biological functions, we selected the Hsp27 upregulated proteins and the PRDX3 downregulated proteins to confirm their expression in 20 patients. The protein and mRNA expressions of Hsp27 were statistically higher in the stenotic colon segment tissues than in the normal colon segment tissues, whereas the protein and mRNA expressions of PRDX3 were statistically lower in the stenotic colon segment tissues than in the normal colon segment tissues. These findings of changes in mRNA and protein in tissues from patients with HSCR provide information which may be helpful in understanding the pathomechanism that is implicated in the disease.     

The expression and clinical significance of TPM4 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is known as the fifth most common cancer in the world for its poor prognosis. New diagnostic markers and treatments are urgent to discover. To evaluate the protein expression of Tropomyosin4 (TPM4) and investigate its prognostic value in HCC, we collected 110 patients with different degrees of HCC and 10 patients with normal hepatic tissues and performed immunohistochemistry. Western bot was used to evaluate the expression of TPM4 in three HCC cell lines (HepG2, Huh7, SMMC-7721) and normal liver cell line LO2, as well as 7 HCC tissues and 7 normal hepatic tissues. The results of TPM4 staining revealed that TPM4 expression in HCC was higher than that in normal hepatic tissues, which was positive in 51.8% (n=57) and negative in 48.2% (n=53) while in normal hepatic tissues positive staining was in 10% (n=1) and negative staining was in 90% (n=9) (P=0.011). And the expression of TPM4 was related to pT status, grade and stage (P<0.001, P=0.015 and P<0.001, respectively). Western blot results indicated that TPM4 was high expressed in HCC cell line and HCC tissues. In conclusion, we believe that TPM4 can be applied as a diagnostic and prognostic marker to assist the management of HCC.     

AROS Is a Significant Biomarker for Tumor Aggressiveness in Non-cirrhotic Hepatocellular Carcinoma

Despite a low risk of liver failure and preserved liver function, non-cirrhotic hepatocellular carcinoma (HCC) has a poor prognosis. In the current study, we evaluated an active regulator of SIRT1 (AROS) as a prognostic biomarker in non-cirrhotic HCC. mRNA levels of AROS were measured in tumor and non-tumor tissues obtained from 283 non-cirrhotic HCC patients. AROS expression was exclusively up-regulated in recurrent tissues from the non-cirrhotic HCC patients (P = 0.015) and also in tumor tissues irrespective of tumor stage (P < 0.001) or BCLC stage (P < 0.001). High mRNA levels of AROS were statistically significantly associated with tumor stage (P < 0.001), BCLC stage (P = 0.007), alpha fetoprotein (AFP) level (P = 0.013), microvascular invasion (P = 0.001), tumor size (P = 0.036), and portal vein invasion (P = 0.005). Kaplan-Meir curve analysis demonstrated that HCC patients with higher AROS levels had shorter disease-free survival (DFS) in both the short-term (P < 0.001) and long-term (P = 0.005) compared to those with low AROS. Cox regression analysis demonstrated that AROS is a significant predictor for DFS along with large tumor size, tumor multiplicity, vascular invasion, and poor tumor differentiation, which are the known prognostic factors. In conclusion, AROS is a significant biomarker for tumor aggressiveness in non-cirrhotic hepatocellular carcinoma.     

Differential proteomic analysis of children infected with respiratory syncytial virus

Respiratory syncytial virus (RSV) infection is the main cause of lower respiratory tract infection in children. However, there is no effective treatment for RSV infection. Here, we aimed to identify potential biomarkers to aid in the treatment of RSV infection. Children in the acute and convalescence phases of RSV infection were recruited and proteomic analysis was performed to identify differentially expressed proteins (DEPs). Subsequently, promising candidate proteins were determined by functional enrichment and protein-protein interaction network analysis, and underwent further validation by western blot both in clinical and mouse model samples. Among the 79 DEPs identified in RSV patient samples, 4 proteins (BPGM, TPI1, PRDX2, and CFL1) were confirmed to be significantly upregulated during RSV infection. Functional analysis showed that BPGM and TPI1 were mainly involved in glycolysis, indicating an association between RSV infection and the glycolysis metabolic pathway. Our findings provide insights into the proteomic profile during RSV infection and indicated that BPGM, TPI1, PRDX2, and CFL1 may be potential therapeutic biomarkers or targets for the treatment of RSV infection.     

Up-regulation of AKAP13 and MAGT1 on cytoplasmic membrane in progressive hepatocellular carcinoma: a novel target for prognosis

Hepatocellular carcinoma (HCC) is one of the most common cancers and is associated with high mortality worldwide. The current gold standards for HCC surveillance are detection of serum α-fetoprotein (AFP) and ultrasonography; however, non-specificity of AFP and ultrasonography has frequently been reported. Therefore, alternative tools, especially novel specific tumor markers, are required. In this study, cytoplasmic membrane proteins were isolated from phorbol 12-myristate 13-acetate (PMA)-induced invasive HepG2 cells and identified using nano-scale liquid chromatographic tandem mass spectrometry (NLC-MS/MS) with comparison to non-treated controls. The results showed that two proteins, magnesium transporter protein 1 (MAGT1) and A-kinase anchor protein 13 (AKAP13), were highly expressed in PMA-treated HepG2 cells. This up-regulation was confirmed by real-time RT-PCR, western blot analysis, and immunofluorescent staining studies. Furthermore, evaluation of MAGT1 and AKAP13 expression in clinical HCC tissues by immunohistochemistry suggested that both proteins were strongly expressed in tumor tissues with significantly higher average immunoreactive scores of Remmele and Stegner (IRS) than in non-tumor tissues (P ≤ 0.005). In conclusion, the expression levels of MAGT1 and AKAP13 in HCC may be potential biomarkers for the diagnosis and prognosis of this cancer.