Secretory Clusterin as a Novel Molecular-targeted Therapy for Inhibiting Hepatocellular Carcinoma Growth.

BACKGROUND: Although secretory clusterin (sCLU) plays a crucial role in Hepatocellular Carcinoma (HCC) cells proliferation, Multiple Drug Resistance (MDR), metastasis and so on, its targeted effects and exact mechanism are still unknown. This review summarizes some new progress in sCLU as a molecular-targeted therapy in the treatment of HCC. METHODS: A systematic review of the published English-language literature about sCLU and HCC has been performed using the PubMed and bibliographic databases. Some valuable studies on sCLU in HCC progression were searched for relevant articles with the keywords: HCC, diagnosis, MDR, as molecular-targeted in treatment, and so on. RESULTS: The incidence of the positive rate of sCLU was significantly higher in HCC tissues as compared to the surrounding tissues at mRNA or protein level, gradually increasing with tumor-nodemetastasis staging (P<0.05). Also, the abnormal level of sCLU was related to poor differentiation degree, and considered as a useful marker for HCC diagnosis or independent prognosis for patients. Hepatic sCLU could be silenced at mRNA level by specific sCLU-shRNA or by OGX-011 to inhibit cancer cell proliferation with an increase in apoptosis, cell cycle arrest, reversal MDR, alteration of cell migration or invasion behaviors, and a decrease in GSK-3ß or AKT phosphorylation in vitro, as well as significant suppression of the xenograft growth by down-regulating ß-catenin, p-GSK3ß, and cyclinD1 expression in vivo. CONCLUSION: Abnormal hepatic sCLU expression should not only be a new diagnostic biomarker but also a novel promising target for inhibiting HCC growth.

Oncogenic Wnt3a: A Candidate Specific Marker and Novel Molecular Target for Hepatocellular Carcinoma.

Background and aim: It is of the utmost importance for the specific diagnosis and effective therapy of hepatocellular carcinoma (HCC). Abnormality of oncogenic Wingless-type MMTV integration site family member 3a (Wnt3a) has been associated with progression of HCC. In this study, we aimed to evaluate Wnt3a as a novel biomarker and target for HCC. Methods: Circulating Wnt3a levels were quantitatively detected in a cohort of chronic liver diseases by an enzyme-linked immune-absorbent assay (ELISA). Hepatic Wnt3a expression in HCC and para-cancerous tissues was analyzed by immunohistochemistry. Prognostic value of Wnt3a for HCC was discovered in the cohort from the Cancer Genome Atlas (TCGA). Dynamic alterations of Wnt3a levels were detected in the hepatocarcinogenesis model induced by 2-acetylaminofluorene. Effects of Wnt3a on biological behaviors were evaluated in vitro and in vivo based on Crispr/Cas9. Results: Up-regulated Wnt3a levels were observed in serum of HCC patients with high specificity and sensitivity for HCC diagnosis. Combination of Wnt3a and AFP could improve sensitivity to 93.9% in serological detection. In addition, Wnt3a expression in HCC tissues was significantly higher than that in para-cancerous tissues. The cohort of TCGA demonstrated that high Wnt3a expression led to a poor survival of HCC patients, especially in cases at advanced stages. Furthermore, the hepatocarcinogenesis model showed that Wnt3a dynamically increased in the development of HCC. Functionally, silencing Wnt3a by Crispr/Cas9 suppressed the proliferation, colony formation, induced cell cycle arrest of HCC cells by de-activating Wnt/ß-catenin pathway in vitro, and inhibited xenograft tumor growth in vivo. Conclusions: Oncogenic Wnt3a could be considered as a candidate biomarker and novel target for HCC.

Alteration of oncogenic IGF-II gene methylation status associates with hepatocyte malignant transformation.

BACKGROUND: Oncogenic insulin-like growth factor-II (IGF-II) is overexpressed in hepatocellular carcinoma (HCC). The present study aimed to analyze the dynamic alteration of IGF-II CpG site methylation status and its molecular mechanism in HCC progression. METHODS: IGF-II alterations were observed in rat hepatocarcinogenesis models induced by 2-acetylaminofluorene. Liver IGF-II expression was compared by immunohistochemistry or tissue IGF-II specific concentration (nmol/mg protein). Status of human IGF-II promoter 3 (P3) or rat IGF-II P2 CpG site methylation was amplified by methylation-specific polymerase chain reaction (MSP). Serum IGF-II levels were quantitatively detected by an enzyme-linked immunosorbent assay. RESULTS: The levels of hepatic IGF-II expression were significantly elevated in the HCC group (P < 0.001). The unmethylation rate of IGF-II P3 CpG sites was 100% in the HCC-, 52.5% in the paracancerous-, and none (0%) in the distal noncancerous-tissues. Abnormal IGF-II expression was related to differentiation degree, tumor invasion, and positive HBV-DNA (all P < 0.001), with a negative correlation between P3 methylation degree and IGF-II expression. There was a positive correlation between liver IGF-II specific concentration and circulating IGF-II level (r = 0.97, P < 0.001). Significantly negative correlation was found between IGF-II P2 CpG site methylation and circulating IGF-II (rs = -0.89, P < 0.001) or liver IGF-II level (rs = -0.84, P < 0.001). CONCLUSIONS: The increase of serum IGF-II and the alteration of oncogenic gene IGF-II methylation may be biomarkers for HCC diagnosis and DNA methylation may be the therapeutic target of HCC.

Insulin-like Growth Factor I Receptor: A Novel Target for Hepatocellular Carcinoma Gene Therapy.

Human insulin-like growth factor (IGF) axis affects the molecular pathogenesis of hepatocellular carcinoma (HCC), especially in the abnormality of hepatic IGF-I receptor (IGF-IR) or IGF-II expression as a key molecule in hepatocarcinogenesis. However, the over-expression of hepatic IGFIR is associated with HCC progression with largely unknown mechanisms. The IGF-IR as one key molecule of the IGF signal pathway plays an important role in the hepatocyte malignant transformation. Attaching importance to IGF-IR might improve the prognostic or the therapeutic technique of HCC. This article reviews IGF-IR alteration during HCC development, and the effects of silencing IGF-IR gene by specific short hairpin RNA on the inhibition of cell proliferation in vitro or HCC xenograft growth in vivo to elucidate it as a novel molecular-targeted therapy for HCC.

High mobility group box 3 as an emerging biomarker in diagnosis and prognosis of hepatocellular carcinoma.

PURPOSE: High mobility group box 3 (HMGB3) is associated with hepatocytes malignant transformation by our previous work. We continued to investigate the diagnostic and prognostic values of HMGB3 for hepatocellular carcinoma (HCC). PATIENTS AND METHODS: Circulating HMGB3 levels were quantitatively detected in a cohort of 225 patients with chronic liver diseases by ELISA and compared with alpha-fetoprotein by the receiver operating characteristic curve. HMGB3 expression in tissues of 170 HCC was detected by tissue microarray and immunohistochemistry. Relationship between HMGB3 level and HCC prognosis was evaluated by the Kaplan-Meier curves and Cox regression model. RESULTS: The incidence of serum HMGB3 >2.0 ng/mL was 75.6% in HCC (96/127), 20.8% in liver cirrhosis (10/48), 16.0% in chronic hepatitis (8/50), and none in healthy controls (0/49). Significant difference (P<0.001) of circulating HMGB3 level was found between HCC and benign liver diseases. Total diagnostic sensitivity of serum HMGB3 plus alpha-fetoprotein was up to 89.0% for HCC. Higher HMGB3 expression was confirmed to be 73.5% in HCC tissues (125/170) >30.6% in their paracancerous tissues (52/170). HMGB3 expression was closely related to tumor size, TNM stage, poor survival, and high recurrence, suggesting an independent prognosis factor for HCC. CONCLUSION: HMGB3 with aberrant expression could be a novel diagnostic and prognostic marker for HCC.

Role of secretory clusterin in hepatocarcinogenesis.

Secretory clusterin (sCLU) is a small stress-induced cytoprotective chaperone protein. Its biological functions are similar to those of a heat-shock protein. The sCLU plays a crucial role in cell proliferation, multiple drug resistance, metastasis, and tumor progression. Abnormal sCLU expression in tumor tissues or sera of patients with primary hepatic cancer has been considered a useful biomarker for diagnosis and surveillance. However, the exact relationship between sCLU overexpression and malignant transformation of hepatocytes is still unknown. The present review examines some novel advances of the knowledge about the oncogenic role of sCLU in hepatocarcinogenesis.

Abnormal expression of HMGB-3 is significantly associated with malignant transformation of hepatocytes.

AIM: To explore the relationship between dynamic expression of high mobility group box-3 (HMGB3) and malignant transformation of hepatocytes. METHODS: Expression of HMGB family proteins were observed in rat hepatocarcinogenesis models induced with 2-acetylaminofluorene. Alterations of HMGB3 were analyzed at the mRNA level by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and at the protein level by immunohistochemistry or Western blotting. HMGB3 in human liver cancer tissues were evaluated using bioinformatics databases from GEO, TCGA, and Oncomine. A specific HMGB3-shRNA was used to knock down HMGB3 expression in order to investigate its effects on proliferation and cell cycle in vitro and in vivo. RESULTS: Elevated HMGB3 levels were first reported in hepatocarcinogenesis, with increasing expression from normal liver to cancer. Bioinformatic databases showed that HMGB3 expression in hepatocellular carcinoma tissues was significantly higher than that in normal liver tissues. Higher HMGB3 expression was discovered in liver cancer cells compared with LO2 cells in vitro. According to gene set enrichment analysis, HMGB3 mRNA levels were correlated with cell cycle and DNA replication pathways. Knocking down HMGB3 by specific shRNA significantly inhibited proliferation of HepG2 cells by cell cycle arrest and downregulating DNA replication related genes (cyclin B1, FEN1, and PCNA) at the mRNA and protein level. Furthermore, silencing HMGB3 significantly inhibited xenograft tumor growth (measured by Ki67) in vivo. CONCLUSION: HMGB3 is involved in malignant transformation of hepatocytes and could be a useful biomarker for diagnosis and a potential target for therapy of liver cancer.

Expression of hepatic Wnt5a and its clinicopathological features in patients with hepatocellular carcinoma.

BACKGROUD: Wingless-type MMTV integration site family member 5a (Wnt5a) is involved in carcinogenesis. However, little data are available in Wnt5a signaling with hepatocellular carcinoma (HCC). In the present study, we investigated the expression of hepatic Wnt5a in HCC and the role of Wnt5a in HCC progression and outcome. METHODS: Wnt5a expression and cellular distribution in HCCs and their matched paracancerous tissues from 87 patients were analyzed with tissue microarray and immunohistochemistry and compared with hepatic Wnt3a signaling. Wnt5a expression was categorized into low or high based on immunohistochemistry. Overall survival rate of HCC patients was estimated in correlation with the hepatic Wnt5a level using Kaplan-Meier method; the survival difference between patients with low and those with high Wnt5a was compared with log-rank test; and prognostic analysis was carried out with Cox regression. RESULTS: Total incidence of Wnt5a expression in the HCC tissues was 70.1%, which was significantly lower (χ2 = 13.585, P < 0.001) than that in their paracancerous tissues (88.5%). Significant difference of Wnt5a intensity was found between HCC and their paracancerous tissues (Z = 8.463, P < 0.001). Wnt5a intensity was inversely correlated with Wnt3a signaling (r = -0.402, P < 0.001) in HCC tissues. A decrease of Wnt5a expression in relation to the clinical staging from stage I to IV and low or no staining at advanced HCC were observed. Wnt5a level was related to periportal embolus (χ2 = 11.069, P < 0.001), TNM staging (χ2 = 8.852, P < 0.05), 5-year survival (χ2 = 4.961, P < 0.05), and confirmed as an independent prognosis factor of HCC patients (hazard ratio: 1.957; 95% confidence interval: 1.109-3.456; P < 0.05). CONCLUSIONS: The decrease of hepatic Wnt5a signaling is associated with HCC progression and poor prognosis.

Ang-2 promotes lung cancer metastasis by increasing epithelial-mesenchymal transition.

Lung cancer is the most common malignant tumor with increasing angiopoietin-2 (Ang-2) and a high rate of metastasis. However, the mechanism of Ang-2 enhancing tumor proliferation and facilitating metastasis remains to be clarified. In this study, Ang-2 expression and its gene transcription on effects of biological behaviors and epithelial-mesenchymal transition (EMT) were investigated in lung cancers. Total incidence of Ang-2 expression in the cancerous tissues was up to 91.8 % (112 of 122) with significantly higher (χ2=103.753, P2=7.883, P=0.005), differentiation degree (χ2=4.554, P=0.033), tumor node metastasis (TNM) staging (χ2=5.039, P=0.025), and 5-year survival rate (χ2 =11.220, P2=18.881, P2=0.81, P=0.776) or III & IV (χ2=1.845, P=0.174). Over-expression of Ang-2 or Ang-2 mRNA in lung A549 and NCI-H1975 cells were identified among different cell lines. When silencing Ang-2 in A549 cells with specific shRNA-1 transfection, the cell proliferation was significantly inhibited in a time-dependent manner, with up-regulating E-cadherin, down-regulating Vimentin, Twist, and Snail expression, and decreasing invasion and metastasis of cancer cell abilities, suggesting that Ang-2 promote tumor metastasis through increasing EMT, and it could be a potential target for lung cancer therapy.

Oncogenic secretory clusterin in hepatocellular carcinoma: Expression at early staging and emerging molecular target.

Secretory clusterin (sCLU) is associated with hepatocellular carcinoma (HCC) progression by contributing to angiogenesis, chemoresistance, cell survival, and metastasis. However, the sCLU expression at early stage of HCC progression remains to be clarified. In this study, the alteration of sCLU oncogenicity was firstly evaluated in HCC- and their para-cancerous- tissues. The incidence of sCLU expression in HCC was significantly higher than that in their non-tumorous tissues at message RNA (mRNA) or protein level, gradually increasing with tumor-node-metastasis (TNM) staging. Abnormal sCLU expression was associated with the poor differentiation, TNM stage, and considered as an independent prognostic factor for HCC patients. Furthermore, silencing sCLU gene transcription inhibited the colony formation and proliferation of HCC cells, with decreasing phosphorylation level of AKT and GSK-3ß in HCCLM3 cells in vitro and significantly suppressed the HCC xenograft growth in vivo, suggesting that sCLU with oncogenicity should be not only an early indicator but also novel potential molecular-targeted therapy for HCC.

Advances in the study of oncofetal antigen glypican-3 expression in HBV-related hepatocellular carcinoma.

Early specific diagnosis and effective treatment of hepatocellular carcinoma (HCC) are crucial. Expression of membrane-associated heparan sulfate proteoglycan glypican-3 (GPC-3) was recently found to increase as part of the malignant transformation of hepatocytes, and this increase is especially marked in patients with hepatitis B virus (HBV) infection, periportal cancerous embolus, or extra-hepatic metastasis. According to data from basic and clinical studies, the oncofetal antigen GPC-3 is a highly specific diagnostic biomarker of HCC and an indicator of its prognosis, and GPC-3 is also a promising target molecule for HCC gene therapy since it may play a crucial role in cell proliferation, metastasis, and invasion and it may mediate oncogenesis and oncogenic signaling pathways. This review summarizes recent advances in the use of oncofetal antigen GPC-3 to diagnose HBV-related HCC, estimate its prognosis, and its targeted therapy.

Expression of oncofetal antigen glypican-3 associates significantly with poor prognosis in HBV-related hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor prognosis. However, its prognostic evaluation is still an urgent problem. The objectives of this present study were to investigate oncofetal antigen glypican-3 (GPC-3) expression in HCC and their match para-cancerous tissues by the array technology with immunohistochemistry and estimate its value as a novel prognostic marker for HCC. The incidence of GPC-3 expression was 95.7 % in the cancerous tissues with significantly higher (χ2 = 33.824, P < 0.001) than that in the para-cancerous tissues (52.2 %). Abnormal expression of GPC-3 in HCC tissues was markedly related to poor or moderate differentiation (P < 0.001), hepatitis B virus (HBV) infection (P = 0.004), periportal cancer embolus (P = 0.043), and tumor-node- metastasis staging (P = 0.038). According to the univariate and multivariate analysis, the overall survival of HCC patients with high GPC-3 level was significantly worse than those with low or without GPC-3 expression (P < 0.001), suggesting that abnormal GPC-3 expression should be an independent prognostic factor for HBV-related HCC patient's survival.

[Abnormal expression of circulating nuclear factor-κB in hepatocellular carcinoma and reversal of multidrug resistance through intervening its gene transcription].

OBJECTIVE: To investigate the expressions of nuclear factor-κB (NF-κB) and P-glycoprotein (P-gp) in patients with liver diseases and the effects and mechanism of intervening NF-κB activation on multiple drug resistance (MDR) reversal. METHODS: The levels of circulating NF-κB and P-gp expression were quantitatively detected in 294 patients with liver diseases by using enzyme-linked immunosorbent assay. NF-κB gene transcription in human HepG2 cells was intervened by specific siRNA with/without doxorubicin treatment. Levels of protein or gene expression were analyzed by Western blotting or fluorescence quantitative polymerase chain reaction (PCR). RESULTS: The dynamic increase of circulating NF-κB and P-gp expressions were observed during patients from chronic hepatitis to cirrhosis to hepatocellular carcinoma (HCC) development. The levels of serum NF-κB and P-gp expression in HCC were significantly higher (P<0.001) than those in cirrhosis, chronic hepatitis, and normal controls. The expression levels of serum NF-κB and P-gp were significantly associated with HBV infection (P<0.05), elevated after interventional chemotherapy with higher frequency or prolonged therapy, and significantly decreased in the post-operative HCC (P<0.001). Both expressions in HepG2 cells were significantly higher after adding doxorubicin in the cell cultures. After the cells were transfected with siRNA, the NF-κB expression was down-regulated at mRNA or protein level with higher sensitivity to doxorubicin. CONCLUSION: The over-expressions of NF-κB and P-gp are closely associated with MDR formation; intervention of NF-κB activation can inhibit P-gp expression, and enhance the sensitivity to anti-cancer drug and reverse MDR of HCC.

[Inhibitory effects of intervention of the TNFa/NF-kappaB signaling pathway activation on hepatoma cell proliferation].

OBJECTIVE: To investigate the inhibitory effects of intervention of the tumor necrosis factor-alpha (TNFa)/nuclear factor-kappa B (NF-kappaB) signaling pathway activation on hepatoma cell proliferation and to explore its mechanism. METHODS: A rodent hepatoma model was established by feeding N-2-fluorenylacetamide (2-N-FAA) to male Sprague-Dawley rats. Human subjects with various liver diseases were enrolled in the study, and serum and peripheral blood nuclear cells were collected for analysis. HepG2 cells were cultured in vitro and treated with anti-TNFa (monoclonal antibody, mAb) to down-regulate its expression or transfected with siRNA targeting the p65 subunit of NF-kappaB to inhibit its activation. The liver cell line L02 was used as a control. Changes in protein and gene expression levels of NF-kappaB and TNFa were analyzed by Western blotting or enzyme-linked immunosorbent assay and real-time PCR, respectively. Changes in the cell cycle or apoptosis were evaluated by flow cytometry or Annexin-V/PI double-labeling assay, respectively. RESULTS: TNFa and NF-kappaB expression showed increasing trends during the malignant transformation of rat hepatocytes, and the differential expression patterns showed association with histopathological alterations in the hepatocytes. Following treatment with the TNFa mAb, the HepG2 cells showed a higher percentage of apoptotic cells than the untreated control cells (21.45% +/- 4.07% vs. 5.63% +/- 0.93%, q =10.07, P less than 0.01).There was a significant difference in the rate of cells in the G0/G1 phase in the p65-siRNA transfected cells (66.23% +/- 1.29% vs. untreated control cells: 59.00% +/- 1.02%, q =10.98, P less than 0.01). The decreased expression of TNFa and NF-kappaB in cell culture supernatants was positively correlated with the dose of treatment (r =0.89, P less than 0.01), with the most robust decreases being achieved with the highest concentrations ( P less than 0.01). NF-kappaB expression was significantly higher in the HepG2 cells than in the L02 cells, and transfection of p65-siRNA reduced the mRNA (93%) and protein (62%) levels and increased the cell apoptosis index (to 85%). CONCLUSION: Proliferation of hepatoma cells may be significantly inhibited by intervening in the activation of the TNFa/NF-kappaB signaling pathway, which promotes cell apoptosis and blocks cell cycling.

Glypican-3 as an emerging molecular target for hepatocellular carcinoma gene therapy.

Glypican-3 (GPC-3), a membrane-associated heparan sulfate proteoglycan, plays a crucial role in cell proliferation and metastasis, particularly in hepatocellular carcinoma (HCC) progression, and perhaps is a valuable target for its gene therapy. However, its mechanism remains to be explored. In the present study, the biological behaviors of HCC cells were investigated by interfering GPC-3 gene transcription. After the cells were transfected with specific GPC-3 short hairpin RNA (shRNA), the inhibition of GPC-3 expression was 75.6 % in MHCC-97H or 73.8 % in Huh7 cells at mRNA level; the rates of proliferation and apoptosis were 53.6 and 60.5 % in MHCC-97H or 54.9 and 54.4 % in Huh7 cells, with the cell cycles arrested in the G1 phase; the incidences of cell migration, metastasis, and invasion inhibition were 80.1, 56.4, and 69.1 % in MHCC-97H or 80.9, 59.6, and 58.3 % in Huh7 cells, respectively. The cell biological behaviors were altered by silencing GPC-3 with down-regulation of ß-catenin, insulin-like growth factor-II and vascular endothelial growth factor, and Gli1 up-regulation. The cell proliferation was significantly inhibited (up to 95.11 %) by shRNA plus anti-cancer drugs, suggesting that GPC-3 gene should be a potential target for promoting hepatoma cell apoptosis and inhibiting metastasis through the Wnt/ß-catenin and Hh singling pathways.

Circulating specific biomarkers in diagnosis of hepatocellular carcinoma and its metastasis monitoring.

Hepatocellular carcinoma (HCC) is one of the most common and rapidly fatal malignancies worldwide with a multifactorial, multistep, complex process and poor prognosis. Its early diagnosis and metastasis monitoring are of the utmost importance. Hepatoma tissues synthesize various tumor-related proteins, genes, enzymes, microRNA, etc. and then secrete into the blood. Detections of circulating biomarkers are useful to find tumor at an early stage or monitor metastasis after postoperative treatment. This paper summarizes recent studies of specific biomarkers at early diagnosis or in monitoring metastasis or postoperative recurrence of HCC.

Down-regulating glypican-3 expression: molecular-targeted therapy for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) prognosis is very poor, its early treatment is of the utmost importance. Glypican-3 (GPC-3), a membrane-associated heparan sulfate proteoglycan, plays a crucial role in cell proliferation and metastasis, particularly in progression. GPC-3 mediated oncogenesis involves signaling pathways during the malignant transformation of hepatocyte carcinogenesis, with an increasing expression of GPC-3 observed from non-cancerous- to cancerous-tissues, with brown granule-like staining localized in tumor parts of atypical hyperplasia and HCC formation. GPC-3 expression in HCC tissues or circulating blood was associated with tumor size or HBV infection. Circulation of GPC-3-mRNA was detected in HCC patients with relation to TNM stage, periportal cancerous embolus, and extra-hepatic metastasis. After hepatoma, cells were transfected with shRNA, GPC-3 expression or proliferation was inhibited with promoting apoptosis, cell cycle arrested in G1 phase, alteration of hepatoma cell migration and invasion behaviors with down-regulation of ß-catenin, IGF-II, and VEGF, and growth of nude mice xenograft tumors was significantly suppressed with the decreases in ß-catenin, p-GSK3ß, and cyclinD1 expression, suggesting that GPC-3 not only is a specific biomarker for HCC diagnosis, but also is a valuable molecular-target for HCC gene therapy.

Inhibition of Annexin A2 gene transcription is a promising molecular target for hepatoma cell proliferation and metastasis.

Hepatocyte Annexin A2 (ANXA2) expression is associated with the progression and metastasis of hepatocellular carcinoma (HCC). Circulating ANXA2 levels in HCC patients are significantly higher compared with that of patients with benign liver disease. ANXA2 levels have been found to correlate with hepatitis B virus infection, extrahepatic metastasis and portal vein thrombus. By contrast, ANXA2 levels do not correlate with tumour size and AFP levels. However, the underlying mechanisms of ANXA2 remain obscure. The results of the current study identified that abnormalities in hepatic ANXA2 expression were localised to the cell membrane and cytoplasm of HCC tissues and mainly in the cytoplasm of para-cancerous tissues. ANXA2 was overexpressed in MHCC97-H cells which have high metastatic potential. Following specific ANXA2-small hairpin RNA (shRNA) transfection in vitro, ANXA-2 was effectively inhibited and the S phase ratio of cells was 27.76%, compared with 36.14% in mock-treated cells. In addition, the invading cell ratio was reduced in the shRNA-treated group (52.16%) compared with the mock-treated group (86.14%). The growth and volume of xenograft tumours in vivo was significantly suppressed (P<0.05) in the shRNA group compared with that of the mock group, indicating that ANXA2 may be a novel and useful target for elucidating molecular mechanisms involving the proliferation and metastasis of HCC.

Overexpression of insulin-like growth factor-I†receptor as a pertinent biomarker for hepatocytes malignant transformation.

AIM: To investigate the dynamic features of insulin-like growth factor-I receptor (IGF-IR) expression in rat hepatocarcinogenesis, and the relationship between IGF-IR and hepatocytes malignant transformation at mRNA or protein level. METHODS: Hepatoma models were made by inducing with 2-fluorenylacetamide (2-FAA) on male Sprague-Dawley rats. Morphological changes of hepatocytes were observed by pathological Hematoxylin and eosin staining, the dynamic expressions of liver and serum IGF-IR were quantitatively analyzed by an enzyme-linked immunosorbent assay. The distribution of hepatic IGF-IR was located by immunohistochemistry. The fragments of IGF-IR gene were amplified by reverse transcription-polymerase chain reaction, and confirmed by sequencing. RESULTS: Rat hepatocytes after induced by 2-FAA were changed dynamically from granule-like degeneration, precancerous to hepatoma formation with the progressing increasing of hepatic mRNA or IGF-IR expression. The incidences of liver IGF-IR, IGF-IR mRNA, specific IGF-IR concentration (ng/mg wet liver), and serum IGF-IR level (ng/mL) were 0.0%, 0.0%, 0.63 ± 0.17, and 1.33 ± 0.47 in the control; 50.0%, 61.1%, 0.65 ± 0.2, and 1.51 ± 0.46 in the degeneration; 88.9%, 100%, 0.66 ± 0.14, and 1.92 ± 0.29 in the precancerosis; and 100%, 100%, 0.96 ± 0.09, and 2.43 ± 0.57 in the cancerous group, respectively. IGF-IR expression in the cancerous group was significantly higher (P < 0.01) than that in any of other groups at mRNA or protein level. The closely positive IGF-IR relationship was found between livers and sera (r = 0.91, t = 14.222, P < 0.01), respectively. CONCLUSION: IGF-IR expression may participate in rat hepatocarcinogenesis and its abnormality should be an early marker for hepatocytes malignant transformation.

[Short hairpin RNA mediated glypican-3 silencing inhibits hepatoma cell invasiveness and disrupts molecular pathways of angiogenesis].

OBJECTIVE: To construct glypican-3 (GPC-3) short hairpin RNA (shRNA) and investigate the effects of GPC-3 transcription silencing on hepatoma cell invasion and angiogenesis mechanisms. METHODS: GPC-3-specific shRNA and non-target control shRNA were constructed and transfected into the human hepatoma cell lines HepG2, MHCC-97H, and Huh7. shRNA-mediated silencing of GPC-3 expression was confirmed at the mRNA and protein levels by fluorescence quantitative reverse transcription (FQRT)-PCR and western blotting, respectively. The effect of silenced GPC-3 expression on cell proliferation was detected by EdU and sulforhodamine B assays, on migration by wound healing (scratch) assay, on invasion by transwell chamber assay, and on apoptosis by luminescence assay of caspase-3/7 activity. The effect of silenced GPC-3 expression on angiogenesis-related signaling factors was detected by FQRT-PCR (for the glioma-associated oncogene homolog-1 hedgehog signaling factor, GLI1, and the beta-catenin Wnt signaling factor, b-catenin), immunofluorescent staining (for the insulin-like growth factor-II, IGF-II), and ELISA (for the vascular endothelial growth factor, VEGF). Pairwise comparisons were made by the independent sample t-test, and multiple comparisons were made by one-way ANOVA. RESULTS: In all cell lines, transfection with the GPC-3-specific shRNA significantly reduced GPC-3 mRNA levels (% reduction as compared to the non-target control shRNA: HepG2, 89.2+/-6.0%, t = -25.753, P less than 0.001; MHCC-97H, 75.3+/-4.9%, t = -26.487, P less than 0.001; Huh7, 73.6+/-4.6%, t = -27.607, P less than 0.001); the GPC-3 protein levels were similarly reduced. The GPC-3 shRNA-silenced cells showed significantly reduced proliferative, migratory and invasive capacities, as well as significantly increased apoptosis. The shRNA-mediated GPC-3 silencing was accompanied by significant down-regulation of b-catenin mRNA (HepG2, 46.9+/-0.6%; MHCC-97H, 67.5+/-2.7%; Huh7, 56.3+/-8.4%) and significant up-regulation of GLI1 mRNA (HepG2, 49.2+/-28.6%; MHCC-97H, 54.6+/-24.4%; Huh7, 31.6+/-15.7%). At 72 h after transfection, the HepG2 cells showed significant down-regulation of VEGF protein (54.3+/-1.5%, t = 46.746, P less than 0.001). CONCLUSION: GPC-3 contributes to migration, invasion, angiogenesis, and apoptosis of hepatoma cells, possibly through its interactions with the Wnt/b-catenin and Hedgehog signaling pathways. GPC-3 may represent a useful target for gene silencing by molecular-based therapies to treat hepatocellular carcinoma.