Oncogenic Wnt3a is a promising sensitive biomarker for monitoring hepatocarcinogenesis.

BACKGROUND: The effective treatment for hepatocellular carcinoma (HCC) depends on early diagnosis. Previously, the abnormal expression of Wnt3a as the key signaling molecule in the Wnt/ß-catenin pathway was found in HCC cells and could be released into the circulation. In this study, we used rat model of hepatocarcinogenesis to dynamically investigate the alteration of oncogenic Wnt3a and to explore its early monitor value for HCC. METHODS: Sprague-Dawley rats (SD) were fed with diet 2-fluorenylacetamide (2-FAA, 0.05%) for inducing hepatocarcinogenesis, and grouped based on liver morphological alteration by Hematoxylin & Eosin (H&E) staining; rats fed with normal chow were used as normal control (NC). Total RNA and protein were purified from rat livers. Differently expressed genes (DEGs) or Wnt3a mRNA, cellular distribution, and Wnt3a protein levels were analyzed by whole genome microarray with signal logarithm ratio (SLR log2cy5/cy3), immunohistochemistry, and enzyme-linked immunosorbent assay, respectively. RESULTS: Models of rat hepatocarcinogenesis were successfully established based on liver histopathological H&E staining. Rats were divided into the cell degeneration (rDeg), precancerosis (rPre-C) and HCC (rHCC) groups. Total numbers of the up- and down-regulated DEGs with SLR ≥ 8 were 55 and 48 in the rDeg group, 268 and 57 in the rPre-C group, and 312 and 201 in the rHCC group, respectively. Significantly altered genes were involved in cell proliferation, signal transduction, tumor metastasis, and apoptosis. Compared with the NC group, Wnt3a mRNA was increased by 4.6 folds (P < 0.001) in the rDeg group, 7.4 folds (P < 0.001) in the rPre-C group, and 10.4 folds (P < 0.001) in the rHCC group; the positive rates of liver Wnt3a were 66.7% (P = 0.001) in the rDeg group, 100% (P < 0.001) in the rPre-C group, and 100% (P < 0.001) in the rHCC group, respectively. Also, there were significant differences of liver Wnt3a (P < 0.001) or serum Wnt3a (P < 0.001) among different groups. CONCLUSIONS: Overexpression of Wnt3a was associated with rat hepatocarcinogenesis and it should be expected to be a promising monitoring biomarker for HCC occurrence at early stage.

[Alteration of Wnt3a overexpression and its early monitoring value during hepatocellular carcinogenesis].

Objective: To investigate the dynamic expression pattern of carcinoembryonic Wnt3a and its early monitoring value using a hepatocellular carcinoma model. Methods: Forty-eight Sprague Dawley (SD) rats were fed with pellet feed containing 2-acetylaminofluorene (2-AAF, 0.05%) to induce hepatocarcinogenesis, and control rats were fed a pellet diet. Liver tissue and blood samples were collected every two weeks. Liver tissues were pathologically examined using HE staining and grouped. The gene and Wnt3a mRNA expression were analyzed by genome-wide microarray. The expression and distribution of Wnt3a in liver tissue were analyzed by immunohistochemistry. Wnt3a concentration in liver tissue and serum was quantified by enzyme-linked immunosorbent assay. Statistical methods such as χ2 test, Mann-Whitney test and analysis of variance were used to analyze the differences between groups. Results: According to the pathological examination results, the rat livers were divided into four groups: control, hepatocyte degeneration, precancerous lesions and hepatocellular carcinoma. Genome-wide expression profiling analysis and comparison with the control group revealed that 268 and 312 genes were up-regulated and 57 and 201 genes were down-regulated in the precancerous and cancerous group when signal logarithm ratio (SLR) was >8 log2cy5/cy3, and these significantly altered genes mainly involved in cell proliferation, signal transduction, tumor metastasis, and apoptosis. The expression of Wnt3a at mRNA level was significantly increased in all stages of cancer induction, including degeneration group (1.15±0.24, q=8.227), precancerous group (1.85±0.18, q=12.361) and cancerous group (2.59±0.55, q=18.082). Compared with the control group (0.25±0.11, F=121.103, P<0.001), the degeneration group, the precancerous group and the liver cancer group were up-regulated by 4.6, 7.4 and 10.4-folds, respectively. Immunohistochemistry showed that compared with the control group, the positive rate of Wnt3a in the degeneration group was 66.7% (12/18, χ2=10.701, P=0.001), and both the precancerous and liver cancer groups were positive (9/9, χ2=17.115, P<0.001). Wnt3a expression was gradually increased in liver and blood samples during the process of carcinogenesis, and the difference between two groups was statistically significant (F=176.711, P<0.001). Wnt3a overexpression was secreted into blood stream via cancerous liver tissue, and there was a linear correlation between Wnt3a levels in blood and liver samples (r=0.732, P<0.001). Conclusions: Wnt3a overexpression is closely related with hepatocellular carcinogenesis, and thus may become a new monitoring marker.

Secretory clusterin promotes hepatocellular carcinoma progression by facilitating cancer stem cell properties via AKT/GSK-3ß/ß-catenin axis.

BACKGROUND: To explore the modulatory effects and mechanism of secretory clusterin (sCLU) on cancer stem cell (CSC) properties in hepatocellular carcinoma (HCC). METHODS: The effects of sCLU repression or overexpression on chemoresistance, migration, invasion, and tumor growth were detected by MTT, wound healing, transwell assays, and xenograft assay, respectively. The tumor sphere assay was performed to evaluate the self-renewal ability of HCC cells. In addition, the molecular regulation between sCLU and AKT/GSK-3ß/ß-catenin axis in HCC cells were discovered by western blotting, quantitative real-time PCR (qRT-PCR), and immunofluorescence. The expression status of sCLU and ß-catenin in HCC tissues were investigated by immunohistochemistry. RESULTS: Knockdown or overexpressing sCLU remarkably inhibited or promoted the chemoresistance against sorafenib/doxorubicin, metastasis, and tumor growth of HCC cells, respectively. HepG2 and HCCLM3-derived spheroids showed higher expression of sCLU than that in attached cells. Additionally, repressing sCLU impaired the self-renewal capacity of HCC cells and CSC-related chemoresistance while overexpression of sCLU enhanced these CSC properties. Knockdown or overexpression of sCLU inhibited or increased the expressions of ß-catenin, cyclinD1, MMP-2 and MMP-9, and the phosphorylation of AKT or GSK3ß signaling, respectively. However, LiCl or LY294002 abrogated the effects mediated by sCLU silencing or overexpression on chemoresistance, metastasis, and CSC phenotype. Furthermore, co-expression of sCLU and ß-catenin in HCC tissues indicated poor prognosis of HCC patients. CONCLUSIONS: Taken together, the oncogenic sCLU might promote CSC phenotype via activating AKT/GSK3ß/ß-catenin axis, suggesting that sCLU was a potential molecular-target for HCC therapy.

Dynamic expression of hepatic GP73 mRNA and protein and circulating GP73 during hepatocytes malignant transformation.

BACKGROUND: Hepatic Golgi protein-73 (GP73) expression is related to hepatocellular carcinoma (HCC) progression. The aim of this study was to investigate the dynamic expression of GP73 mRNA and protein during hepatocytes malignant transformation. METHODS: Human GP73 expressions in 88 HCC tissues and their self-control surrounding tissues were examined by immunohistochemistry, and survival time of HCC patients was evaluated by the Kaplan-Meier method. HCC model of Sprague-Dawley rats was made by diet containing 2-fluorenylacetamide. The rats were divided into the control, hepatocyte degeneration, precanceration, and HCC groups to observe GP73 protein and mRNA alterations during hepatocytes malignant transformation. RESULTS: The GP73 expression was significantly higher in the cancerous tissues than that in the surrounding tissues, with shorter survival time, and the positive rates of GP73 protein in human HCC tissues were 53.3% at stage I, 84.0% at stage II, 84.6% at stage III, and 60.0% at stage IV, respectively. The positive rates of hepatic GP73 protein and mRNA in the rat models were none in the control group, 66.7% and 44.4% in the hepatocytes degeneration group, 88.9% and 77.8% in the hepatocytes precanceration group, and 100% in the HCC group, respectively. There was a positive correlation (r = 0.91, P<0.01) between hepatic GP73 and serum GP73 during rat hepatocytes malignant transformation. CONCLUSIONS: Abnormal GP73 expression may be a sensitive and valuable biomarker in hepatocarcinogensis.

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.

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.

IGF-I receptor as an emerging potential molecular-targeted for hepatocellular carcinoma in vitro and in vivo.

Abnormal expression of insulin-like growth factor I receptor (IGF-IR) is associated with hepatocellular carcinoma (HCC) progression with largely unknown mechanisms. In this study, IGF-IR expression among different HCC cell lines and silencing its gene transcription on effects of HCC were investigated by short hairpin RNA (shRNA). Specific shRNA was successfully transfected into Bel-7404 or PLC/PRF/5 cells with 90 or 71 % efficiency. The inhibiting rate of IGF-IR at mRNA level were 54.9 % in Bel-7404 or 59.6 % in PLC/PRF/5 cells in accordance with its protein suppression, with the cell cycles at the G1 phase arrest and decreasing cyclinD1 via promoting apoptosis in vitro. With the xenograft models of PLC/PRF/5 cells inserted specific shRNA in vivo, the tumor-forming time (14.0 ± 1.1 days) or tumor volume (143 ± 24 mm3) in the shRNA group was significantly lengthened or smaller than those in the control group (7.2 ± 0.8 days or 372 ± 46 mm3, P < 0.001) or in the neg-shRNA group (7.5 ± 1.0 days or 350 ± 50 mm3, P < 0.001). Silencing the IGF-IR gene transcription inhibited cell proliferation or xenograft tumor growth of HCC, suggesting that IGF-IR might be a novel potential target for HCC gene therapy.

Abnormality of Wnt3a expression as novel specific biomarker for diagnosis and differentiation of hepatocellular carcinoma.

The member 3a of Wingless-type MMTV integration site family (Wnt3a) as an oncogene is overexpressed in many kinds of tumors with a worse outcome. However, the mechanism and alteration of Wnt3a expression in hepatocellular carcinoma (HCC) have not been clarified. In this study, the levels of Wnt3a expression were investigated in 80 HCC tissues or sera of 186 patients with chronic liver diseases. The incidence of hepatic Wnt3a expression in HCC tissues was 96.25 % and significantly higher (χ (2) = 48.818, P < 0.001) than that in their surrounding tissues (46.25 %). The higher level (>800 ng/L) of circulating Wnt3a expression was found in 92.5 % HCC patients and significantly related (P < 0.05) to alpha-fetoprotein (AFP) level, liver cirrhosis, hepatitis B virus infection, poor differentiation, tumor node metastasis, and extra-hepatic metastasis. The level of Wnt3a expression in HCC patients was obviously higher (P < 0.001) than that in any group of cases with benign liver diseases. The diagnostic specificity or the area under the receiver operating characteristic curve was 94.34 % or 0.994 in Wnt3a and 69.81 % or 0.710 in AFP for HCC, respectively. The present data suggested that Wnt3a expression associated with tumor progression should be a novel specific biomarker for diagnosis and differentiation of HCC.

Diagnostic and prognostic significance of secretory clusterin expression in patients with hepatocellular carcinoma.

The upregulation of secretory clusterin (sCLU) is associated with tumor progression by contributing to angiogenesis, chemo-resistance, cell survival, and metastasis. However, its diagnostic or prognostic values for hepatocellular carcinoma (HCC) still remain to be clarified. The average serum sCLU level analyzed by an enzyme-linked immunosorbent assay was significantly higher (P < 0.001) in HCC patients than that in any of cases with cirrhosis, chronic hepatitis, or healthy control. The area under receiver operating characteristic curve and diagnostic sensitivity were 0.75 and 74.7 % in sCLU, and 0.74 and 58.7 % in α-fetoprotein (AFP), respectively. The combining detections of sCLU and AFP rose up to 90.7 % for HCC diagnosis. In liver, sCLU by immunohistochemistry was significantly higher (P < 0.001) in the HCC (77.3 %) group than that in their para-cancerous group (33.3 %). Abnormal serum or tissue sCLU expression was closely associated with tumor-node-metastasis (TNM) classification of malignant tumors and lymph node metastasis, as an independent prognosis factor (hazard ratio, 2.287; 95 % confidence interval, 1.044-5.007; P = 0.039), and higher sCLU expression significantly correlated (χ (2) = 4.252, P = 0.039) with poor survival of HCC patients analyzed by multivariate Cox regression or Kaplan-Meier method, suggesting that abnormal sCLU expression associated with tumor progression could be a potential diagnostic and prognostic biomarker for HCC.

Effective targeting of colorectal cancer cells using TORC1/2 kinase inhibitors in vitro and in vivo.

AIM: We investigated the effects of TORC1/2 kinase inhibitors on colorectal cancer (CRC) cell lines. MATERIALS & METHODS: Using selective TORC1/2 inhibitors, rapamycin and PP242, we assessed their effect on the growth of CRC cells in vitro and tumor growth in vivo. RESULTS: Rapamycin and PP242 inhibit proliferation and induce apoptosis of CRC cells. They also enhance proapoptotic effect of conventional chemo drug doxorubicin in CRC cells in vitro. When combined with doxorubicin, rapamycin and PP242 almost completely inhibit tumor growth in vivo. Rapamycin and PP242 inhibit phosphorylation of Akt, ribosomal S6 kinase, 4EBP1 and mTOR. CONCLUSION: Our study suggests rapamycin and PP242 may be a useful therapeutic agent and inhibiting mTOR signaling pathway represents a new targeted therapy for CRC.

[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.

Up-regulation of annexin A2 expression predicates advanced clinicopathological features and poor prognosis in hepatocellular carcinoma.

Hepatic annexin A2 (ANXA2) orchestrates multiple biologic processes and clinical symptoms and plays a key role in development, metastasis, and drug resistance of lethal hepatocellular carcinoma (HCC). However, the prognostic significance of ANXA2 for HCC has not been elucidated up to now. In this study, ANXA2 was frequently found to be up-regulated in HCC tissues compared with benign liver disease (BLD) tissues, which was consistent with the results in serum samples and tissue specimens of patients with HCC. Furthermore, ANXA2 expression was significantly correlated with differentiated degree, intrahepatic metastasis, portal vein thrombus, and tumor node metastasis (TNM) staging. More importantly, increased ANXA2 level was first confirmed to be closely associated with shortened overall survival of HCC (χ (2) = 12.872, P = 0.005) and identified as an independent prognostic factor (hazard ratio 1.338, 95 % confidence interval (CI) 1.013 ~ 1.766, P = 0.040), suggesting that ANXA2 up-regulation might represent an acquired metastasis phenotype of HCC, help to screen out high-risk population for HCC, or more effectively treat a subset of postsurgical HCC patients positive for ANXA2.

Silencing clusterin gene transcription on effects of multidrug resistance reversing of human hepatoma HepG2/ADM cells.

Abnormal clusterin (CLU) expression is associated with multidrug resistance (MDR) of hepatocellular carcinoma (HCC). In the present study, the CLU expression was analyzed in human hepatoma cells and chemoresistant counterpart HepG2/ADM cells. Compared with L02 cells, the overexpression of cellular CLU was identified in HepG2, HepG2/ADM, SMMC7721, Hep3B ,and PLC cells and relatively lower expression in Bel-7404, SNU-739, and MHCC97H cells. Specific short hairpin RNAs (shRNAs) to silence CLU gene transcription were designed, and the most effective sequences were screened. After the HepG2/ADM cells transfected with shRNA-1, the inhibition of CLU expression was 73.68 % at messenger RNA (mRNA) level by real-time quantitative RT-PCR with obvious enhancement in cell chemosensitivity, increasing apoptosis induced by doxorubicin using fluorescence kit, and Rh-123 retention qualified with flow cytometry. Knockdown CLU also significantly decreased the drug efflux pump activity through the depression of MDR1/P-glycoprotein (q = 11.739, P < 0.001). Moreover, silencing CLU led to downregulation of ß-catenin (q = 13.544, P = 0.001), suggesting that downregulation of CLU might be a key point to reverse multidrug resistance of HepG2/ADM cells.

Glypican-3 is a biomarker and a therapeutic target of hepatocellular carcinoma.

BACKGROUND: The carcinogenesis of hepatocellular carcinoma (HCC) is a multi-factorial, multi-step and complex process. Early diagnosis and effective treatments are of utmost importance. This review summarized the recent studies of oncofetal glypican-3 (GPC-3), a membrane-associated heparan sulfate proteoglycan, in the diagnosis and treatment of HCC. DATA SOURCES: English-language reports published from June 2001 to September 2014 were searched from MEDLINE. The key words searched included: GPC-3, biomarker, target and HCC. The sensitivity, specificity, positive and negative predictive values were extracted, and the effect of GPC-3 targeted therapy on HCC was also evaluated. RESULTS: GPC-3 plays a crucial role in HCC cell proliferation and metastasis. It mediates oncogenesis involving signaling pathways during hepatocyte malignant transformation. GPC-3 expression is increased in atypical hyperplasia and cancerous tissues. GPC-3 levels in HCC patients are related to HBV infection, TNM stage, periportal cancerous embolus, and extrahepatic metastasis. The diagnostic accuracy of the combination of serum GPC-3 and alpha-fetoprotein in HCC is up to 94.3%. Down-regulation of GPC-3 with specific siRNA or anti-GPC-3 antibody alters cell migration, metastasis and invasion behaviors. The nude mice xenograft tumor growth is inhibited by silencing GPC-3 gene transcription. CONCLUSION: Oncofetal GPC-3 is a highly specific biomarker for the diagnosis of HCC and a promising target molecule for HCC gene therapy.

[Down-regulated clusterin expression enhances sensitivity of hepatoma cells to anti-cancer drugs].

OBJECTIVE: To investigate the relationship between and underlying mechanistic pathway of clusterin (CLU) and chemo-resistance ofhepatocellular carcinoma (HCC) cells. METHODS: CLU protein expression in HCC cell lines (Hep3B, SMMC7721, PLC, and HepG2) and HepG2/ADM cells was quantified by western blotting. Four short-hairpin (sh)RNAs designed to block CLU-mRNA were generated, screened by RT-PCR, and transfected into the cells to determine effects of CLU on cell viability and apoptosis. Effects of CLU blockade on drug efflux pump activity were measured by flow cytometry. RESULTS: CLU was found to be over-expressed in HCC cell lines and HepG2/ADM cells. The four shRNAs inhibited CLU-mRNA as follows (vs. levels in untransfected cells): shRNA-1: 73.68% (q =23.011, P < 0.01), shRNA-2: 39.26% (q =11.991, P < 0.01), shRNA-3: 62.36% (q =19.392, P < 0.01), and shRNA-4: 55.35% (q =17.149, P < 0.01). shRNA-mediated depletion of CLU led to increased sensitivity to anti-cancer drugs and increased doxorubicin-induced apoptosis in HepG2/ADM cells, as evidenced by the apoptosis ratio of the shRNA-1 group of 39.28% vs. the apoptosis ratio of the untransfected control group of 4.92%. Silencing of CLU also decreased drug etflux pump activity, and the level of MDR1/P-gp expression was significantly reduced (shRNA-1 group vs.untransfected control group: q =14.604, P < 0.01). CONCLUSION: CLU repression may enhance sensitivity of HCC cells to anti-cancers drugs and represents a potential molecular-target for reversal of multidrug-resistant HCC.

Abnormal expressions of inflammatory-related mediators and inhibition of fat metabolism in mice infected with influenza a virus.

The pathophysiological role of influenza infection is poorly understood. In this study, one non-neurovirulent virus (IAV/Aichi/2/68/H3N2) strain was used to infect intra-nasally mice at different age to investigate the mechanism of cerebral edema formation and lower activities of mitochondria enzymes after influenza A virus (IAV) infection. Mice suffered 46.4% mortality in newborn compared with 96.0% in weanling, 100% in adult on day 7, respectively. IAV-RNA was easily detected in the brain of newborn mice. Significant production of endothelin-1 and inducible nitric oxide syntheses were increased on the 3rd and 5th day after IAV infection, associated with increasing blood-brain barrier permeability, brain edema formation and the higher mortality of animals. Production of tumor necrosis factor-α was related to inhibition of mitochondrial enzyme activities, suggesting that over expression of inflammatory cytokines and lower enzyme activities in mitochondria after IAV infection.

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.

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.

Expression of SRC suppressed C kinase substrate in rat neural tissues during inflammation.

Src-suppressed C kinase substrate (SSeCKS), an in vivo and in vitro protein kinase C substrate, is a major lipopolysaccharide (LPS) response protein which markedly upregulated in several organs, including brain, lung, heart, kidney etc., indicating a possible role of SSeCKS in inflammatory process. However, the expression and biological function of SSeCKS during neuronal inflammation remains to be elucidated, so we established an inflammatory model injected with LPS to investigate the gene expression patterns of SSeCKS in neural tissues by using TaqMan quantitative real-time PCR and immunohistochemistry in rat. Real-time PCR showed that LPS stimulated the expression of SSeCKS mRNA in a dose- and time-dependent manner in sciatic nerves, spinal cords and dorsal root ganglions. Immunohistochemistry showed that SSeCKS colocalized with nerve fibers in sciatic nerve after LPS administration, but there was no colocalization between SSeCKS and Schwann cells. In addition, SSeCKS colocalized with neurons which existed in dorsal root ganglions and spinal cords. These findings indicated that SSeCKS might play some important roles in sciatic nerve fibers and neurons in spinal cords and dorsal root ganglions after LPS injection.