Early monitoring values of oncogenic signalling molecules for hepatocellular carcinoma

The prevention and early diagnosis of liver cancer remains a global medical challenge.During the malignant transformation of hepatocytes,a variety of oncogenic cellular signalling molecules,such as novel high mobility group-Box 3,angiopoietin-2,Golgi protein 73,glypican-3,Wnt3a(a signalling molecule in the Wnt/β-catenin pathway),and secretory clusterin,can be expressed and secreted into the blood.These signalling molecules are derived from different signalling pathways and may not only participate in the malignant transformation of hepatocytes but also become early diagnostic indicators of hepatocarcinogenesis or specific targeted molecules for hepatocellular carcinoma therapy.This article reviews recent progress in the study of several signalling molecules as sensitive biomarkers for monitoring hepatocarcinogenesis.

Female hepatology:Favorable role of estrogen in chronic liver disease with hepatitis B virus infection

Chronic hepatitis B virus (HBV) infection is the most common cause of hepatic fibrosis and hepatocellular carcinoma (HCC),mainly as a result of chronic necroinflammatory liver disease. A characteristic feature of chronic hepatitis B infection,alcoholic liver disease and nonalcoholic fatty liver disease (NAFLD) is hepatic steatosis. Hepatic steatosis leads to an increase in lipid peroxidation in hepatocytes,which,in turn,activates hepatic stellate cells (HSCs). HSCs are the primary target cells for inflammatory and oxidative stimuli,and these cells produce extracellular matrix components. Chronic hepatitis B appears to progress more rapidly in males than in females,and NAFLD,cirrhosis and HCC are predominately diseases that tend to occur in men and postmenopausal women. Premenopausal women have lower hepatic iron stores and a decreased production of proinflammatory cytokines. Hepatic steatosis has been observed in aromatase-deficient mice,and has been shown to decrease in animals after estradiol treatment. Estradiol is a potent endogenous antioxidant which suppresses hepatic fibrosis in animal models,and attenuates induction of redox sensitive transcription factors,hepatocyte apoptosis and HSC activation by inhibiting a generation of reactive oxygen species in primary cultures. Variant estrogen receptors are expressed to a greater extent in male patients with chronic liver disease than in females. These lines of evidence suggest that the greater progression of hepatic fibrosis and HCC in men and postmenopausal women may be due,at least in part,to lower production of estradiol and a reduced response to the action of estradiol. A better understanding of the basic mechanisms underlying the sex-associated differences in hepatic fibrogenesis and carciogenesis may open up new avenues for the prevention and treatment of chronic liver disease.

Expression characteristics and diagnostic value of annexin A2 in hepatocellular carcinoma

AIM： TO investigate the characteristics and diagnostic value of annexin A2 （ANXA2） expression in cancerous tissues and sera of patients with hepatitis B virus （HBV）-related hepatocellular carcinoma （HCC）. METHODS： Levels of liver ANXA2 gene transcription or protein expression were analyzed in HCC-, their self- controlled precancerous-, and distant cancerous- tissues from 30 HCC. Serum levels of ANXA2 expression in 115 patients with HCC, 25 with metastatic liver can cer, 35 with chronic hepatitis, 28 with acute hepatitis, 38 with cirrhosis, and 30 healthy controls were deter- mined. Clinicopathological characteristics of circulating ANXA2 expression were analyzed, and its diagnostic efficiency and clinical values in HCC were evaluated. RESULTS： ANXA2 expression was localized in both cell membrane and cytoplasm in HCC tissue, mainly in the cytoplasm of matched adjacent cancerous tissue, and there was almost no positive staining in matched distant cancerous tissue. Abnormal expression of liver ANXA2 was present in HCC tissues compared with self-con- trolled adjacent- and distant-cancerous tissues at pro- tein or mRNA level. Circulating ANXA2 in HCC patients was significantly higher than that of other liver diseases （P 〈 0.01） except metastatic liver cancer. If the diag- nostic cutoff value of ANXA2 level was more than 18 ng/ mL, the incidence of serum ANXA2 was 86.96% in the HCC group, 80% in the metastatic liver cancer group, 31.58% in the liver cirrhosis group, none in the chronic hepatitis or acute hepatitis or normal control group, respectively. Serum ANXA2 expression in HCC patients was correlated with HBV infection （27.38 ± 5.67 ng/mL vs 18.58 ± 7.83 ng/mL, P 〈 0.01）, extrahepatic metas- tasis （26.11±5.43 ng/mL ys 22.79 ± 5.64 ng/mL, P 〈 0.01）, and portal vein thrombus （26.03 ± 5.99 ng/mL vs 23.06 ± 5.03 ng/mL, P 〈 0.01）, and was significantly higher （P 〈 0.01） in the moderately- （26.19±5.34 ng/ mL） or the poorly- differentiated group （27.05 ± 5.13 ng/mL） than in the well differentiated group （20.43 ± 4.97 ng/mL）, and in the tumor node metastasis stages Ⅲ-Ⅳ（P 〈 0.01） than in stages Ⅰ-Ⅱ. ANXA2 was not correlated with patient sex, age, size or α-fetoprotein （AFP） level. Area under the receiver operating charac- teristic curve for the whole range of sensitivities and specificities was 0.796 for ANXA2 and 0.782 for AFP. Combining detection of serum ANXA2 and AFP substan- tially improved the diagnostic efficiency （96.52%） and the neclative predictive value （＇96.61%） for HCC.of ANXA2 expression has good diagnostic potential for HCC diagnosis.

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.

Annexin A2 silencing inhibits invasion, migration, and tumorigenic potential of hepatoma cells

AIM: To investigate the effects of Annexin A2 (ANXA2) silencing on invasion, migration, and tumorigenic potential of hepatoma cells. METHODS: Human hepatoma cell lines [HepG2, SMMC-7721, SMMC-7402, and MHCC97-H, a novel human hepatocellular carcinoma (HCC) cell line with high metastasis potential] and a normal hepatocyte cell line(LO2) were used in this study. The protein and mRNA expression levels of ANXA2 were analysed by western blotting and real-time polymerase chain reaction, re-spectively. The intracellular distribution profile of ANXA2 expression was determined by immunofluorescence and immunohistochemistry. Short hairpin RNA target-ing ANXA2 was designed and stably transfected into MHCC97-H cells. Cells were cultured for in vitro analy-ses or subcutaneously injected as xenografts in mice for in vivo analyses. Effects of ANXA2 silencing on cell growth were assessed by cell counting kit-8 (CCK-8) as-say (in vitro ) and tumour-growth assay (in vivo ), on cell cycling was assessed by flow cytometry and propidium iodide staining (in vitro ), and on invasion and migration potential were assessed by transwell assay and wound-healing assay, respectively (both in vitro ). RESULTS: The MHCC97-H cells, which are known to have high metastasis potential, showed the highest lev-el of ANXA2 expression among the four HCC cell types examined; compared to the LO2 cells, the MHCC97-H expression level was 8-times higher. The ANXA2 expres-sion was effectively inhibited (about 80%) by ANXA2-specific small hairpin RNA (shRNA). ANXA2 expression in the MHCC97-H cells was mainly localized to the cel-lular membrane and cytoplasm, and some localization was detected in the nucleus. Moreover, the proliferation of MHCC97-H cells was obviously suppressed by shR-NA-mediated ANXA2 silencing in vitro , and the tumour growth inhibition rate was 38.24% in vivo . The per-centage of MHCC97-H cells in S phase dramatically de-creased (to 27.76%) under ANXA2-silenced conditions. Furthermore, ANXA2-silenced MHCC97-H cells showed lower invasiveness (percentage of invading cells de-creased to 52.16%) and suppressed migratory capacity (migration distance decreased to 63.49%). It is also worth noting that shRNA-mediated silencing of ANXA2 in the MHCC97-H cells led to abnormal apoptosis. CONCLUSION: shRNA-mediated silencing of ANXA2suppresses the invasion, migration, and tumorigenic potential of hepatoma cells, and may represent a useful target of future molecular therapies.

Values of circulating GPC-3 mRNA and alpha-fetoprotein in detecting patients with hepatocellular carcinoma

BACKGROUND: The prognosis of hepatocellular carcinoma (HCC) is poor and its early diagnosis is of the utmost importance. This study aimed to investigate the values of glypican-3 (GPC-3) expression in the liver and sera and its gene transcription for diagnosis and monitoring of metastasis of HCC. METHODS: Liver GPC-3 was analyzed in HCC tissues from 36 patients by immunohistochemistry and Western blotting. GPC-3 mRNA from circulating peripheral blood mononuclear cells from 123 HCC patients or 246 patients with other diseases or 36 HCC tissues was amplified by RT-PCR, quantitative realtime PCR, and confirmed by DNA sequencing. Circulating GPC-3 level was detected by ELISA. RESULTS: The increasing expression of GPC-3 was observed from non-cancerous to cancerous tissues, with brown granule-like staining localized in tumor parts of atypical hyperplasia and HCC formation. The positive rate of GPC-3 was 80.6% in HCC, 41.7% in their paracancerous tissues, and none in distal cancerous tissues (P【0.001), with no significant difference in differentiation grade and tumor number except for size (Z=2.941, P=0.003). Serum GPC-3 was detected only in HCC (52.8%) and significant difference was found between GPC-3 and tumor size (χ2 =6.318, P=0.012) or HBV infection (χ2 =23.362, P【0.001). Circulating GPC-3 mRNA was detected in 70.7% of HCC tissues, with relation to TNM stage, periportal cancerous embolus, and extra-hepatic metastasis (P【0.001). The combination ofcirculating GPC-3, GPC-3 mRNA and alpha-fetoprotein is of complementary value for HCC diagnosis (94.3%). CONCLUSION: Both GPC-3 overexpression and GPC-3 mRNA abnormality could be used as markers for the diagnosis of HCC and monitoring its metastasis.

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

BACKGROUND： The carcinogenesis of hepatocellular car- cinoma （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 on- cofetal glypican-3 （GPC-3）, a membrane-associated heparan sulfate proteoglycan, in the diagnosis and treatment of HCC. DATA SOURCES： English-language reports published from Iune 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 tar- geted therapy on HCC was also evaluated. RESULTS： GPC-3 plays a crucial role in HCC cell prolifera- tion and metastasis. It mediates oncogenesis involving signal- ing 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 biomark- er for the diagnosis of HCC and a promising target molecule for HCC gene therapy.

Evaluation of HCPTd_1, d_(14)-double passaged intervening chemotherapy protocol for hepatocellular carcinoma

AIM. To establish a kind of standardization of the clinical chemotherapeutic prototypes for unresectable hepatocellular carcinomas （HCC）. METHODS： 10-Hydroxycamptothecin （HCPT） was applied through transcatheter arterial embolization （TAE） to HCC patients who were categorized into three groups： （1） test group： treatment with HCPT twice （HCPT dl and 14） through TAE and portal venous embolization. （2） Control Ⅰ： treatment with anticancer drugs without HCPT. （3） Control Ⅱ： treatment with HCPT as a major component in anticancer drugs once （HCPT dl）. A set of comparisons between test groups and control Ⅰ and Ⅱ groups were performed before and after the treatment to study the effectiveness of each treatment, in terms of tumor volumes, dynamic variations in serum alpha-fetoprotein （AFP）, gamma-glutamyl transferase hepatoma-specific band （GGT-Ⅱ）, patient survival and adverse events. RESULTS： The general effectiveness rate of the test group reached 62.1% （72/116）, remarkably higher than that of control Ⅰ （32.1%, 40/124） and control Ⅱ （54.7%, 47/56）, （P〈0.01 and P〈0.05, respectively）. Especially, the reduction rate or disappearance of the portal vein tumor emboli was as high as 88.4% （61/69） in the test group, in contrast with 13.9% （10/72） in control Ⅰ and 35.9% （18/51） in control Ⅱ （P〈0.01 and P〈0.01, respectively）. After treatment, AFP decreased or turned to negative levels at 52.3% （34/65） in control Ⅰ, 67.3% （35/52） in control Ⅱ, and 96.8% （60/62） in the test group. Also GGT-Ⅱ declined or became negative at 37.8% （28/74） in control Ⅰ, 69.5% （57/82） in control Ⅱ, and 94.7% （89/94） in test group （P〈0.01 and P〈0.05, respectively）. CONCLUSION： We have designed a good protocol （test group） to treat HCC with excellent advantages of high efficiency, low cost, low toxicity and low adverse events and easy application. It could be recommended as one of the standardizations for HCC treatment in clinical practice.

Expression and alteration of insulin-like growth factor II-messenger RNA in hepatoma tissues and peripheral blood of patients with hepatocellular carcinoma

AIM: To investigate the clinical values of serum free insulin-like growth factor Ⅱ (IGF-Ⅱ) levels and IGF-Ⅱ mRNA in hepatocellular carcinoma (HCC) tissues and peripheral blood for diagnosis of HCC and monitoring of extrahepatic metastasis.METHODS: Total RNAs were extracted from HCC tissues or peripheral blood mononuclear cells from patients with HCC, liver diseases devoid of cancer, non-hepatic tumors,and healthy controls, respectively. IGF-Ⅱ cDNAs were synthesized through random primers and reversetranscriptase, amplified by polymerase chain reaction (PCR), and confirmed by DNA sequencing analysis. Serum free IGF-Ⅱ levels in patients with different liver diseases were analyzed by an enzyme-linked immunosorbent assay.RESULTS: The amplified fragments of IGF-Ⅱ mRNA by RT-PCR were identical to originally designed ones with a size of 170 bp and confirmed by sequencing analysis.The dilution experiments revealed that the lowest sensitivity of our system was 2 ng/L of total RNA. The positive frequencies of IGF-Ⅱ mRNA were 100% in HCC tissues,53.3% in para-cancerous tissues, and 0% in non-cancerous tissues, respectively. The serum free IGF-Ⅱ levels were significantly higher in HCC than those in chronic hepatitis or liver cirrhosis. The positive frequency of circulating IGF-Ⅱ mRNA was 34.2% in HCC, no amplified fragment was found in other liver diseases, extrahepatic tumors,and normal controls, respectively. The circulating IGF-Ⅱ mRNA correlated with the stage of HCC, and its positive rate was 100% in HCC with extrahepatic metastasis and 35.5% in HCC with AFP-negative. No significant correlation was found between tumor sizes and circulating IGF-Ⅱ mRNA fragment.CONCLUSION: The abnormal expressions of free IGF-Ⅱ and IGF-Ⅱ mRNA are useful tumor markers for HCC diagnosis, differentiation of extrahepatic metastasis and monitoring postoperative recurrence.

Dynamic alteration of telomerase expression and its diagnostic significance in liver or peripheral blood for hepatocellular carcinoma

AIM： To investigate the dynamic alteration of telomerase expression during development of hepatocellular carcinoma （HCC） and its diagnostic implications in liver tissues or peripheral blood mononuclear cells for HCC. METHODS： Dynamic expressions of liver telomerase during malignant transformation of hepatocytes were observed in Sprague-Dawly （SD） rats fed with 0.05% of 2-fluoenyacetamide （2-FAA）. Total RNA and telomerase were extracted from rat or human liver tissues. The telomerase activities in livers and in circulating blood were detected by a telomeric repeat amplification protocol-enzyme-linked immunosorbent assay （TRAP- ELISA）, and its diagnostic value was investigated in patients with benign or malignant liver diseases. RESULTS： The hepatoma model displayed the dynamic expression of hepatic telomerase during HCC development. The telomerase activities were consistent with liver total RNA levels （r = 0.83, P 〈 0.01） at the stages of degeneration, precancerosis, and cancerization of hepatocytes. In HCC patients, the telomerase levels in HCC tissues were significantly higher than in their adjacent non-cancerous tissues, but liver total RNA levels were lower in the former than in the latter. Although the circulating telomerase of HCC patients was abnormally expressed among patients with chronic liver diseases, the telomerase activity was a non-specific marker for HCC diagnosis, because the incidence was 15.7% in normal control, 25% in chronic hepatitis, 45.9% in liver cirrhosis, and 85.2% in HCC, respectively when absorbance value of telomerase activity was more than 0.2. If the value was over 0.6, the incidence was 60% in HCC group and 0% in any of the others （P 〈 0.01） except in two cases with liver cirrhosis. However, the combination of circulating telomerase with serum alpha-fetoprotein level could increase the positive rate and the accuracy （92.6%, 125 of 135） of HCC diagnosis. CONCLUSION： The overexpression of telomerase is associated with HCC development, and its abnormality in liver tissues or in peripheral blood could be a useful marker for diagnosis and prognosis of HCC.

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 m RNA level by reverse transcription-quantitative polymerase chain reaction(RT-q PCR) 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-sh RNA was used to knockdown 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 m RNA levels were correlated with cell cycle and DNA replication pathways. Knocking down HMGB3 by specific sh RNA significantly inhibited proliferation of Hep G2 cells by cell cycle arrest and downregulating DNA replication related genes(cyclin B1, FEN1, and PCNA) at the m RNA 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.

Mitochondrial carnitine palmitoyl transferase-Ⅱ inactivity aggravates lipid accumulation in rat hepatocarcinogenesis

AIM To investigate the dynamic alteration of mitochondrial carnitine palmitoyl transferase Ⅱ(CPT-Ⅱ) expression during malignant transformation of rat hepatocytes.METHODS Sprague-Dawley male rats were fed with normal, high fat(HF), and HF containing 2-fluorenylacetamide(2-FAA) diet, respectively. According to the Hematoxylin and Eosin staining of livers, rats were divided into control, fatty liver, degeneration, pre-cancerous, and cancerous groups. Liver lipids were dyed with Oil Red O, CPT-Ⅱ alterations were analyzed by immunohistochemistry, and compared with CPT-Ⅱ specific concentration(μg/mg protein). Levels of total cholesterol(Tch), triglyceride(TG), and aminotransferases [alanine aminotransferase(ALT), aspartate aminotransferase(AST)] were determined by the routine methods.RESULTS After intake of HF and/or HF+2-FAA diets, the rat livers showed mass lipid accumulation. The lipid level in the control group was significantly lower than that in other groups. The changes of serum TG and Tch levels were abnormally increasing, 2-3 times more than those in the controls(P < 0.05). During the rat liver morphological changes from normal to cancer development process with hepatocyte injury, serum AST and ALT levels were significantly higher(4-8 times, P < 0.05) than those in the control group. The specific concentration of CPT-Ⅱ in liver tissues progressively decreased during hepatocyte malignant transformation, with the lowest CPT-Ⅱ levels in the cancer group than in any of the other groups(P < 0.05).CONCLUSION Low CPT-Ⅱ expression might lead to abnormal hepatic lipid accumulation, which should promote the malignant transformation of hepatocytes.

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( r s =-0.89, P < 0.001) or liver IGF-II level( r s =-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.

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.

Oncogenic Wnt3a expression as an estimable prognostic marker for hepatocellular carcinoma

AIM: To investigate member 3a of Wingless-type MMTV integration site family (Wnt3a) expression in cancerous and surrounding tissues and the relationship between clinicopathologic features of hepatocellular carcinoma (HCC) and Wnt3a expression.METHODS: Wnt3a expression and cellular distribution and clinicopathologic characteristics in cancerous tissue and matched surrounding tissues were analyzed in 80 HCC patients from January 2006 to August 2008 by tissue microarrays and immunohistochemistry. The overall and disease-free survival rates were estimated using the Kaplan-Meier method and compared with the log-rank test. The prognostic analysis was carried out with univariate and multivariate Cox regressions models.RESULTS: The incidence of oncogenic Wnt3a expression in the cancerous group was up to 96.25% (77 of 80), which was significantly higher (χ2 = 48.818, P < 0.001) than that in the surrounding group (46.25%, 37 of 80). Brown Wnt3a staining gradually increased with clinical staging that showed very strong staining in advanced HCC. The clinicopathologic features of high Wnt3a expression in HCC were related to poorly-differentiated grade (χ2 = 20.211, P < 0.001), liver cirrhosis (χ2 = 8.467, P < 0.004), hepatitis B virus (HBV) infection (χ2 = 12.957, P < 0.001), higher tumor-node-metastasis stage (χ2 = 22.960, P < 0.001), and 5-year survival rate (χ2 = 15.469, P < 0.001).CONCLUSION: Oncogenic Wnt3a expression associated with HBV infection and cirrhotic liver might be an independent prognostic factor for HCC.

Reversal of multidrug resistance of hepatocellular carcinoma cells by metformin through inhibiting NF-κB gene transcription

AIM: To interfere with the activation of nuclear factor-κB(NF-κB) with metformin and explore its effect in reversing multidrug resistance(MDR) of hepatocellular carcinoma(HCC) cells.METHODS: Expression of P-glycoprotein(P-gp) and NF-κB in human HepG 2 or HepG 2/adriamycin(ADM) cells treated with pC MV-NF-κB-small interference RNA(siR NA) with or without metformin, was analyzed by Western blot or fluorescence quantitative PCR. Cell viability was tested by CCK-8 assay. Cell cycle and apoptosis were measured by flow cytometry and Annexin-V-PE/7-AnnexinV apoptosis detection double staining assay, respectively. RESULTS: P-gp overexpression in HepG 2 and HepG 2/ADM cells was closely related to mdr1 mR NA(3.310 ± 0.154) and NF-κB mR NA(2.580 ± 0.040) expression. NF-κB gene transcription was inhibited by specific siR NA with significant down-regulation of P-gp and enhanced HCC cell chemosensitivity to doxorubicin. After pretreatment with metformin, Hep G2/ADM cells were sensitized to doxorubicin and P-gp was decreased through the NF-κB signaling pathway. The synergistic effect of metformin and NF-κB siR NA were found in HepG 2/ADM cells with regard to proliferation inhibition, cell cycle arrest and inducing cell apoptosis. CONCLUSION: Metformin via silencing NF-κB signaling could effectively reverse MDR of HCC cells by downregulating MDR1/P-gp expression.

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 thepresent study, we investigated the expression of hepatic Wnt5a in HCC and the role of Wnt5a in HCCprogression and outcome.

Chemosensitization of HepG2 cells by suppression of NF-κB/p65 gene transcription with specific-si RNA

AIM: To investigate small interfering RNA(si RNA)-mediated inhibition of nuclear factor-kappa B(NF-κB) activation and multidrug-resistant(MDR) phenotype formation in human Hep G2 cells. METHODS: Total RNA was extracted from human Hep G2 or LO2 cells. NF-κB/p65 m RNA was amplified by nested reverse transcription polymerase chain reaction and confirmed by sequencing. NF-κB/p65 was analyzed by immunohistochemistry. Specific-si RNA was transfected to Hep G2 cells to knock down NF-κB/p65 expression. The effects on cell proliferation, survival, and apoptosis were assessed, and the level of NF-κB/p65 or P-glycoprotein(P-gp) was quantitatively analyzed by enzyme-linked immunosorbent assay.RESULTS: Hep G2 cells express NF-κB/p65 and express relatively less phosphorylated p65(P-p65) and little P-gp. After treatment of Hep G2 cells with different doses of doxorubicin, the expression of NF-κB/p65, P-p65, and especially P-gp were dose-dependently upregulated. After Hep G2 cells were transfected with NF-κB/p65 si RNA(100 nmol/L), the expression of NF-κB/p65, P-p65, and P-gp were downregulatedsignificantly and dose-dependently. The viability of Hep G2 cells was decreased to 23% in the combination NF-κB/p65 si RNA(100 nmol/L) and doxorubicin(0.5 μmol/L) group and 47% in the doxorubicin(0.5 μmol/L) group(t = 7.043, P < 0.001). CONCLUSION: Knockdown of NF-κB/p65 with si RNA is an effective strategy for inhibiting Hep G2 cell growth by downregulating P-gp expression associated chemosensitization and apoptosis induction.

Abnormal CD44 activation of hepatocytes with nonalcoholic fatty accumulation in rat hepatocarcinogenesis

BACKGROUND Prevalence of nonalcoholic fatty liver disease(NAFLD)is rapidly increasing,and NAFLD has become one of the most common chronic liver diseases worldwide.With abnormal CD44 activation,the severe form of NAFLD can progress to liver cirrhosis and hepatocellular carcinoma(HCC).Thus,the molecular mechanism of CD44 in NAFLD needs to be identified.AIM To investigate the relationship between CD44 activation and malignant transformation of rat hepatocytes under nonalcoholic lipid accumulation.METHODS Sprague-Dawley rats were fed a high-fat(HF)for 12 wk to entice NAFLD and then with HF plus 2-fluorenylacetamide(0.05%)to induce HCC.Rats were sacrificed every 2 wk,and subsequently divided into the groups based on liver pathological examination(hematoxylin and eosin staining):NAFLD,denaturation,precancerosis,HCC,and control.Liver CD44 mRNA was detected by OneArray.Liver fat as assessed by Oil red O staining or CD44 by immunohistochemical assay was compared with their integral optic density.Serum CD44,alanine aminotransferase,aspartate aminotransferase,triglyceride,total cholesterol,and AFP levels were quantitatively tested.RESULTS Elevated CD44 was first reported in hepatocarcinogenesis,with increasing expression from NAFLD to HCC at the protein or mRNA level.The CD44 integral optic density values were significantly different between the control group and the NAFLD(t=25.433,P<0.001),denaturation(t=48.822,P<0.001),precancerosis(t=27.751,P<0.001),and HCC(t=16.239,P<0.001)groups,respectively.Hepatic CD44 can be secreted into the blood,and serum CD44 levels in HCC or precancerous rats were significantly higher(P<0.001)than those in any of the other rats.Positive correlations were found between liver CD44 and CD44 mRNA(rs=0.373,P=0.043)and serum CD44(rs=0.541,P=0.002)and between liver CD44 mRNA and serum CD44(rs=0.507,P=0.004).Moreover,significant correlations were found between liver CD44 and liver AFP(rs=0.572,P=0.001),between serum CD44 and serum AFP(rs=0.608,P<0.001),and between CD44 mRNA and AFP mRNA(rs=0.370,P=0.044).CONCLUSION The data suggested that increasing CD44 expression is associated with the malignant transformation of hepatocytes in NAFLD.

Oncogenic tuftelin 1 as a potential molecular-targeted for inhibiting hepatocellular carcinoma growth

BACKGROUND Abnormal tuftelin 1(TUFT1)has been reported in multiple cancers and exhibits oncogenic roles in tumor progression.However,limited data are available on the relationship between TUFT1 and hepatocellular carcinoma(HCC),and the exact biological mechanism of TUFT1 is still poorly understood in HCC.AIM To investigate TUFT1 expression in HCC and how interfering TUFT1 transcription affects HCC growth.METHODS TUFT1 in HCC and non-HCC tissues based on databases of the Cancer Genome Atlas and Oncomine were analyzed,and TUFT1 in human HCC tissues on microarray were detected by immunohistochemistry for clinicopathological features,overall survival,and disease-free survival.HCC cells were transfected with constructed vectors of TUFT1 that interfere or over-express TUFT1 for analyzing the biological behaviors of HCC cells.Proliferation,invasion,migration,and apoptosis of cells were detected by cell counting kit-8,scratch assay,transwell tests,and flow cytometry and confirmed by Western blotting,respectively.RESULTS Abnormal TUFT1 levels in databases expressed in HCC at messenger RNA(mRNA)level and HCC tissues were mainly located in cytoplasm and membrane.The level of TUFT1 expression in the HCC group was significantly higher(χ2=18.563,P<0.001)than that in the non-cancerous group,closely related to clinical staging,size,vascular invasion of tumor,hepatitis B e-antigen positive,and ascites(P<0.01)of HCC patients,and negatively to HCC patients’overall survival and disease-free survival(P<0.001).After interfering with TUFT1 transcription at mRNA level in the MHCC-97H cells by the specific TUFT1-short hairpin RNA,cell proliferation,invasion,and metastasis were significantly inhibited with increasing apoptosis rate.In contrast,proliferation,invasion,and migration were significantly enhanced after over-expression of TUFT1 mRNA in Hep3B cells in vitro.CONCLUSION Oncogenic TUFT1 was associated with the progression of HCC and could be a potential molecular-target for inhibiting HCC growth.