Prognostic nomogram for hepatocellular carcinoma patients after transarterial chemoembolization based on des-γ-carboxy prothrombin reactivity and modified Response Evaluation Criteria in Solid Tumors.

AIMS: The aim of this study was to construct a nomogram that will predict the overall survival (OS) of hepatocellular carcinoma (HCC) patients after transarterial chemoembolization (TACE). MATERIALS AND METHODS: Imaging data, clinical characteristics, and serum des-γ-carboxy prothrombin (DCP) levels of 93 HCC patients treated with TACE were collected. Lasso regression, random forest, and other methods were used to screen the OS-related variables and construct the Cox prognosis model. The model was visualized by nomogram, and the net benefit of the clinical decision was assessed by decision curve analysis (DCA). RESULTS: It was found that DCP level after TACE was an important predictor of OS in HCC patients. The OS of the patients with lower serum DCP levels after TACE was significantly better than the group with higher levels (P = 0.003). The Cox prognostic model was constructed using four predictors including DCP reactivity (P = 0.001), modified Response Evaluation Criteria in Solid Tumors (mRECIST, P = 0.005), Child-Pugh class (P = 0.018), and portal vein thrombosis (P = 0.039). The C-index of the nomogram for OS of patients after TACE was 0.813. The clinical decision-making net benefits based on the nomogram were better than the decision-making based on the TNM stage system. CONCLUSION: DCP reactivity and mRECIST are the key predictors of prognosis in HCC patients that received TACE as their initial treatment. The nomogram constructed with these two indicators as the core could predict the OS of HCC patients after TACE and help in clinical decision-making.

Annexin A2 promotion of hepatocellular carcinoma tumorigenesis via the immune microenvironment.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer with a dismal prognosis, especially when diagnosed at advanced stages. Annexin A2 (ANXA2), is found to promote cancer progression and therapeutic resistance. However, the underlining mechanisms of ANXA2 in immune escape of HCC remain poorly understood up to now. Herein, we summarized the molecular function of ANXA2 in HCC and its relationship with prognosis. Furthermore, we tentatively elucidated the underlying mechanism of ANXA2 immune escape of HCC by upregulating the proportion of regulatory T cells and the expression of several inhibitory molecules, and by downregulating the proportion of natural killer cells and dendritic cells and the expression of several inhibitory molecules or effector molecules. We expect a lot of in-depth studies to further reveal the underlying mechanism of ANXA2 in immune escape of HCC in the future.

MicroRNA-30c inhibits metastasis of ovarian cancer by targeting metastasis-associated gene 1.

BACKGROUND: It is important to find reliable molecular markers or biological targets that associate with ovarian cancer (OC) metastasis for diagnosis and treatment. In this study, researchers investigated the regulated chain of microRNA-30c (miR-30c) and metastasis-associated gene 1 (MTA1) in OC tissues and cells. MATERIALS AND METHODS: Expression of miR-30c and MTA1 was detected with quantitative real-time polymerase chain reaction and immunohistochemistry in 33 OC and matched adjacent tissues. MiR-30c mimics were synthetized and transfected into SKOV3 cells to target MTA1. The wound healing and transwell assays were detected to observe migration and invasion of transfected OC cells. RESULTS: Compared with matching normal ovarian tissues, the MTA1 expression was upregulated and localized in the cytoplasm, and the expression of miR-30c was significantly reduced. The expression intensity of MTA1 was correlated with the Federation of Gynecology and Obstetrics stage, tumor grade, and metastasis of OC. Transfecting miR-30c mimics could significantly reduce the expression of MTA1 in SKOV3 cells and obviously inhibit the migration and invasion of SKOV3 cells. CONCLUSION: MiR-30c and MTA1 abnormally expressed in OC, which may be related to metastasis of OC. In MiR-30c as a tumor suppressor gene, its expression in OC could lead to reduced expression of MTA1, which may be one of the mechanisms of metastasis of OC cells.

[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.

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 real-time 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 (X2=6.318, P=0.012) or HBV infection (X2=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 of circulating 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.

[Correlation between epigenetic alterations in the insulin growth factor-II gene and hepatocellular carcinoma].

To investigate whether epigenetic alterations in the insulin-like growth factor-II (IGF-II) gene that cause differential transcription or expression are correlated with onset and severity of hepatocellular carcinoma (HCC). Patient-matched specimens of HCC, paracancerous, and non-cancerous tissues were collected from 40 primary liver cancer patients. Epigenetic alterations in the promoter (P3) sequence of the IGF-II gene were analyzed by methylation-specific PCR (MSP) and IGF-II transcription was measured by RT-PCR. IGF-II protein expression and clinicopathological features were assessed by immunohistochemistry and microscopic observation. The rate of IGF-II P3 methylation was significantly lower in HCC tissues (0%) than in paracancerous tissues (vs. 47.5%; x2 = 24.918, P less than 0.001) and non-cancerous tissues (vs. 100%; x2 = 80.000, P less than 0.001). IGF-II mRNA expression was significantly higher in HCC tissues (100%) than in paracancerous tissues (vs. 52.5%; x2 = 24.918, P less than 0.001) and non-cancerous tissues (vs. 0%; x2 = 80.000, P less than 0.001). IGF-II protein expression was significantly higher in HCC tissues (82.5%) than in paracancerous tissues (vs. 45.0%; x2 = 12.170, P less than 0.001) and non-cancerous tissues (vs. 0%; x2 = 56.170, P less than 0.001). IGF-II overexpression in HCC was significantly associated with degree of differentiation, extent of infiltrated serosa, size of tumor, and HBV-positive infection status. Epigenetic alterations in the IGF-II gene regulate its transcription and expression and are closely associated with HCC development and progression.

Oncofetal antigen glypican-3 as a promising early diagnostic marker for hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is characterized by a multi-cause, multi-stage and multi-focus process of tumor progression. Its prognosis is poor and early diagnosis is of utmost importance. This study was undertaken to investigate the dynamic expression of oncofetal antigen glypican-3 (GPC-3) and GPC-3 mRNA in hepatocarcinogenesis and to explore their early diagnostic value for HCC. METHODS: A hepatoma model was induced in male Sprague-Dawley rats with 0.05% 2-fluorenylacetamide and confirmed by hematoxylin and eosin staining and gamma-glutamyltransferase (GGT) expression. Total RNA was purified and transcribed into cDNA by reverse transcription. Fragments of the GPC-3 gene were amplified by nested RT-PCR, and confirmed by sequencing. GPC-3 was analyzed by immunohistochemistry, Western blotting or ELISA. RESULTS: Positive GPC-3 expression showed as brown granule-like staining localized in the cytoplasm. Histological examination of hepatocytes revealed three morphological stages of granule-like degeneration, atypical hyperplasia (precancerous), and cancer formation, with a progressive increase of liver total RNA and GGT expression. The incidence of liver GPC-3 mRNA and GPC-3, and serum GPC-3 was 100%, 100% and 77.8% in the HCC group, 100%, 100%, and 66.7% in the precancerous group, 83.3%, 83.3%, and 38.9% in the degeneration group, and no expression in the liver or blood of the control group, respectively. There was a positive correlation between liver GPC-3 mRNA and total RNA level (r=0.475, P<0.05) or liver GPC-3 (r=1.0, P<0.001) or serum GPC-3 (r=0.994, P<0.001). CONCLUSION: Abnormal oncofetal antigen GPC-3 and GPC-3 mRNA expression in hepatocarcinogenesis may be promising molecular markers for early diagnosis of HCC.

[Expression features of glypican-3 and its diagnostic and differential values in hepatocellular carcinoma].

OBJECTIVE: To investigate the expression features of glypican-3 (GPC-3) and its diagnostic and differential values in hepatocellular carcinoma (HCC). METHODS: Rat hepatoma models were made and the dynamic expression features of GPC-3 protein and its gene were investigated by Western blotting and RT-PCR respectively. Liver specimens from 36 HCC patients were collected by self-control method and the expression and clinicopathological features of GPC-3 were analyzed by immunohistochemistry. Serum GPC-3 levels were quantitatively detected by ELISA and its efficiency for HCC diagnosis was evaluated in patients with liver diseases. RESULTS: The incidence of GPC-3 was 0% in control, 83.3% in degeneration, 100% in precancerosis and 100% in canceration during dynamic formation of rat hepatoma, respectively. The positive GPC-3 was brown granule- like staining localized in membrane and cytoplasm in human HCC. CONCLUSIONS: The GPC-3 positive rates were 80.6% in HCC, 41.7% in surrounding tissues and none in distal tissues (P < 0.01), respectively. No positive relationship presented between GPC-3 and differentiation grade or the number of tumor except of tumor size (Z = 2.941, P < 0.01). The incidence of serum GPC-3 was 52.8% in HCC patients except of one patient with cirrhosis. No significant differences were found between GPC-3 and sex, age, AFP, tumor number, Child classification or extrahepatic metastasis except of tumor size (χ² = 6.318, P < 0.05) and HBV infection (χ² = 23.362, P < 0.01). Combined detection of GPC-3 and AFP could rise up diagnosis of HCC. GPC-3 expression closely associated with HCC and might be useful for early diagnosis of HCC.

[Inhibitory effect of miRNA silencing hypoxia-inducible factor alpha subunit gene on the proliferation of HepG2 cells].

OBJECTIVE: To investigate the effect of miRNA silencing HIF-1α gene on the proliferation of HepG2 cells. METHODS: The eukaryotic expression plasmids of HIF-1α miRNA and report gene containing hypoxia-reponse element were constructed and transfected into HepG2 cells. The expressions of HIF-1α gene and protein were determined by real time-PCR and Western blotting. The expressions of HIF-1α, vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) were quantitatively detected by ELISA. The alterations of cell cycles and apoptosis rate were quantitatively measured by flow cytometry and Annexin V-FITC/PI double dyeing assay. RESULTS: 72 h after transfection the down regulations of HIF-1α mRNA and protein were 87% and 56% respectively, and the decrease of target gene was 46% in the report gene, 54% in VEGF and 36% in Ang-2, respectively. The apoptotic ratio of HepG2 cells was 22.46+/-0.61% (P < 0.01). The cell cycle changed greatly at the ratio of G1 (61.49+/-1.12%) and S (22.40+/-0.58%, P < 0.01). After being combined with doxorubicin, the apoptotic ratio increased to 36.99+/-0.88% and the ratios of G1 and S phases were upregulated to 65.68+/-0.91% and 19.47+/-1.34% respectively. CONCLUSIONS: HIF-1α miRNA or / and doxorubicin can regulate the growth cycles of HepG2 cells, promote the cell apoptosis and inhibit the cell proliferation.

[Effect of siRNA-mediated inhibition of nuclear transcription factor-kappa B on apoptosis of hepatocarcinoma cells].

OBJECTIVE: To investigate the effect of siRNA-mediated inhibition of NF-κB on apoptosis of hepatocarcinoma cells. METHODS: Specific small interfering RNA Targeting NF-κB gene was synthesized and transfected into HepG2 cells by liposomes. Nested RT-PCR and quantitative RT-PCR were used to detect the mRNA expression of NF-κB. Immunohistochemistry, enzyme-linked immunosorbent assay and Western blot were performed to examine the protein expression of NF-κB. Annexin V-FITC was used to test cell apoptosis. RESULTS: The expression of NF-κB in HepG2 cells (1.13+/-0.03) was significantly higher (t=27.02, P<0.05) than that in normal hepatocytes (0.29+/-0.07). The down-regulation of NF-κB expression was depended on the dosage of siRNA and the time after transfection. 72 h after siRNA transfection, NF-κB expression reduced by 93% and 62% at the mRNA and protein levels, respectively. The apoptosis of HepG2 cells increased by 85% with NF-κB inhibition. CONCLUSIONS: NF-κB is abnormally active in HepG2 cells and NF-κB inhibition mediated by siRNA promotes HepG2 cells apoptosis. It suggested that NF-κB could be a potential target for HCC prevention gene therapy.

[Expression characteristics of hypoxia inducible factor-1a and its clinical values in hepatocellular carcinoma].

OBJECTIVE: To investigate the dynamic expression of hypoxia inducible factor-1alpha (HIF-1alpha) and its clinical values in hepatocellular carcinoma (HCC). METHODS: The dynamic changes of liver pathology, HIF-1alpha transcription and expression were observed through the hepatoma model. The self-control specimens from 35 human HCC patients were collected and the expression, cellular distribution, and clinicopathological features of HIF-1alpha and its gene was analyzed by immunohistochemistry, western blotting and nested- PCR, respectively. RESULTS: Both levels of hepatic HIF-1alpha and HIF-1alpha mRNA expression increased during the HCC development course. The incidence of HIF-1alpha and the ratio of HIF-1alpha to beta-actin was 0% and 0.16+/-0.02 in the control rats, 77.8% and 0.29+/-0.04 in the denatured rats, 88.9% and 0.52+/-0.03 in the precancerous rats, and 100% and 0.84+/-0.02 in the cancerous rats respectively, with significant difference between the control group and any of the experimental groups (P = 0.000). The positive HIF-1alpha was brown and granule-like and mainly presented in cytoplasm and few in nucleus. The incidence of HIF-1alpha was 80% (28/35) in HCC and 100% (35/35) in its surrounding tissues. The clinical pathological features indicated HIF-1alpha expression associated with tumor size and differentiation degree the of HCC. No correlation was found between HIF-1alpha and tumor numbers or positive-HBsAg. CONCLUSIONS: HIF-1alpha expression is associated with occurrence and development of HCC, and is perhaps a target molecule for HCC therapy.

Delayed hepatocarcinogenesis through antiangiogenic intervention in the nuclear factor-kappa B activation pathway in rats.

BACKGROUND: The active form of nuclear factor-kappa B (NF-kappaB) is involved in the initiation, generation, and development of hepatocellular carcinoma (HCC), and is up-regulated in inflammation-associated malignancies. We investigated the dynamic expression of NF-kappaB and its influences on the occurrence of HCC through antiangiogenic (thalidomide) intervention in NF-kappaB activation. METHODS: Hepatoma models were induced with 2-fluorenylacetamide (2-FAA, 0.05%) in male Sprague-Dawley rats, and thalidomide (100 mg/kg body weight) was administered intragastrically to intervene in NF-kappaB activation. The pathological changes in the liver of sacrificed rats were assessed after hematoxylin and eosin staining. NF-kappaB mRNA was amplified by RT-nested PCR. The alterations of NF-kappaB and vascular endothelial growth factor (VEGF) expression were analyzed by enzyme-linked immunosorbent assay, immunohistochemistry, and Western blotting. RESULTS: Rat hepatocytes showed denatured, precancerous, and cancerous stages in hepatocarcinogenesis, with an increasing tendency of hepatic NF-kappaB, NF-kappaB mRNA, and VEGF expression, and their values in the HCC group were higher than those in controls (P<0.001). In the thalidomide-treated group, the morphologic changes generated only punctiform denaturation and necrosis at the early or middle stages, and nodular hyperplasia or a little atypical hyperplasia at the final stages, with the expression of NF-kappaB (X2=9.93, P<0.001) and VEGF (X2=8.024, P<0.001) lower than that in the 2-FAA group. CONCLUSION: NF-kappaB is overexpressed in hepatocarcinogenesis and antiangiogenic treatment down-regulates the expression of NF-kappaB and VEGF, and delays the occurrence of HCC.

[Dynamic alterations of VEGF and intervention of its expression on effect of hepatocyte malignant transformation].

OBJECTIVE: To investigate the influences of VEGF expression through the intervention of thalidomide in malignant transformation of hepatocytes. METHODS: Hepatoma model was induced with 2-fluorenyl-acetamide (2-FAA, 0.05%) in male SD rats. And thalidomide was administered intragastrically to block the progress of hepatoma. Some rats were sacrificed at a fortnightly interval. Morphological changes were observed by pathological examinations (HE staining). The VEGF expressions in rat liver tissues were detected by ELISA and immunohistochemistry respectively. RESULTS: Hepatocytes in rats fed with 2-FAA showed vacuole-like denaturations at an early stage, dysplastic nodules appeared at a middle stage and finally progressed to tubercles of cancerous nest. All were the manifestations of highly differentiated hepatocellular carcinoma (HCC). An increasing tendency of hepatic VEGF protein was found for normal liver to precancerous to cancerous tissues during the development of hepatoma. The VEGF levels in hepatoma were significantly higher than those in normal ones. Thalidomide repressed the morphologic change of hepatic cells. And the VEGF level of thalidomide group was lower than those in 2-FAA group. CONCLUSION: Thalidomide can inhibit the hepatic VEGF expression and arrest the development of rat hepatoma.

Characteristics of hepatic nuclear-transcription factor-kappa B expression and quantitative analysis in rat hepatocarcinogenesis.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. We analyzed the expression of nuclear-transcription factor-kappa B (NF-kappaB) during hepatocarcinogenesis in order to evaluate its dynamic expression and its clinical value in the development and diagnosis of HCC. METHODS: Hepatoma models were induced by oral administration of 2-acetamidoflurene (2-FAA) to male Sprague-Dawley rats. Morphological changes were observed after hematoxylin and eosin staining. The cellular distribution of NF-kappaB expression during different stages of cancer development was investigated by immunohistochemistry, and the level of NF-kappaB expression in liver tissues was quantitatively analyzed by ELISA. The gene fragments of hepatic NF-kappaB were amplified by nested-polymerase chain reaction assay. RESULTS: Hepatocytes showed vacuole-like degeneration during the early stages, then had a hyperplastic nodal appearance during the middle stages, and finally progressed to tubercles of cancerous nests with high differentiation. The NF-kappaB-positive material was buff-colored, fine particles localized in the nucleus, and the incidence of NF-kappaB-positive cells was 81.8% in degeneration, 83.3% in precancerous lesions, and 100% in cancerous tissues. All of these values were higher than those in controls (P<0.01). Hepatic NF-kappaB expression and hepatic NF-kappaB-mRNA were also higher during the course of HCC development (P<0.01). CONCLUSION: The NF-kappaB signal transduction pathway is activated during the early stages of HCC development, and its abnormal expression may be associated with the occurrence of HCC.

Quantitative analysis of hepatic hypoxia-inducible factor-1alpha and its abnormal gene expression during the formation of hepatocellular carcinoma.

BACKGROUND: Hepatic hypoxia-inducible factor-1 (HIF-1) is activated in the progression of hepatocellular carcinoma (HCC). This study aimed to investigate the dynamic alterations of HIF-1alpha and its gene expression so as to explore the relationship between HIF-1alpha expression and hepatocarcinogenesis at the early stage of HCC. METHODS: A hepatoma model was made with 2-fluorenyl-acetamide (2-FAA) in male Sprague-Dawley rats. Morphological changes of rat hepatocytes were assessed pathologically (HE staining). The dynamic expression of hepatic and circulating HIF-1alpha was quantitatively analyzed by ELISA. The gene fragments of hepatic HIF-1alpha mRNA were amplified by RT-PCR and confirmed by sequencing. The cellular distribution of hepatic HIF-1alpha expression was confirmed by immunohistochemistry. RESULTS: Histological examination confirmed granule-like degeneration to atypical hyperplasia and HCC development in rat hepatocytes and progressive increases in the levels of hepatic and circulating HIF-1alpha and its gene expression during the course. The levels of HIF-1alpha expression in the liver and blood of rats with hepatoma were significantly higher than those in normal rats and those with degeneration. Immunohistochemical analysis confirmed the positive expression and hepatocyte distribution of HIF-1alpha in the development of rat hepatoma. A positive relationship was found between HIF-1alpha expression in the liver and blood (P<0.01). CONCLUSIONS: The above observations support the hypothesis that the overexpression of HIF-1alpha and its gene are closely associated with the malignant transformation of hepatocytes and play an important role at the stage of hepatocarcinogenesis.

Abnormal expression of insulin-like growth factor-II and its dynamic quantitative analysis at different stages of hepatocellular carcinoma development.

BACKGROUND: Hepatocellular carcinoma (HCC) is characterized by multiple causes, clear multiple stages and a multifocal process of tumor progression related intimately to the overexpression of many cellular factors. The aim of this study was to investigate the dynamic expression of insulin-like growth factor-II (IGF-II) and its abnormal alteration in the early stages of HCC development. METHODS: Hepatoma models were induced by 2-fluorenylacetamide (2-FAA) in male Sprague-Dawley rats. Morphological changes of rat livers were assessed by pathological examination (HE staining). The levels of IGF-II expression in the livers and sera of rats were quantitatively detected by an enzyme-linked immunosorbent assay (ELISA). Simultaneously, the expression and cellular distribution of liver IGF-II were analyzed by immunohistochemistry. RESULTS: Histological examination confirmed that rat hepatocytes showed changes from granule-like degeneration to atypical hyperplasia to HCC, and progressively increasing hepatic IGF-II levels during HCC development. The levels of hepatic or serum IGF-II in HCC tissues and sera were significantly higher than those in normals and rats with degeneration. The immunohistochemical evidence indicated the positive expression and hepatocyte distribution of IGF-II in rat hepatoma. A positive relationship of IGF-II levels was found between liver tissues and sera of experimental rats (P<0.01). CONCLUSION: Hepatic IGF-II may participate in hepatocyte cancer development and detection of IGF-II expression during HCC development could be a useful molecular marker for its early diagnosis.

Clinical impact of plasma TGF-beta1 and circulating TGF-beta1 mRNA in diagnosis of hepatocellular carcinoma.

BACKGROUND: Transforming growth factor-beta (TGF-beta) plays an important role in the regulation of cell growth and differentiation, angiogenesis, extracellular matrix formation, immunosuppression and cancer development. In this study, we investigated the levels of TGF-beta1 and TGF-beta1 mRNA expression, their relationship with HBV replication, and their diagnostic value for hepatocellular carcinoma (HCC). METHODS: Total RNAs were extracted from HCC samples and matched non-tumor tissues, and from peripheral blood mononuclear cells in HCC patients. TGF-beta1 mRNA was amplified by RT-PCR and confirmed by DNA sequencing. The distribution of TGF-beta1 expression was assessed by immunohistochemistry. The clinical characteristics were analyzed between TGF-beta1 and HBV replication. The diagnostic value of circulating TGF-beta1 and TGF-beta1 mRNA levels were investigated in HCC patients. RESULTS: The incidence of hepatic TGF-beta1 expression was 83.3% in HCC samples, 43.3% in the surrounding tissues, 94.7% in the HBV DNA-positive group, and 63.6% in the HBV DNA-negative group. Liver TGF-beta1 expression was associated with the degree of HCC differentiation and the status of HBV replication, but not with the size or number of tumors. Circulating TGF-beta1 level and incidence of TGF-beta1 mRNA were significantly higher in the HCC group than in any group of patients with benign liver disease, with a higher sensitivity of 89.5% and a specificity of 94.0% for HCC diagnosis when circulating TGF-beta1 levels were >1.2 microg/L. No significant correlation was found between TGF-beta1 expression and AFP level or tumor size. Combining TGF-beta1 level and serum AFP raised the detection rate to 97.4%. CONCLUSIONS: Abnormal expression of hepatic TGF-beta1 is associated with the degree of HCC differentiation and HBV replication. Both circulating TGF-beta1 and TGF-beta1 mRNA can be used as sensitive biomarkers for the diagnosis and prognosis of HBV-induced HCC.