Abnormal Expression of Fibrinogen Gamma (FGG) and Plasma Level of Fibrinogen in Patients with Hepatocellular Carcinoma

Materials and Methods

Tissue specimens and cell lines used. HCC tissues and adjacent non-cancerous tissues were collected fresh from 37 surgical patients with HCC examined by pathology. All tissues were snap frozen in liquid nitrogen and stored at -80°C. The HCC cell lines SMMC-7721 (Institute of cell biology of CAS, Shanghai, P. R. China) and HepG2 (ATCC, Manassas, USA) were cultured in RPMI-1640 (Gibco BRL, Grand Island, USA) supplemented with 10% fetal bovine serum (Gibco BRL), 100 U/ml penicillin G and 100 μg/ml streptomycin. Cells were incubated at 37°C in a humidified atmosphere with 5% CO₂ in air.

Blood samples and measurement. The blood samples mixed with the anticoagulant sodium citrate (9:1) were collected from 114 HCC patients (92 male, 22 female, aged from 19 to 76 years, mean age 52.5 years) diagnosed histologically and 30 healthy volunteers served as controls (15 male, 15 female, mean age 41.0 years). All samples were centrifuged for 10 minutes at 3,000 rpm and the supernatants were stored at -80°C. Fibrinogen levels were measured in all samples by COULTER ACL-200 automated coagulant analyzer. The details of clinical features of patients are given in Table I.

Clinical stage and tumor thrombosis. The Classification of Malignant Tumours (TNM) developed and maintained by the International Union Against Cancer (UICC) was referred to as our tumor clinical stage classifying criterion for 114 HCC patients: I: solitary tumor without vascular invasion; II: solitary tumor with vascular invasion, III: multiple tumors hepatic invasion and lymphoid metastasis. The presence of tumor thrombosis in a major branch of the portal or the hepatic vein was verified by computed tomography with or without angiography.

Northern blot and semiquantitative RT-PCR. The mRNA of SMMC-7721, HepG2 cells and normal hepatocytes were isolated and purified using an mRNA kit (Qiagen, Santa Clarita, USA) as described by the manufacturer. The detailed method of Northern blot was referred to in our previous work (11). Total RNA of tissues was isolated using RNeasy kit (Qiagen) as described by the manufacturer. The PCR primer of FGG was 5′-CTT AGA TGT AAG ATT CGG TAG-3′ and 5′-TGT TCT GCG AGT AGG AG-3′. In parallel, human β-actin was amplified to serve as a control. All PCR products were analyzed on 1.0% agarose gel followed by quantitative analysis using densitometry (UVIPro analysis system; UVtec, Cambridge, UK).

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Figure 1.

Northern blot analysis of FGG mRNA expression in HCC cell lines. Lane 1, normal hepatocyte; 2, SMMC-7721; 3, HepG2.

Statistical analysis. The results of fibrinogen analysis are reported as mean values ± standard deviation. Comparisons of plasma fibrinogen in different clinical stages were evaluated by Student's t-test. Relationships between categorical variables were compared using the chi-square test. Plasma level of fibrinogen was divided into two groups (positive and negative) according to the normal upper limit (3.4 g l^-1). Odds ratio (OR) as a measure of the relative risk of tumor thrombosis was estimated by means of multivariate logistic regression analysis. Age, gender, HBsAg, Child-Pugh class and α-fetal protein (AFP) were included as covariates. P-values <0.05 were considered significant. All these tests were performed using SPSS 10 software (Chicago, USA).