Gene silencing of heparanase results in suppression of invasion and migration of hepatoma cells.

BACKGROUND: This study investigated the effect of transcriptional gene silencing (TGS) of the heparanase gene on hepatoma SMCC-7721 cells. METHODS: SiRNAs targeting the promoter region and coding region of the heparanase gene were designed and synthesized. Then the siRNAs were transfected into hepatoma SMCC-7721 cells by nuclear transfection or cytoplasmic transfection. The expression of heparanase was detected by RT-PCR and Western blotting 48 h, 72 h and 96 h post-transfection. In addition, wound healing and invasion assays were performed to estimate the effect of TGS of the heparanase gene on the migration and invasion of hepatoma SMCC-7721 cells. RESULTS: Protein and mRNA expression of the heparanase gene were interfered with by TGS or post-transcriptional gene silencing (PTGS) 48 h after transfection. At 72 h post-transfection, the expression of the PTGS group of genes had recovered unlike the TGS group. At 96 h post-transfection, the expression of the heparanase gene had recovered in both the TGS group and PTGS group. Invasion and wound healing assays showed that both TGS and PTGS of the heparanase gene could inhibit invasion and migration of hepatoma SMCC-7721 cells, especially the TGS group. CONCLUSIONS: TGS can effectively interfere with the heparanase gene to reduce the invasion and migration of hepatoma SMCC-7721 cells.

Identification of matrix metalloproteinase 11 as a predictive tumor marker in serum based on gene expression profiling.

PURPOSE: Prognostic markers discovery is a strategy for early diagnosis and individualization therapy for human cancer. In this study, we focus to integrate different methods to identify specific biomarker and elucidate its clinical significance. EXPERIMENTAL DESIGN: A powerful tool named Digital Gene Expression Display online was applied to isolate differentially expressed genes correlated with gastric cancer. Matrix metalloproteinase 11 (MMP11) was selected and confirmed at both mRNA and protein level in 10 cell lines, 123 cases of tumor tissues, and 305 cases of gastric cancer serum specimen by semiquantitative PCR, immunohistochemistry staining, and ELISA techniques, respectively. RESULTS: Our data showed that overexpression of MMP11 at mRNA and protein level was consistently detected in cell lines and primary tumors compared with matched normal tissues. Importantly, serum MMP11 levels were also significantly elevated in gastric cancer patients compared with those of the control subjects (P < 0.001), and the positive expression was well correlated with metastasis in gastric cancer patients (P = 0.009). Furthermore, we have shown that overexpression of MMP11 was associated with the malignant proliferation of AGS cells. CONCLUSIONS: Combination of gene expression profiling and specific clinical resource is a promising approach to validate gene expression patterns associated with malignant phenotype. As a secreted protein, MMP11 may play an important role in carcinogenesis and has potential implication as a biomarker for the diagnosis and prognosis of human cancers including gastric cancer.

[Prognostic role of apoptosis-related gene Fas-1377 and -670 polymorphisms in breast cancer].

OBJECTIVE: To investigate the correlations between Fas-1377 and -670 polymorphisms and survival in Chinese women with breast cancer. METHODS: Polymerase chain reaction-restriction fragment length polymorphism assay (PCR-RFLP) was used to detect the polymorphism of Fas gene in 310 breast cancer patients with a long-term follow-up (median 10.5 years, range 0.2 - 16.1 years). Survival curves were analyzed by Kaplan-Meier method. RESULTS: The polymorphism of neither Fas-1377 nor Fas-670 was significantly correlated with the overall survival in this series of 310 cases (P > 0.05). However, among 146 patients without lymph node metastasis, the 5-year overall survival (OS) rate was significantly lower in the patients with Fas-1377 AA genotype than that in the patients with Fas-1377 GA or GG genotype (OS: 66.7% vs. 95.4%, P = 0.03). Among 117 patients with lymph node metastasis, both the Fas-1377 and Fas-670 polymorphisms were not significantly correlated with OS (P = 0.42). CONCLUSION: Among breast cancer patients without lymph node metastasis, patients with Fas-1377 AA genotype may have a worse survival, while patients with Fas-1377 GA or GG genotype may not be so.

[Alteration of early growth response 1 expression in gastroenterological cancers and its biological significance].

OBJECTIVE: To investigate the expression of early growth response 1 (EGR1) in gastroenterological cancers and its significance in the pathogenesis. METHODS: RT-PCR was used to determine the expression of EGR1 in normal gastric mucosa tissues from 20 non-tumor patients, gastroenterological tumor tissues and matched para-cancer tissues normal morphologically. RT-PCR and Western blotting were used to analyze the mRNA and protein expression of EGR1 in 20 cancer cell lines. Immunohistochemistry (IHC) was preformed to measure the expression level of EGR1 protein on tissue microarray including 179 tumors and 159 normal tissues. RESULTS: EGR1 was overexpressed in gastric cancer (GC) and its matched adjacent normal tissue (ANT), but not expressed or expressed at a low level in the normal gastric mucosa from the non-tumor patients, which was consistent with the GC gene expression profiling data. Overexpression of EGR1 was seen in the 20 cancer cell lines at both mRNA and protein levels. IHC showed strong positive staining of EGR1 protein in the cytoplasm of both tumor tissues and matched normal tissues and showed negative or weaker nuclear staining in the normal gastric mucosa tissues from non-tumor patients. Overexpression of EGR1 was detected in 87% (49/56) of the GC tissues and 79% (43/54) of their ANTs; 83% (43/52) of the hepatocellular carcinoma tissues and 79% (32/42) of their ANTs; 78% (41/52) of colorectal cancer tissues and 63% (22/35) of their ANTs; and 79% (15/19) of the squamous cell carcinoma tissues and 78% (14/18) of their ANTs. CONCLUSION: EGR1 may be correlated with the abnormal proliferation of cells at the early stage of malignant transformation.

Effects of the epigenetic drug MS-275 on the release and function of exosome-related immune molecules in hepatocellular carcinoma cells.

BACKGROUND: Tumor-derived exosomes have been viewed as a source of tumor antigens that can be used to induce anti-tumor immune responses. In the current study, we aim to investigate the regulatory effect of the epigenetic drug MS-275 on hepatoma G2 (HepG2) cell-derived exosomes, especially for their immunostimulatory properties and alteration of some non-specific immune protein expression, such as heat shock protein (HSP) 70, major histocompatibility complex (MHC) class I polypeptide-related sequence A (MICA) and MICB. METHODS: MS-275 was used to modulate the secretion of exosomes in human HepG2 cells, and exosomes from untreated HepG2 cells served as negative controls. RT-PCR was used to test the expression of HSP70, MICA and MICB in HepG2 cells. Immunogold labeling of exosomes and western blotting analysis were carried out to compare the expression of HSP70, MICA and MICB proteins in exosomes with or without MS-275 treatment. A natural killer (NK) cell cytotoxicity assay and peripheral blood mononuclear cell (PBMC) proliferation assay were used to evaluate the effect of MS-275 on the immunostimulatory ability of exosomes. RESULTS: Immunogold labeling and western blot analysis showed that modification of MS-275 increased the expression of HSP70 and MICB in exosomes. RT-PCR showed the mRNA levels of HSP70 and MICB were upregulated in HepG2 cells and were consistent with their protein levels in exosomes. The exosomes modified by MS-275 could significantly increase the cytotoxicity of NK cells and proliferation of PBMC (P < 0.05). CONCLUSIONS: The non-specific immune response of exosomes derived from HepG2 cells could be enhanced with treatment by the histone deacetylase inhibitor (HDACi) drug MS-275; this could provide a potential tumor vaccine strategy against liver cancer.

Prostate stem cell antigen rs2294008 polymorphism differentially contributes to Helicobacter pylori-negative gastric cancer among various populations in China.

Gastric cancer is a lethal disease with a high mortality rate. Studies have suggested that prostate stem cell antigen (PSCA) rs2294008 polymorphism is associated with gastric cancer (GC). In this case-control study, we investigated rs2294008 polymorphism in the Tibet, Hui and Han nationalities in the Qinghai area of China. Genomic DNA was extracted from the peripheral blood of 286, 315 and 350 healthy volunteers and from 219, 233 and 265 Helicobacter pylori-negative non-cardia GC patients from the Tibet, Hui and Han populations, respectively. The rs2294008 polymorphism was analyzed by denaturing high-performance liquid chromatography. rs2294008 CT and TT genotypes were associated with GC both in the Tibet and Han populations (adjusted OR=1.51, 1.47, 2.01, 1.85; 95% CI, 1.04-2.19, 1.05-2.06, 1.04-3.88, 1.03-3.34; P=0.030, 0.025, 0.039, 0.040, respectively). rs2294008 TT genotype was associated with GC in the Hui population (adjusted OR=2.14; 95% CI, 1.29-3.55; P=0.003). Furthermore, when stratified by histopathology, the rs2294008 CT and TT genotypes were associated with diffuse GC in the Tibet and Han nationalities (adjusted OR=1.93, 1.73, 2.69, 2.86; 95% CI, 1.09-3.44, 1.01-2.95, 1.06-6.84, 1.27-6.46; P=0.025, 0.045, 0.038, 0.011, respectively). However, the rs2294008 TT genotype was associated with both intestinal and diffuse types of GC (adjusted OR=2.10, 2.21; 95% CI, 1.17-3.75, 1.12-4.38; P=0.012, 0.023, respectively) and the rs2294008 CT genotype was only associated with intestinal-type GC in the Hui nationalitiy group (adjusted OR=1.60; 95% CI, 1.04-2.47; P=0.034). The results therefore showed that rs2294008 may differentially contribute to GC among different nationalities in one area and its role is independent from Helicobacter pylori-infection.

Associations between interleukin-1 polymorphisms and gastric cancers among three ethnicities.

AIM: To investigate the associations between interleukin (IL)-1B and IL-1RN polymorphisms and gastric cancers among the Tibet, Hui and Han ethnicities. METHODS: Genomic DNA was extracted from peripheral blood of 210, 205, and 202 healthy volunteers and from 155, 158, and 197 gastric cancer patients from the Tibet, Hui, and Han populations, respectively. Polymorphisms in IL-1B and IL-1RN were analyzed by denaturing high-performance liquid chromatography. RESULTS: Carriers of the IL-1B-31 CC genotype had an increased risk of intestinal type gastric cancer [odds ratio (OR) = 2.17, P = 0.037] in the Tibet ethnicity. Carriers of the IL-1B 2/L genotype had an increased risk of both intestinal and diffuse types of gastric cancer (OR = 2.08, 2.31, P = 0.007, 0.016, respectively) in the Hui ethnicity. In the Han population, carriers of the IL-1B-31 CC, IL-1B-511CT, TT genotypes had increased risk of intestinal type gastric cancer (OR = 2.51, 2.74, 5.66, P = 0.005, 0.002, 0.000, respectively). CONCLUSION: IL-1B and IL-RN genotypes may differentially contribute to gastric cancer among the Tibet, Hui, and Han ethnicities in the Qinghai area of China.