【 Abstract 】 Objective Overexpressions of epidermal growth factor (EGF) and EGF receptor have been associated with progression and invasive phenotype of pancreatic cancer. However, the underlying molecular mechanism by which EGF worked in pancreatic cancer cells has not been completely understood. In this study, effect of EGF on the invasion and metastasis of pancreatic cancer cells and its regulatory mechanism were investigated. Methods The effects of EGF on the proliferation, adhesion and invasion of pancreatic cancer cells were detected by WST-1 proliferation assay, adhesion assay and invasive assay, respectively. The activity and expression of MMP-2 and MMP-9 were examined by zymography, Western blot and RT-PCR, respectively. The activity of NF- κ B was examined by EMSA. Results EGF could significantly promote the invasiveness of pancreatic cancer cells but did not affect cell proliferation or adhesion. The expressions of NF- κ B and MMP-9 were significantly increased by EGF, but EGF did not affect the activity and expression of MMP-2. Furthermore, EGF stimulated the NF- κ B binding activity. Pretreatment with NF- κ B inhibitors, pyrrolidine dithiocarbamate (PDTC), could significantly inhibit the activity of NF- κ B induced by EGF. Meanwhile, the EGF-induced expression and activity of MMP-9, as well as cell invasiveness were also inhibited by NF- κ B inhibitor. Conclusion EGF could increase the expression and promote the invasiveness of MMP-9 via the activation of NF- κ B in pancreatic cancer cells, which implies that NF- κ B inhibitant, such as PDTC, may diminish the invasiveness of pancreatic cancer cells.
ObjectiveTo explore the expression of MSI2 in gastric cancer and its association with clinical significance.
MethodsThe expression level of MSI2 mRNA in gastric cancer tissues and para-carcinoma tissues was detected by Real-time PCR to explore its clinical significance. The expression level of MSI2 protein was detected by Western blotting. The prognosis of patients with the ratio of MSI2 mRNA expression level in gastric cancer tissues and adjacent normal tissues was more than 2 times and below 2 times were compared.
ResultsThere was no significant difference between the expressions of MSI2 mRNA in cancer tissues and para-carcinoma tissues(P > 0.05). The expression level of MSI2 mRNA were associated with the invasion depth (P=0.017), TNM stage (P=0.028), differentiation (P=0.020), and size (P=0.030) of tumor, but no association with other clinical factors such as gender, age, and location were found. The overall survival of the patients with high expression of MSI2 mRNA was significantly shorter than that of the patients with low expression of MSI2 mRNA (χ2=4.221, P=0.040).
ConclusionMSI2 expression is associated with the gastric cancer invasion, TNM stage and differentiation, and the patients with higher expression of MSI2 mRNA have poor prognosis, which makes it possible to be a potential therapeutic target.
For an advanced elucidation of mechanisms of nm23-H1 suppressive effects on metastasis of primary hepatocellular carcinoma (HCC), it is necessary to investigate the correlation between nm23-H1 expression and relative factors involved in the HCC invasion. In present report, full-length cDNA of nm23-H1 was subcloned into pBKCMV vector and transfected into HCC cell line to observe its effects on invasion, cytosolic free Ca2+ and Nras mRNA expression. The results showed that lower expression of N-ras and higher cytosolic free Ca2+ in transfected cell line were detected, while the potential of invasion was depressed. It suggests that the suppressive effects on HCC metastasis might interact with intracellular signal transduction which is essential for stimulating cell invasion.
Objective To detect the expression of KiSS-1 protein in papillary thyroid carcinoma, and to analyze its significance. Methods Paraffin-embedded specimens of 32 patients with thyroid papillary carcinoma and its adjacent cancer tissues were included in this study. Then the expression of KiSS-1 protein was detected by munohistochemistry and its relationship with clinical pathological features was analyzed. Results KiSS-1 protein mainly expressed in the cell membrane and cytoplasm. The expression of KiSS-1 protein was positive in adjacent tissues, but decreased or absent in cancer tissues in 32 patients. In the latter, there were 11 cases with positive expression (34.4%) and 21 cases with negative expression (65.6%), and the difference was statistically significant (χ2=31.256, Plt;0.001). The average value of KiSS-1 protein expression represented by absorbance (A) value (119.595 2) in cancer tissues was higher than that in adjacent tissues (174.805 0), t=34.429, Plt;0.001. The expression of KiSS-1 protein in cancer tissues was not related to patient gender (P=0.618) and age (P=0.061), but except TNM staging (P=0.034). The expression rate of KiSS-1 protein in cancer tissues with lymph node metastasis (4/4, 100%) was significantly higher than that without lymph node metastasis (7/28, 25.0%)， P=0.003. Conclusion The expression of KiSS-1 protein is decreased or absent in papillary thyroid carcinoma, which may be involved in tumorigenesis, invasion, and metastasis.
Objective To investigate the expression of c-met in tall cell variant of papillary thyroid carcinoma, and to compare it with other types of thyroid carcinoma and benign thyroid tissue. Methods The expressions of c-met in 60 cases of thyroid specimens were tested by immunohistochemical staining. Results The levels of expressed c-met in tall cell variant specimens were significantly higher than those in other types of papillary thyroid carcinoma and benign thyroid tissue. c-met expressions were significantly different in the following pairs of types: tall cell variant vs common papillary carcinoma of thyroid (P=0.000 1), tall cell variant vs follicular variant papillary thyroid carcinoma (P=0.000 1), and tall cell variant vs benign thyroid tissue (P=0.000 1). In addition, for all types of papillary carcinomas evaluated, c-met expression was significantly higher in specimens with extracapsular spread (P=0.010 0) and skeletal muscle invasion (P=0.020 0). Conclusion The high expression of c-met is a significant marker for tall cell variant papillary carcinoma of thyroid and its invasive behavior. This finding may explain the unusually aggressive behavior of this tumor and suggest a role for c-met in the early identification of patients with tall cell variant thyroid carcinoma.
ObjectiveTo investigate the influence of EZH2 gene down-regulation by RNA interference on the proliferation and invasion of human glioma cell line U251.
MethodsThe recombinant plasmid of small hairpin RNA targeting EZH2 gene was constructed, and transfected into gioma U251 cells by electroporation. The expression of EZH2 mRNA and protein in the cells was detected by using reverse transcriptase-polymerase chain reaction and Western blot respectively; the viability of cells was determined by using methyl thiazol tetrazo1ium assay; and the invasiveness of U251 cells was tested by Transwell cabin.
ResultsThe expression levels of EZH2 mRNA in U251 cells were detected in a significantly lower proportion in the EZH2-shRNA group (0.19±0.02) than that in the untransfected group (1.13±0.05) and the control-shRNA-GFP group (1.15±0.05). The expression levels of EZH2 protein in U251 cells were detected in a significantly lower proportion in the EZH2-shRNA group (0.20±0.02) than that in the untransfected group (1.03±0.03) and the control-shRNA-GFP group (0.97±0.06). The proliferation rates in EZH2-shRNA group were significantly decreased as compared with those in the untransfected group and control-shRNA-GFP group (24 hours after transfection:60.13%±3.15%, 100.00%±9.31%, 100.03%±9.35%; 48 hours after transfection:53.01%±3.14%, 100.00%±9.13%, 99.58%±9.27%; P<0.05) and Transwell cabin suggested that the invasiveness of U251 cells was significantly decreased (46.00±2.82, 60.67±5.71, 61.00±2.48; P<0.01).
ConclusionEZH2-targeted RNA interference can reduce the proliferation and invasion of human glioma cells, which suggests that EZH2 shRNA may be a potential gene therapeutic target of human glioma.
Objective To examine the effects of newly designed LY52 on the expression of matrix metalloproteinases and invasive ability of hepatocellular carcinoma HepG2 cells. Methods The effects of LY52 on the proliferations of HepG2 cells were detected by MTT assay. Gelatin zymography and Western blot were used to detect the effects of LY52 on matrix metalloproteinase-2 expression in the cell line. Transwell chamber assay was used to detect the effects of LY52 on the invasion of the cells. Results No obvious inhibitory or cytotoxicity effects of LY52 was found in lower concentrations (lt;200 μg/ml) of LY52. Gelatin zymography and Western blot showed that matrix metalloproteinase-2 expression were inhibited by LY52 in a dose-dependent manner in HepG2 cells. Furthermore, transwell chamber assay showed that LY52 could significantly inhibit the invasion of the cell line in a dose-dependent manner.Conclusion The results suggest that LY52 may inhibit the invasion of hepatocellular carcinoma cells by suppressing the matrix metalloproteinase-2 activity.
ObjectiveTo investigate effect of small interfering RNA (siRNA) targeting inhibition of growth hormone receptor (GHR) on proliferation and invasion of human liver cancer line SMMC-7721.
MethodsSMMC-7721 cells were transfected with siRNA targeting human GHR by GenMuteTM transfection regent.The cells were divided into three groups:blank control group (non-transfected siRNA),negative control group (transfected with non-specific siRNA),and specificity transfected group (transfected with expression specifically interfere by GHR siRNA).the relative expression of GHR mRNA was detected by using real-time PCR.the expression of GHR protein was detected by using Western blot.The cell proliferation was assessed by CCK-8 assay.And the ability of invasion was examined by Transwell assay.
ResultsThe expressions of GHR mRNA and GHR protein in the specificity transfected group were significantly lower than those in the blank control group (P<0.05) and the negative control group (P<0.05).Compared with the blank control group and the negative control group,the absorbance value and the number of migrating cells of SMMC-7721 cells were decreased obviously (P<0.05) in the specificity transfected group.
ConclusionsiRNA targeting human GHR could reduce capability of proliferation migration and invasion of SMMC-7721 cells.
ObjectiveTo investigate the effect and mechanism of silent information regulator 1 (SIRT1)in invasion of human gastric cancer.
MethodsThe expressions of SIRT1 protein and vascular endothelial growth factor A (VEGF-A) protein in 46 cases of gastric cancer were tested by immunohistochemical SP method; the effect of expressions of SRIT1 and VEGF-A protein on prognosis of gastric cancer was analyzed by Kaplan-Meier test; the expressions of SRIT1 and VEGF-A protein in human gastric mucosa GES-1 cells and SGC7901 cells were tested by Western blot method; after the interference of siRNA on SIRT1 gene, expressions of SRIT1 and VEGF-A protein were also tested by Western method, and the invasion ability was determined by Transwell test.
ResultsCompared with normal gastric mucosa tissues, expression levels of SIRT1 and VEGF-A protein of gastric tissues were both higher (P < 0.050), and survival situation of patients with SIRT1-positive (P=0.001) or SIRT1-positive and VEGF-A-positive (P=0.006) were both bad, but there was no significant difference on the relationship between prognosis of gastric cancer and expression of VEGF-A protein (P=0.091). Expression levels of SITR1 protein (P=0.010) and VEGF-A protein (P=0.020) in GES-1 cells were both higher than those of SGC7901 cells. In siRNA positive group, expressions of SIRT1 and VEGF-A protein (P=0.010) of SGC7901 cells down-regulated, and invasion ability decreased (P=0.000).
ConclusionsSIRT1 gene may promote the expression of VEGF-A protein and the invasion ability of gastric cancer, it may be a therapeutic target of invasion inhibition for gastric cancer.
Objective To observe the effect of epidermal growth factor (EGF) on the proliferation, adhesion, invasiveness and the activation of nuclear factor-κB (NF-κB), matrix metalloproteinases (MMPs) expression and explore related mechanisms in pancreatic cancer cells. Methods Cell invasion assay, proliferation assay and adhesion assay were used to examine the proliferation, adhesion and invasiveness of pancreatic cancer cells, respectively. NF-κB activity was detected by electrophoretic mobility shift assay (EMSA), and MMPs protein and mRNA expressions were investigated by gelatin zymography, Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR). Results EGF increased the invasiveness of pancreatic cancer cell in a dose-dependent manner (P＜0.05), but did not affect cell proliferation or adhesion. The expressions of MMP-9 mRNA and protein significantly increased after induction by EGF and were highest when EGF concentration was 50 ng/ml, while there was no effect on the expressions of MMP-2 mRNA and protein. Furthermore, NF-κB activity increased with increased concentration of EGF in a concentration-dependent manner (P＜0.05). In addition, NF-κB activity and the expressions of MMP-9 mRNA and protein by pretreatment with both pyrrolidine dithiocarbamate (PDTC) and EGF decreased when compared that by pretreatment with EGF alone. The invasiveness of pancreatic cancer cell by pretreatment with both PDTC and EGF decreased when compared that by pretreatment with EGF alone and nothing (P＜0.05).Conclusion The findings indicate that the NF-κB-mediated MMP-9 induction is essential for EGF-induced invasiveness in pancreatic cancer cells, which can be inhibited by PDTC.
