| Part One The Expression of CXCR4 in Ovarian Cancer and its Clinical RelationObjectiveTo examine the expression of CXCR4 in malignant epithelial ovarian tumors and ovarian cancer cell lines, to investigate its relationship to clinicopathological features and the expression ofβ-catenin.Methods1. Immunohistochemical analysis was used to detect the expression of CXCR4 andβ-catenin in 49 ovarian cancer tissues and 7 normal ovarian tissues and to analyze the relationship between CXCR4 and clinicopathological features in ovarian cancer. Pearson's analysis was applied to study the correlation of CXCR4 andβ-catenin expression.2. Real-Time PCR method was used to analyze the mRNA expression of CXCR4 in normal ovarian tissues, malignant epithelial ovarian tumors and ovarian cancer cell lines.3. Western blot were used to analyze the protein expression of CXCR4 in normal ovarian tissues, and ovarian cancer cell lines.Results1. Abnormal expression of CXCR4 andβ-catenin were detected in ovarian cancer tissues. There was significant correlation between CXCR4 andβ-catenin expression (r=0.628, P<0.001). The expression of CXCR4 was also correlated to the lymphatic metastasis (P=0.016)2. The mRNA expression of CXCR4 was overexpressed by (6.10±2.4), (6.01±0.3),(4.33±0.3) and(3.62±0.6)times in malignant ovarian tumors and ovarian cancer cell lines CAOV3,SKOV3 and A2780 respectively compared with normal ovarian tissue, the differences were Statistically significant (P<0.001).3. The protein expression of CXCR4 in ovarian cancer cell lines CAOV3,SKOV3 and A2780 were (3.51±0.08),(2.95±0.14) and (2.82±0.10) times higher than in normal ovarian tissue, the differences were Statistically significant (P<0.001).ConclusionCXCR4 was highly expressed in malignant ovarian tumors and ovarian cancer cell lines, correlated to the lymphatic metastasis and expression ofβ-catenin. Part Two The effect of CXCR4 shRNA on the invasion and metastasis in ovarian cancer cell lines CAOV3ObjectiveTo investigate the effect of CXCR4 silencing by RNAi on cell proliferation and invasive ability in ovarian cancer cell lines CAOV3.Methods1. RNAi technique was applied to construct four CXCR4 shRNA expression vector. Lipofectamine 2000 was used to transfect ovarian cancer cell lines CAOV3. Real-Time PCR and Western blot were used to observe the inhibitory effect of RNAi on CXCR4 expression.2. MTT assay was used to detect the influence of specific CXCR4 shRNA on CAOV3 cell proliferation.3. Transwell migration assay was used to detect the effect of CXCR4 knockdown on the invasion ability of CAOV3 cells.Results1. Four CXCR4 shRNA plasmid expression vectors were constructed successfully. Compared with shNC-transfected cell, the expression of CXCR4 mRNA was decreased by 88% (P=0.005),69%(P=0.019) and 91% (P=0.003) in CAOV3 cells tranfected with shRNA-151, shRNA-701 and shRNA-868, respectively, and shRNA-868 was the most effective one among the four shRNAs. Meanwhile, Western blot results showed that shRNA-868 inhibited CXCR4 protein expression significantly, P<0.01.2. MTT results showed that the CXCR4 shRNA inhibited the proliferation of CAOV3 cells significantly, P<0.05.3. Transwell migration assay results showed that CXCR4 silencing inhibited the invasive ability of CAOV3 cells significantly, P<0.01.ConclusionKnockdown of CXCR4 suppressed proliferation and invasive capability of ovarian cancer cells CAOV3. CXCR4 played a critical role in the metastasis of human ovarian cancer.Part Three The Study of CXCR4 Silencing on Modulating Wnt/β-catenin PathwayObjectiveTo investigate the effect of CXCR4 knockdown on expression of Wnt/B-catenin pathway related genes, to reveal the effective mechanism of CXCR4 on ovarian cancer metastasis.Methods1. Real-Time PCR was used to detect the mRNA expression of CTNNB1, a critical gene of Wnt/β-catenin pathway encodingβ-catenin, three Wnt target genes C-MYC, MMP-9 and CD44, two EMT-related genes SLUG and vimentin in CXCR4 knockdown cells and shNC cells.2. Western blot was applied to observe the influence of specific CXCR4 shRNA on invasion-related genes MMP-9 and Vimentin protein expression.Results1. Real-Time PCR results showed that the expression of CTNNB1, MMP-9, CD44, C-MYC, SLUG and vimentin was decreased 90.5%,26.1%,35.4%,93.5%,91.5% and 78.3% in CXCR4 knockdown cells compared with that in shNC cells, respectively,all P<0.05.2. Western blot results indicated that specific CXCR4 shRNA effectively inhibited protein expression of MMP-9 and Vimentin, P<0.01.ConclusionCXCR4 silencing inhibited the expression of Wnt/β-catenin pathway related genes. CXCR4 plays a critical role in the metastasis of human ovarian cancer possibly through modulating the Wnt/β-catenin pathway.
|
PDF Full Text Request