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Plexin-B1 silencing inhibits ovarian cancer cell migration and invasion.

Ye S, Hao X, Zhou T, Wu M, Wei J, Wang Y, Zhou L, Jiang X, Ji L, Chen Y, You L, Zhang Y, Xu G, Zhou J, Ma D, Wang S - BMC Cancer (2010)

Bottom Line: Elevated Plexin-B1 expression has been found in diverse human cancers and in non-neoplastic tissues, and it mediates diverse biological and pathological activities.Expression levels of Plexin-B1 protein were significantly higher in serous ovarian carcinomas than in normal ovaries or benign ovarian neoplasms, and in the former, Plexin-B1 expression was positively correlated with lymphatic metastasis, and the membrane and cytoplasm of cancer cells stained positively.Plexin-B1 siRNA significantly suppressed phosphorylation of AKT at Ser473 in SKOV3 cells, but it did not alter total AKT expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China.

ABSTRACT

Background: Elevated Plexin-B1 expression has been found in diverse human cancers and in non-neoplastic tissues, and it mediates diverse biological and pathological activities. However, whether or not Plexin-B1 expression is involved in human ovarian tumors remains unclear. In the present study, Plexin-B1 expression was explored in benign and malignant human ovarian tumor tissues. In addition, the impact of Plexin-B1 expression on ovarian cancer cell proliferation, migration and invasion were investigated in vitro.

Methods: Plexin-B1 expression was analyzed in normal and benign ovarian tissues and serous ovarian tumors (both borderline and malignant) by immunohistochemical staining, as well as in four human ovarian cancer cell lines (A2780, C13*, SKOV3, and OV2008) by RT-PCR and western blot analyses. Furthermore, endogenous Plexin-B1 expression was suppressed by Plexin-B1 siRNA in SKOV3 cells, which overexpress Plexin-B1. Protein levels of Plexin-B1, AKT and AKTSer473 were examined by western blot analysis. Cell proliferation, migration and invasion were measured with MTT, wound healing and boyden chamber assays, respectively, and the cytoskeleton was monitored via F-actin staining.

Results: Expression levels of Plexin-B1 protein were significantly higher in serous ovarian carcinomas than in normal ovaries or benign ovarian neoplasms, and in the former, Plexin-B1 expression was positively correlated with lymphatic metastasis, and the membrane and cytoplasm of cancer cells stained positively. SKOV3 cells displayed the highest Plexin-B1 expression at both the mRNA and protein levels among the four tested human ovarian cancer cell lines and was selected as a cell model for further in vitro experiments. Plexin-B1 siRNA significantly suppressed phosphorylation of AKT at Ser473 in SKOV3 cells, but it did not alter total AKT expression. In addition, silencing of Plexin-B1 in SKOV3 cells inhibited cell migration and invasion and reorganized the cytoskeleton, whereas cell proliferation was not affected.

Conclusion: Plexin-B1 expression correlates with malignant phenotypes of serous ovarian tumors, probably via phosphorylation of AKT at Ser473, suggesting that Plexin-B1 might be a useful biomarker and/or a novel therapeutic target.

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Effects of Plexin-B1 siRNA on SKOV3 cells. (A) Real-time PCR was performed to detect Plexin-B1 mRNA levels in non-transfected cells (Blank control) and cells at 24 h after a 5 h exposure to negative control siRNA or one of the three different Plexin-B1 siRNAs (Plexin-B1 siRNA1-3). The mean relative level ± SD of each group is shown. Plexin-B1 mRNA levels in three Plexin-B1 siRNA groups were significantly down-regulated (*P < 0.01), while the negative control siRNA did not cause an obvious change. The bar graph shows the results of three independent experiments. (B) Western blot analysis of lysates from untransfected (Blank control) SKOV3 cells and SKOV3 cells at 24 h after a 5-h exposure to one of the three different Plexin-B1 siRNAs (Plexin-B1 siRNA1-3) or to negative control siRNA. The cells were analyzed by immunoblotting with specific antibodies to Plexin-B1, p-AKT (s473), AKT and β-actin. Plexin-B1 expression was significantly down-regulated in each of the three Plexin-B1 siRNA groups relative to the blank control group or the negative control group. p-AKT (s473) expression sequentially decreased in the three Plexin-B1 siRNA groups, and AKT changed impalpably at the protein level. β-actin was the internal loading control. (C) The western blots were scanned and quantified. Data present densitometric analyses of Plexin-B1, p-AKT (s473) or AKT relative to β-actin for n = 5 independent experiments. * indicates P < 0.01 when compared to the blank control.
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Figure 2: Effects of Plexin-B1 siRNA on SKOV3 cells. (A) Real-time PCR was performed to detect Plexin-B1 mRNA levels in non-transfected cells (Blank control) and cells at 24 h after a 5 h exposure to negative control siRNA or one of the three different Plexin-B1 siRNAs (Plexin-B1 siRNA1-3). The mean relative level ± SD of each group is shown. Plexin-B1 mRNA levels in three Plexin-B1 siRNA groups were significantly down-regulated (*P < 0.01), while the negative control siRNA did not cause an obvious change. The bar graph shows the results of three independent experiments. (B) Western blot analysis of lysates from untransfected (Blank control) SKOV3 cells and SKOV3 cells at 24 h after a 5-h exposure to one of the three different Plexin-B1 siRNAs (Plexin-B1 siRNA1-3) or to negative control siRNA. The cells were analyzed by immunoblotting with specific antibodies to Plexin-B1, p-AKT (s473), AKT and β-actin. Plexin-B1 expression was significantly down-regulated in each of the three Plexin-B1 siRNA groups relative to the blank control group or the negative control group. p-AKT (s473) expression sequentially decreased in the three Plexin-B1 siRNA groups, and AKT changed impalpably at the protein level. β-actin was the internal loading control. (C) The western blots were scanned and quantified. Data present densitometric analyses of Plexin-B1, p-AKT (s473) or AKT relative to β-actin for n = 5 independent experiments. * indicates P < 0.01 when compared to the blank control.

Mentions: Three different siRNA duplexes targeting Plexin-B1 and a negative control siRNA were separately transferred into SKOV3 cells. At 24 h after transfection, Plexin-B1 mRNA and protein levels were assessed by real-time PCR and western blot analysis, respectively. As shown in Figure 2A-C, each of the three Plexin-B1 siRNA duplexes significantly reduced the amount of Plexin-B1 mRNA and protein. The data are from densitometric analyses of Plexin-B1: β-actin; n = 5 independent experiments.


Plexin-B1 silencing inhibits ovarian cancer cell migration and invasion.

Ye S, Hao X, Zhou T, Wu M, Wei J, Wang Y, Zhou L, Jiang X, Ji L, Chen Y, You L, Zhang Y, Xu G, Zhou J, Ma D, Wang S - BMC Cancer (2010)

Effects of Plexin-B1 siRNA on SKOV3 cells. (A) Real-time PCR was performed to detect Plexin-B1 mRNA levels in non-transfected cells (Blank control) and cells at 24 h after a 5 h exposure to negative control siRNA or one of the three different Plexin-B1 siRNAs (Plexin-B1 siRNA1-3). The mean relative level ± SD of each group is shown. Plexin-B1 mRNA levels in three Plexin-B1 siRNA groups were significantly down-regulated (*P < 0.01), while the negative control siRNA did not cause an obvious change. The bar graph shows the results of three independent experiments. (B) Western blot analysis of lysates from untransfected (Blank control) SKOV3 cells and SKOV3 cells at 24 h after a 5-h exposure to one of the three different Plexin-B1 siRNAs (Plexin-B1 siRNA1-3) or to negative control siRNA. The cells were analyzed by immunoblotting with specific antibodies to Plexin-B1, p-AKT (s473), AKT and β-actin. Plexin-B1 expression was significantly down-regulated in each of the three Plexin-B1 siRNA groups relative to the blank control group or the negative control group. p-AKT (s473) expression sequentially decreased in the three Plexin-B1 siRNA groups, and AKT changed impalpably at the protein level. β-actin was the internal loading control. (C) The western blots were scanned and quantified. Data present densitometric analyses of Plexin-B1, p-AKT (s473) or AKT relative to β-actin for n = 5 independent experiments. * indicates P < 0.01 when compared to the blank control.
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Figure 2: Effects of Plexin-B1 siRNA on SKOV3 cells. (A) Real-time PCR was performed to detect Plexin-B1 mRNA levels in non-transfected cells (Blank control) and cells at 24 h after a 5 h exposure to negative control siRNA or one of the three different Plexin-B1 siRNAs (Plexin-B1 siRNA1-3). The mean relative level ± SD of each group is shown. Plexin-B1 mRNA levels in three Plexin-B1 siRNA groups were significantly down-regulated (*P < 0.01), while the negative control siRNA did not cause an obvious change. The bar graph shows the results of three independent experiments. (B) Western blot analysis of lysates from untransfected (Blank control) SKOV3 cells and SKOV3 cells at 24 h after a 5-h exposure to one of the three different Plexin-B1 siRNAs (Plexin-B1 siRNA1-3) or to negative control siRNA. The cells were analyzed by immunoblotting with specific antibodies to Plexin-B1, p-AKT (s473), AKT and β-actin. Plexin-B1 expression was significantly down-regulated in each of the three Plexin-B1 siRNA groups relative to the blank control group or the negative control group. p-AKT (s473) expression sequentially decreased in the three Plexin-B1 siRNA groups, and AKT changed impalpably at the protein level. β-actin was the internal loading control. (C) The western blots were scanned and quantified. Data present densitometric analyses of Plexin-B1, p-AKT (s473) or AKT relative to β-actin for n = 5 independent experiments. * indicates P < 0.01 when compared to the blank control.
Mentions: Three different siRNA duplexes targeting Plexin-B1 and a negative control siRNA were separately transferred into SKOV3 cells. At 24 h after transfection, Plexin-B1 mRNA and protein levels were assessed by real-time PCR and western blot analysis, respectively. As shown in Figure 2A-C, each of the three Plexin-B1 siRNA duplexes significantly reduced the amount of Plexin-B1 mRNA and protein. The data are from densitometric analyses of Plexin-B1: β-actin; n = 5 independent experiments.

Bottom Line: Elevated Plexin-B1 expression has been found in diverse human cancers and in non-neoplastic tissues, and it mediates diverse biological and pathological activities.Expression levels of Plexin-B1 protein were significantly higher in serous ovarian carcinomas than in normal ovaries or benign ovarian neoplasms, and in the former, Plexin-B1 expression was positively correlated with lymphatic metastasis, and the membrane and cytoplasm of cancer cells stained positively.Plexin-B1 siRNA significantly suppressed phosphorylation of AKT at Ser473 in SKOV3 cells, but it did not alter total AKT expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China.

ABSTRACT

Background: Elevated Plexin-B1 expression has been found in diverse human cancers and in non-neoplastic tissues, and it mediates diverse biological and pathological activities. However, whether or not Plexin-B1 expression is involved in human ovarian tumors remains unclear. In the present study, Plexin-B1 expression was explored in benign and malignant human ovarian tumor tissues. In addition, the impact of Plexin-B1 expression on ovarian cancer cell proliferation, migration and invasion were investigated in vitro.

Methods: Plexin-B1 expression was analyzed in normal and benign ovarian tissues and serous ovarian tumors (both borderline and malignant) by immunohistochemical staining, as well as in four human ovarian cancer cell lines (A2780, C13*, SKOV3, and OV2008) by RT-PCR and western blot analyses. Furthermore, endogenous Plexin-B1 expression was suppressed by Plexin-B1 siRNA in SKOV3 cells, which overexpress Plexin-B1. Protein levels of Plexin-B1, AKT and AKTSer473 were examined by western blot analysis. Cell proliferation, migration and invasion were measured with MTT, wound healing and boyden chamber assays, respectively, and the cytoskeleton was monitored via F-actin staining.

Results: Expression levels of Plexin-B1 protein were significantly higher in serous ovarian carcinomas than in normal ovaries or benign ovarian neoplasms, and in the former, Plexin-B1 expression was positively correlated with lymphatic metastasis, and the membrane and cytoplasm of cancer cells stained positively. SKOV3 cells displayed the highest Plexin-B1 expression at both the mRNA and protein levels among the four tested human ovarian cancer cell lines and was selected as a cell model for further in vitro experiments. Plexin-B1 siRNA significantly suppressed phosphorylation of AKT at Ser473 in SKOV3 cells, but it did not alter total AKT expression. In addition, silencing of Plexin-B1 in SKOV3 cells inhibited cell migration and invasion and reorganized the cytoskeleton, whereas cell proliferation was not affected.

Conclusion: Plexin-B1 expression correlates with malignant phenotypes of serous ovarian tumors, probably via phosphorylation of AKT at Ser473, suggesting that Plexin-B1 might be a useful biomarker and/or a novel therapeutic target.

Show MeSH
Related in: MedlinePlus