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Adenovirus-mediated siRNA targeting Bcl-xL inhibits proliferation, reduces invasion and enhances radiosensitivity of human colorectal cancer cells.

Yang J, Sun M, Zhang A, Lv C, De W, Wang Z - World J Surg Oncol (2011)

Bottom Line: Ad/shBcl-xL could significantly reduce the expression of Bcl-xL protein in CRC cells.Also, we showed that adenovirus-mediated siRNA targeting Bcl-xL could significantly inhibit proliferation and colony formation of CRC cells.Targeting Bcl-xL will be a promising strategy to inhibit the metastatic potential and reverse the radioresistance of human CRC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing, Jiangsu, People's Republic of China.

ABSTRACT

Introduction: Bcl-xL, an important member of anti-apoptotic Bcl-2 family, plays critical roles in tumor progression and development. Previously, we have reported that overexpression of Bcl-xL was correlated with prognosis of colorectal cancer (CRC) patients. The aim of this study was to investigate the association of Bcl-xL expression with invasion and radiosensitivity of human CRC cells.

Methods: RT-PCR and Western blot assays were performed to determine the expression of Bcl-xL mRNA and protein in CRC cells and normal human intestinal epithelial cell line. Then, adenovirus-mediated RNA interference technique was employed to inhibit the expression of Bcl-xL gene in CRC cells. The proliferation of CRC cells was analyzed by MTT and colony formation assay. The migration and invasion of CRC cells was determined by wound-healing and tranwell invasion assays. Additionally, the in vitro and in vivo radiosensitivity of CRC cells was determined by clonogenic cell survival assay and murine xnograft model, respectively.

Results: The levels of Bcl-xL mRNA and protein expression were significantly higher in human CRC cells than in normal human intestinal epithelial cell line. Ad/shBcl-xL could significantly reduce the expression of Bcl-xL protein in CRC cells. Also, we showed that adenovirus-mediated siRNA targeting Bcl-xL could significantly inhibit proliferation and colony formation of CRC cells. Ad/shBcl-xL could significantly suppress migration and invasion of CRC cells. Moreover, Ad/shBcl-xL could enhance in vitro and in vivo radiosensitivity of CRC cells by increasing caspase-dependent apoptosis.

Conclusions: Targeting Bcl-xL will be a promising strategy to inhibit the metastatic potential and reverse the radioresistance of human CRC.

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Related in: MedlinePlus

The expression of Bcl-xL protein in CRC cells was significantly downregualted after adenovirus infection. A. Western blot analysis of Bcl-xL protein expression in mock LoVo or LoVo infected with Ad/shcontrol or Ad/shBcl-xL. B. Western blot analysis of Bcl-xL protein expression in mock LoVo or LoVo transfected with siRNA/control or siRNA/Bcl-xL. C. Western blot analysis of Bcl-2 and Mcl-1 protein expression in mock LoVo or LoVo infected with Ad/shcontrol or Ad/shBcl-xL. GAPDH was used to an internal control. Each assay was performed at least in triplicate. **P < 0.01 and *P < 0.05 vs mock.
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Figure 2: The expression of Bcl-xL protein in CRC cells was significantly downregualted after adenovirus infection. A. Western blot analysis of Bcl-xL protein expression in mock LoVo or LoVo infected with Ad/shcontrol or Ad/shBcl-xL. B. Western blot analysis of Bcl-xL protein expression in mock LoVo or LoVo transfected with siRNA/control or siRNA/Bcl-xL. C. Western blot analysis of Bcl-2 and Mcl-1 protein expression in mock LoVo or LoVo infected with Ad/shcontrol or Ad/shBcl-xL. GAPDH was used to an internal control. Each assay was performed at least in triplicate. **P < 0.01 and *P < 0.05 vs mock.

Mentions: To determine the optimal MOI for a maximal transgene expression, LoVo cells were infected with Ad/shcontrol or Ad/shBcl-xl at various MOIs (0, 40, 80) and examined by fluorescence microscopy. Approximately 90% of GFP expression could be observed in LoVo cells infected with Ad/shcontrol or Ad/shBcl-xL at 80 MOI (data not shown). Thus, a MOI of 80 was selected as an optimal dose for infection of CRC cells. To testify the effect of adenovirus-mediated siRNA targeting Bcl-xL on the expression of Bcl-xL gene in CRC cell line, Western blot assay was performed to detect the expression of Bcl-xL protein. The expression level of Bcl-xL protein in Ad/shBcl-xL-infected LoVo cells was decreased by approximately 76.3% by compared to that in mock or Ad/shcontrol-infected cells (P < 0.01; Figure 2A). Also, at 48 h after transfection by Lipofectamine 2000, we analyzed the expression of Bcl-xL protein in the siRNA/Bcl-xL-transfected LoVo cells. Compared with mock or siRNA/control-transfected LoVo cells, the expression of Bcl-xL protein was decreased by only 34.6% (P < 0.05; Figure 2B). Thus, the knockdown effect of adenovirus-mediated siRNA showed more efficient than that of siRNA oligonucleotide transfected by lipofection. Next, we analyzed the effects of adenovirus-mediated siRNA targeting Bcl-xL on the expression of other apoptosis relevant proteins including Bcl-2 and Mcl-1. As shown in Figure 2C, the levels of Bcl-2 and Mcl-1 protein expression in Ad/shBcl-xL-infected LoVo cells showed no difference compared with those in mock or Ad/shcontrol-infected cells (P > 0.05; Figure 2C). These data showed that adenovirus-mediated siRNA targeting Bcl-xL could specifically and significantly inhibit the expression of Bcl-xL gene in CRC cells.


Adenovirus-mediated siRNA targeting Bcl-xL inhibits proliferation, reduces invasion and enhances radiosensitivity of human colorectal cancer cells.

Yang J, Sun M, Zhang A, Lv C, De W, Wang Z - World J Surg Oncol (2011)

The expression of Bcl-xL protein in CRC cells was significantly downregualted after adenovirus infection. A. Western blot analysis of Bcl-xL protein expression in mock LoVo or LoVo infected with Ad/shcontrol or Ad/shBcl-xL. B. Western blot analysis of Bcl-xL protein expression in mock LoVo or LoVo transfected with siRNA/control or siRNA/Bcl-xL. C. Western blot analysis of Bcl-2 and Mcl-1 protein expression in mock LoVo or LoVo infected with Ad/shcontrol or Ad/shBcl-xL. GAPDH was used to an internal control. Each assay was performed at least in triplicate. **P < 0.01 and *P < 0.05 vs mock.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198948&req=5

Figure 2: The expression of Bcl-xL protein in CRC cells was significantly downregualted after adenovirus infection. A. Western blot analysis of Bcl-xL protein expression in mock LoVo or LoVo infected with Ad/shcontrol or Ad/shBcl-xL. B. Western blot analysis of Bcl-xL protein expression in mock LoVo or LoVo transfected with siRNA/control or siRNA/Bcl-xL. C. Western blot analysis of Bcl-2 and Mcl-1 protein expression in mock LoVo or LoVo infected with Ad/shcontrol or Ad/shBcl-xL. GAPDH was used to an internal control. Each assay was performed at least in triplicate. **P < 0.01 and *P < 0.05 vs mock.
Mentions: To determine the optimal MOI for a maximal transgene expression, LoVo cells were infected with Ad/shcontrol or Ad/shBcl-xl at various MOIs (0, 40, 80) and examined by fluorescence microscopy. Approximately 90% of GFP expression could be observed in LoVo cells infected with Ad/shcontrol or Ad/shBcl-xL at 80 MOI (data not shown). Thus, a MOI of 80 was selected as an optimal dose for infection of CRC cells. To testify the effect of adenovirus-mediated siRNA targeting Bcl-xL on the expression of Bcl-xL gene in CRC cell line, Western blot assay was performed to detect the expression of Bcl-xL protein. The expression level of Bcl-xL protein in Ad/shBcl-xL-infected LoVo cells was decreased by approximately 76.3% by compared to that in mock or Ad/shcontrol-infected cells (P < 0.01; Figure 2A). Also, at 48 h after transfection by Lipofectamine 2000, we analyzed the expression of Bcl-xL protein in the siRNA/Bcl-xL-transfected LoVo cells. Compared with mock or siRNA/control-transfected LoVo cells, the expression of Bcl-xL protein was decreased by only 34.6% (P < 0.05; Figure 2B). Thus, the knockdown effect of adenovirus-mediated siRNA showed more efficient than that of siRNA oligonucleotide transfected by lipofection. Next, we analyzed the effects of adenovirus-mediated siRNA targeting Bcl-xL on the expression of other apoptosis relevant proteins including Bcl-2 and Mcl-1. As shown in Figure 2C, the levels of Bcl-2 and Mcl-1 protein expression in Ad/shBcl-xL-infected LoVo cells showed no difference compared with those in mock or Ad/shcontrol-infected cells (P > 0.05; Figure 2C). These data showed that adenovirus-mediated siRNA targeting Bcl-xL could specifically and significantly inhibit the expression of Bcl-xL gene in CRC cells.

Bottom Line: Ad/shBcl-xL could significantly reduce the expression of Bcl-xL protein in CRC cells.Also, we showed that adenovirus-mediated siRNA targeting Bcl-xL could significantly inhibit proliferation and colony formation of CRC cells.Targeting Bcl-xL will be a promising strategy to inhibit the metastatic potential and reverse the radioresistance of human CRC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing, Jiangsu, People's Republic of China.

ABSTRACT

Introduction: Bcl-xL, an important member of anti-apoptotic Bcl-2 family, plays critical roles in tumor progression and development. Previously, we have reported that overexpression of Bcl-xL was correlated with prognosis of colorectal cancer (CRC) patients. The aim of this study was to investigate the association of Bcl-xL expression with invasion and radiosensitivity of human CRC cells.

Methods: RT-PCR and Western blot assays were performed to determine the expression of Bcl-xL mRNA and protein in CRC cells and normal human intestinal epithelial cell line. Then, adenovirus-mediated RNA interference technique was employed to inhibit the expression of Bcl-xL gene in CRC cells. The proliferation of CRC cells was analyzed by MTT and colony formation assay. The migration and invasion of CRC cells was determined by wound-healing and tranwell invasion assays. Additionally, the in vitro and in vivo radiosensitivity of CRC cells was determined by clonogenic cell survival assay and murine xnograft model, respectively.

Results: The levels of Bcl-xL mRNA and protein expression were significantly higher in human CRC cells than in normal human intestinal epithelial cell line. Ad/shBcl-xL could significantly reduce the expression of Bcl-xL protein in CRC cells. Also, we showed that adenovirus-mediated siRNA targeting Bcl-xL could significantly inhibit proliferation and colony formation of CRC cells. Ad/shBcl-xL could significantly suppress migration and invasion of CRC cells. Moreover, Ad/shBcl-xL could enhance in vitro and in vivo radiosensitivity of CRC cells by increasing caspase-dependent apoptosis.

Conclusions: Targeting Bcl-xL will be a promising strategy to inhibit the metastatic potential and reverse the radioresistance of human CRC.

Show MeSH
Related in: MedlinePlus