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Id2 promotes the invasive growth of MCF-7 and SKOV-3 cells by a novel mechanism independent of dimerization to basic helix-loop-helix factors.

Meng Y, Gu C, Wu Z, Zhao Y, Si Y, Fu X, Han W - BMC Cancer (2009)

Bottom Line: The ectopic expression of Id2 or its mutants did not alter proliferation of either MCF-7 or SKOV-3 cells.Transfection of the wild-type Id2 significantly induced the invasion potential and migratory capacity of cells, which was further augmented by transfection with the degradation-resistant full-length or HLH-deleted Id2.E-cadherin contributes only in part to Id2-induced cell invasion when Id2 is accumulated to a higher level in some specific cell types.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, Institute of Basic Medicine, Chinese PLA General Hospital, 28 Fu Xing Road, Beijing, 100853, PR China. mengyg6512@vipsina.com

ABSTRACT

Background: Inhibitor of differentiation 2 (Id2) is a critical factor for cell proliferation and differentiation in normal vertebrate development. Most of the biological function of Id2 has been ascribed to its helix-loop-helix motif. Overexpression of Id2 is frequently observed in various human tumors, but its role for invasion potential in tumor cells is dispute. We aimed to reveal the role of Id2 in invasion potential in poorly invasive and estrogen receptor alpha (ERalpha)-positive MCF-7 and SKOV-3 cancer cells.

Methods: MCF-7 and SKOV-3 cells were stably transfected with the wild-type, degradation-resistant full-length or helix-loop-helix (HLH)-deleted Id2, respectively. Protein levels of Id2 and its mutants and E-cadherin were determined by western blot analysis and mRNA levels of Id2 and its mutants were determined by RT-PCR. The effects of Id2 and its mutants on cell proliferation were determined by [3H]-thymidine incorporation assay and the 3- [4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) dye method. The in vitro invasion potential of cells was evaluated by Transwell assay. Cell motility was assessed by scratch wound assay. The promoter activity of E-cadherin was determined by cotransfection and luciferase assays.

Results: Ectopic transfection of the wild-type Id2 markedly increased the protein and mRNA expression of Id2 in MCF-7 and SKOV-3 cells; the protein level but not mRNA level was further increased by transfection with the degradation-resistant Id2 form. The ectopic expression of Id2 or its mutants did not alter proliferation of either MCF-7 or SKOV-3 cells. Transfection of the wild-type Id2 significantly induced the invasion potential and migratory capacity of cells, which was further augmented by transfection with the degradation-resistant full-length or HLH-deleted Id2. E-cadherin protein expression and transactivation of the proximal E-cadherin promoter were markedly suppressed by the degradation-resistant full-length or HLH-deleted Id2 but not wild-type Id2. Ectopic expression of E-cadherin in MCF-7 and SKOV-3 cells only partially blunted the invasion potential induced by the degradation-resistant HLH-deleted Id2.

Conclusion: Overexpression of Id2 in ERalpha-positive epithelial tumor cells indeed increases the cells' invasive potential through a novel mechanism independent of dimerization to basic helix-loop-helix factors. E-cadherin contributes only in part to Id2-induced cell invasion when Id2 is accumulated to a higher level in some specific cell types.

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(A) Effect of Id2-specific siRNA transfection on the expression of the exogenous Id2-DBM protein in MCF-7 cells. The indicated siRNA oligonucleotides were transfected into the indicated MCF-7 cells. 48 h after transfection, total proteins were extracted and subjected to immunoblotting analysis with Id2-specific antibody. β-actin was used as the loading control. (B) Effect of knock down of exogenous Id2-DBM by RNA interference on in vitro invasion of MCF-7 cells. The indicated siRNA oligonucleotides were transfected as described in (A). 48 h after transfection, MCF-7 cells were subjected to performing Transwell experiments. The top panel shows representative pictures of Transwell assays, as described in "Methods". Each data was performed in triplicate and was repeated three times. Data are expressed as the percentage of the control cells (mean ± SEM, the bottom panel). (# P <0.01; N P > 0.05).
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Figure 4: (A) Effect of Id2-specific siRNA transfection on the expression of the exogenous Id2-DBM protein in MCF-7 cells. The indicated siRNA oligonucleotides were transfected into the indicated MCF-7 cells. 48 h after transfection, total proteins were extracted and subjected to immunoblotting analysis with Id2-specific antibody. β-actin was used as the loading control. (B) Effect of knock down of exogenous Id2-DBM by RNA interference on in vitro invasion of MCF-7 cells. The indicated siRNA oligonucleotides were transfected as described in (A). 48 h after transfection, MCF-7 cells were subjected to performing Transwell experiments. The top panel shows representative pictures of Transwell assays, as described in "Methods". Each data was performed in triplicate and was repeated three times. Data are expressed as the percentage of the control cells (mean ± SEM, the bottom panel). (# P <0.01; N P > 0.05).

Mentions: To further address the promotion role of Id2 overexpression on cell invasiveness, Id2-specific siRNA or control-siRNA oligonucleotides was transiently transfected into MCF-7 cells stably expressing Id2-DBM or pcDNA3.1 empty vector. Forty-eight hours later, cells were harvested and subjected to western blot analysis and Transwell experiments. The expression of ectopic Id2-DBM protein was largely inhibited by the Id2-specific siRNA but not by control-siRNA (Figure 4A). Knock down of exogenous Id2-DBM in MCF-7 cells by Id2-specific siRNA cotransfection reduced the cell invasiveness enhanced by Id2-DBM to that of the control cells (Figure 4B).


Id2 promotes the invasive growth of MCF-7 and SKOV-3 cells by a novel mechanism independent of dimerization to basic helix-loop-helix factors.

Meng Y, Gu C, Wu Z, Zhao Y, Si Y, Fu X, Han W - BMC Cancer (2009)

(A) Effect of Id2-specific siRNA transfection on the expression of the exogenous Id2-DBM protein in MCF-7 cells. The indicated siRNA oligonucleotides were transfected into the indicated MCF-7 cells. 48 h after transfection, total proteins were extracted and subjected to immunoblotting analysis with Id2-specific antibody. β-actin was used as the loading control. (B) Effect of knock down of exogenous Id2-DBM by RNA interference on in vitro invasion of MCF-7 cells. The indicated siRNA oligonucleotides were transfected as described in (A). 48 h after transfection, MCF-7 cells were subjected to performing Transwell experiments. The top panel shows representative pictures of Transwell assays, as described in "Methods". Each data was performed in triplicate and was repeated three times. Data are expressed as the percentage of the control cells (mean ± SEM, the bottom panel). (# P <0.01; N P > 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654660&req=5

Figure 4: (A) Effect of Id2-specific siRNA transfection on the expression of the exogenous Id2-DBM protein in MCF-7 cells. The indicated siRNA oligonucleotides were transfected into the indicated MCF-7 cells. 48 h after transfection, total proteins were extracted and subjected to immunoblotting analysis with Id2-specific antibody. β-actin was used as the loading control. (B) Effect of knock down of exogenous Id2-DBM by RNA interference on in vitro invasion of MCF-7 cells. The indicated siRNA oligonucleotides were transfected as described in (A). 48 h after transfection, MCF-7 cells were subjected to performing Transwell experiments. The top panel shows representative pictures of Transwell assays, as described in "Methods". Each data was performed in triplicate and was repeated three times. Data are expressed as the percentage of the control cells (mean ± SEM, the bottom panel). (# P <0.01; N P > 0.05).
Mentions: To further address the promotion role of Id2 overexpression on cell invasiveness, Id2-specific siRNA or control-siRNA oligonucleotides was transiently transfected into MCF-7 cells stably expressing Id2-DBM or pcDNA3.1 empty vector. Forty-eight hours later, cells were harvested and subjected to western blot analysis and Transwell experiments. The expression of ectopic Id2-DBM protein was largely inhibited by the Id2-specific siRNA but not by control-siRNA (Figure 4A). Knock down of exogenous Id2-DBM in MCF-7 cells by Id2-specific siRNA cotransfection reduced the cell invasiveness enhanced by Id2-DBM to that of the control cells (Figure 4B).

Bottom Line: The ectopic expression of Id2 or its mutants did not alter proliferation of either MCF-7 or SKOV-3 cells.Transfection of the wild-type Id2 significantly induced the invasion potential and migratory capacity of cells, which was further augmented by transfection with the degradation-resistant full-length or HLH-deleted Id2.E-cadherin contributes only in part to Id2-induced cell invasion when Id2 is accumulated to a higher level in some specific cell types.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, Institute of Basic Medicine, Chinese PLA General Hospital, 28 Fu Xing Road, Beijing, 100853, PR China. mengyg6512@vipsina.com

ABSTRACT

Background: Inhibitor of differentiation 2 (Id2) is a critical factor for cell proliferation and differentiation in normal vertebrate development. Most of the biological function of Id2 has been ascribed to its helix-loop-helix motif. Overexpression of Id2 is frequently observed in various human tumors, but its role for invasion potential in tumor cells is dispute. We aimed to reveal the role of Id2 in invasion potential in poorly invasive and estrogen receptor alpha (ERalpha)-positive MCF-7 and SKOV-3 cancer cells.

Methods: MCF-7 and SKOV-3 cells were stably transfected with the wild-type, degradation-resistant full-length or helix-loop-helix (HLH)-deleted Id2, respectively. Protein levels of Id2 and its mutants and E-cadherin were determined by western blot analysis and mRNA levels of Id2 and its mutants were determined by RT-PCR. The effects of Id2 and its mutants on cell proliferation were determined by [3H]-thymidine incorporation assay and the 3- [4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) dye method. The in vitro invasion potential of cells was evaluated by Transwell assay. Cell motility was assessed by scratch wound assay. The promoter activity of E-cadherin was determined by cotransfection and luciferase assays.

Results: Ectopic transfection of the wild-type Id2 markedly increased the protein and mRNA expression of Id2 in MCF-7 and SKOV-3 cells; the protein level but not mRNA level was further increased by transfection with the degradation-resistant Id2 form. The ectopic expression of Id2 or its mutants did not alter proliferation of either MCF-7 or SKOV-3 cells. Transfection of the wild-type Id2 significantly induced the invasion potential and migratory capacity of cells, which was further augmented by transfection with the degradation-resistant full-length or HLH-deleted Id2. E-cadherin protein expression and transactivation of the proximal E-cadherin promoter were markedly suppressed by the degradation-resistant full-length or HLH-deleted Id2 but not wild-type Id2. Ectopic expression of E-cadherin in MCF-7 and SKOV-3 cells only partially blunted the invasion potential induced by the degradation-resistant HLH-deleted Id2.

Conclusion: Overexpression of Id2 in ERalpha-positive epithelial tumor cells indeed increases the cells' invasive potential through a novel mechanism independent of dimerization to basic helix-loop-helix factors. E-cadherin contributes only in part to Id2-induced cell invasion when Id2 is accumulated to a higher level in some specific cell types.

Show MeSH
Related in: MedlinePlus