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ShRNA-mediated gene silencing of MTA1 influenced on protein expression of ER alpha, MMP-9, CyclinD1 and invasiveness, proliferation in breast cancer cell lines MDA-MB-231 and MCF-7 in vitro.

Jiang Q, Zhang H, Zhang P - J. Exp. Clin. Cancer Res. (2011)

Bottom Line: Tumor cells invasive ability were evaluated by Boyden chamber assay, the cells proliferation were evaluated using cell growth curve and MTT analysis, the cell cycle analysis was performed using flow cytometry.Down-regulation of MTA1 by RNAi approach led to re-expression of ER alpha in ER-negative breast cancer cell lines MDA-MB-231, and reduced protein levels of MMP-9 and CyclinD1, as well as decreased tumor cell invasion and proliferation, more cells were blocked in G0/G1 stage(P < 0.05).Silencing effect of MTA1 could efficiently inhibit the invasion and proliferation in MDA-MB-231 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, School of Basic Medicine Sciences, Chong Qing University of Medical Sciences, Chong Qing, 400016, China.

ABSTRACT

Background: MTA1(metastasis associated-1) is a tumor metastasis associated candidate gene and overexpression in many human tumors, including breast cancer. In this study, we investigated depressive effect on MTA1 by MTA1-specific short hairpin RNA(shRNA) expression plasmids in human breast cancer cell lines MDA-MB-231 and MCF-7, and effect on protein levels of ER alpha, MMP-9, cyclinD1, and tumor cell invasion, proliferation.

Methods: ShRNA expression vectors targeting MTA1 was constructed and transfected into human breast cancer cell lines MDA-MB-231 and MCF-7. The transfection efficiency was evaluated by fluorescence microscopy, mRNA levels of MTA1 were detected by reverse transcription-polymerase chain reaction (RT-PCR), protein levels of ER alpha, MMP-9 and cyclinD1 were detected by Western blotting, respectively. Tumor cells invasive ability were evaluated by Boyden chamber assay, the cells proliferation were evaluated using cell growth curve and MTT analysis, the cell cycle analysis was performed using flow cytometry.

Results: Down-regulation of MTA1 by RNAi approach led to re-expression of ER alpha in ER-negative breast cancer cell lines MDA-MB-231, and reduced protein levels of MMP-9 and CyclinD1, as well as decreased tumor cell invasion and proliferation, more cells were blocked in G0/G1 stage(P < 0.05). However, after inhibiting mRNA levels of MTA1, protein expression of ER alpha, MMP-9, cyclinD1 and the changes of cancer cells invasiveness, proliferation, cells cycle were no statistical difference in ER-positive human breast cancer cell lines MCF-7 (P > 0.05).

Conclusions: ShRNA targeted against MTA1 could specifically mediate the MTA1 gene silencing and consequentially recover the protein expression of ER alpha, resulting in increase sensitivity of antiestrogens, as well as suppress the protein levels of MMP-9 and cyclinD1 in ER-negative human breast cancer cell lines MDA-MB-231. Silencing effect of MTA1 could efficiently inhibit the invasion and proliferation in MDA-MB-231 cells. The shRNA interference targeted against MTA1 may have potential therapeutic utility in human breast cancer.

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Restrictive enzyme incision analysis for pGensil-1/MTA1 shRNA plasmid using RT-PCR. M: DNA Marker. lane 1: pGenesil-1/MTA1 shRNA(pGM1) plasmid was cut off by BamHI and HindIII. lane 2: pGenesil-1/MTA1 shRNA(pGM1) plasmid was cut off by BamHI and XbaI.lane 3: pGenesil-1/MTA1 shRNA(pGM1) plasmid was cut off by HindIII and XbaI. lane 4: pGenesil-1/MTA1 shRNA(pGM2) plasmid was cut off by BamHI and HindIII. lane 5: pGenesil-1/MTA1 shRNA(pGM2) plasmid was cut off by BamHI and XbaI. lane 6: pGenesil-1/MTA1 shRNA(pGM2) plasmid was cut off by HindIII and XbaI.
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Figure 1: Restrictive enzyme incision analysis for pGensil-1/MTA1 shRNA plasmid using RT-PCR. M: DNA Marker. lane 1: pGenesil-1/MTA1 shRNA(pGM1) plasmid was cut off by BamHI and HindIII. lane 2: pGenesil-1/MTA1 shRNA(pGM1) plasmid was cut off by BamHI and XbaI.lane 3: pGenesil-1/MTA1 shRNA(pGM1) plasmid was cut off by HindIII and XbaI. lane 4: pGenesil-1/MTA1 shRNA(pGM2) plasmid was cut off by BamHI and HindIII. lane 5: pGenesil-1/MTA1 shRNA(pGM2) plasmid was cut off by BamHI and XbaI. lane 6: pGenesil-1/MTA1 shRNA(pGM2) plasmid was cut off by HindIII and XbaI.

Mentions: The recombinant plasmids were cut off by restriction enzyme Xba, BamHⅠand HindⅢ, The band about 66 bp was cut off using BamHⅠand HindⅢ on 0.8% agarose gel electrophoresis, the band about 342 bp was cut off using XbaⅠand BamHⅠ, the band about 408 bp was cut off using XbaⅠand HindⅢ (Figure 1). The results of incision with restriction endonucleases and sequencing showed correct plasmids.


ShRNA-mediated gene silencing of MTA1 influenced on protein expression of ER alpha, MMP-9, CyclinD1 and invasiveness, proliferation in breast cancer cell lines MDA-MB-231 and MCF-7 in vitro.

Jiang Q, Zhang H, Zhang P - J. Exp. Clin. Cancer Res. (2011)

Restrictive enzyme incision analysis for pGensil-1/MTA1 shRNA plasmid using RT-PCR. M: DNA Marker. lane 1: pGenesil-1/MTA1 shRNA(pGM1) plasmid was cut off by BamHI and HindIII. lane 2: pGenesil-1/MTA1 shRNA(pGM1) plasmid was cut off by BamHI and XbaI.lane 3: pGenesil-1/MTA1 shRNA(pGM1) plasmid was cut off by HindIII and XbaI. lane 4: pGenesil-1/MTA1 shRNA(pGM2) plasmid was cut off by BamHI and HindIII. lane 5: pGenesil-1/MTA1 shRNA(pGM2) plasmid was cut off by BamHI and XbaI. lane 6: pGenesil-1/MTA1 shRNA(pGM2) plasmid was cut off by HindIII and XbaI.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113748&req=5

Figure 1: Restrictive enzyme incision analysis for pGensil-1/MTA1 shRNA plasmid using RT-PCR. M: DNA Marker. lane 1: pGenesil-1/MTA1 shRNA(pGM1) plasmid was cut off by BamHI and HindIII. lane 2: pGenesil-1/MTA1 shRNA(pGM1) plasmid was cut off by BamHI and XbaI.lane 3: pGenesil-1/MTA1 shRNA(pGM1) plasmid was cut off by HindIII and XbaI. lane 4: pGenesil-1/MTA1 shRNA(pGM2) plasmid was cut off by BamHI and HindIII. lane 5: pGenesil-1/MTA1 shRNA(pGM2) plasmid was cut off by BamHI and XbaI. lane 6: pGenesil-1/MTA1 shRNA(pGM2) plasmid was cut off by HindIII and XbaI.
Mentions: The recombinant plasmids were cut off by restriction enzyme Xba, BamHⅠand HindⅢ, The band about 66 bp was cut off using BamHⅠand HindⅢ on 0.8% agarose gel electrophoresis, the band about 342 bp was cut off using XbaⅠand BamHⅠ, the band about 408 bp was cut off using XbaⅠand HindⅢ (Figure 1). The results of incision with restriction endonucleases and sequencing showed correct plasmids.

Bottom Line: Tumor cells invasive ability were evaluated by Boyden chamber assay, the cells proliferation were evaluated using cell growth curve and MTT analysis, the cell cycle analysis was performed using flow cytometry.Down-regulation of MTA1 by RNAi approach led to re-expression of ER alpha in ER-negative breast cancer cell lines MDA-MB-231, and reduced protein levels of MMP-9 and CyclinD1, as well as decreased tumor cell invasion and proliferation, more cells were blocked in G0/G1 stage(P < 0.05).Silencing effect of MTA1 could efficiently inhibit the invasion and proliferation in MDA-MB-231 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, School of Basic Medicine Sciences, Chong Qing University of Medical Sciences, Chong Qing, 400016, China.

ABSTRACT

Background: MTA1(metastasis associated-1) is a tumor metastasis associated candidate gene and overexpression in many human tumors, including breast cancer. In this study, we investigated depressive effect on MTA1 by MTA1-specific short hairpin RNA(shRNA) expression plasmids in human breast cancer cell lines MDA-MB-231 and MCF-7, and effect on protein levels of ER alpha, MMP-9, cyclinD1, and tumor cell invasion, proliferation.

Methods: ShRNA expression vectors targeting MTA1 was constructed and transfected into human breast cancer cell lines MDA-MB-231 and MCF-7. The transfection efficiency was evaluated by fluorescence microscopy, mRNA levels of MTA1 were detected by reverse transcription-polymerase chain reaction (RT-PCR), protein levels of ER alpha, MMP-9 and cyclinD1 were detected by Western blotting, respectively. Tumor cells invasive ability were evaluated by Boyden chamber assay, the cells proliferation were evaluated using cell growth curve and MTT analysis, the cell cycle analysis was performed using flow cytometry.

Results: Down-regulation of MTA1 by RNAi approach led to re-expression of ER alpha in ER-negative breast cancer cell lines MDA-MB-231, and reduced protein levels of MMP-9 and CyclinD1, as well as decreased tumor cell invasion and proliferation, more cells were blocked in G0/G1 stage(P < 0.05). However, after inhibiting mRNA levels of MTA1, protein expression of ER alpha, MMP-9, cyclinD1 and the changes of cancer cells invasiveness, proliferation, cells cycle were no statistical difference in ER-positive human breast cancer cell lines MCF-7 (P > 0.05).

Conclusions: ShRNA targeted against MTA1 could specifically mediate the MTA1 gene silencing and consequentially recover the protein expression of ER alpha, resulting in increase sensitivity of antiestrogens, as well as suppress the protein levels of MMP-9 and cyclinD1 in ER-negative human breast cancer cell lines MDA-MB-231. Silencing effect of MTA1 could efficiently inhibit the invasion and proliferation in MDA-MB-231 cells. The shRNA interference targeted against MTA1 may have potential therapeutic utility in human breast cancer.

Show MeSH
Related in: MedlinePlus