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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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Effects of MTA1 specific shRNA on invasion in MDA-MB-231 and MCF-7 cells. A: MDA-MB-231 cells passed through the filter and attached to the lower side of the filter (400×)before silencing MTA1. B: MDA-MB-231 cells passed through the filter and attached to the lower side of the filter (400×) after silencing MTA1 C: MCF-7 cells passed through the filter and attached to the lower side of the filter (400×) before silencing MTA1. D: MCF-7 cells passed through the filter and attached to the lower side of the filter (400×) after silencing MTA1.
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Figure 5: Effects of MTA1 specific shRNA on invasion in MDA-MB-231 and MCF-7 cells. A: MDA-MB-231 cells passed through the filter and attached to the lower side of the filter (400×)before silencing MTA1. B: MDA-MB-231 cells passed through the filter and attached to the lower side of the filter (400×) after silencing MTA1 C: MCF-7 cells passed through the filter and attached to the lower side of the filter (400×) before silencing MTA1. D: MCF-7 cells passed through the filter and attached to the lower side of the filter (400×) after silencing MTA1.

Mentions: The effects of inhibiting MTA1 gene on invasion of breast cancer cells were evaluated by Boyden chamber migration assay. The invasion index before silencing MTA1 in MDA-MB-231 and MCF-7 cells were 76.3 ± 2.4%, 25.6 ± 1.9%, respectively, the difference was obvious(P < 0.05). After silencing MTA1 gene in MDA-MB-231 cells, the invasion index was 27.2 ± 2.1%, compared to before transfection, the statistics difference was obvious(P < 0.05). But in MCF-7 cells, invasion index was 23.3 ± 1.6% after silencing MTA1, compared to blank control, it's no statistics difference(P > 0.05). The invasion index in MDA-MB-231 and MCF-7 cells treated with empty vector were 73.2 ± 2.0%, 23.1 ± 2.1%, compared to blank control, its' no statistics difference(P > 0.05), respectively. (Figure 5)


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)

Effects of MTA1 specific shRNA on invasion in MDA-MB-231 and MCF-7 cells. A: MDA-MB-231 cells passed through the filter and attached to the lower side of the filter (400×)before silencing MTA1. B: MDA-MB-231 cells passed through the filter and attached to the lower side of the filter (400×) after silencing MTA1 C: MCF-7 cells passed through the filter and attached to the lower side of the filter (400×) before silencing MTA1. D: MCF-7 cells passed through the filter and attached to the lower side of the filter (400×) after silencing MTA1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Effects of MTA1 specific shRNA on invasion in MDA-MB-231 and MCF-7 cells. A: MDA-MB-231 cells passed through the filter and attached to the lower side of the filter (400×)before silencing MTA1. B: MDA-MB-231 cells passed through the filter and attached to the lower side of the filter (400×) after silencing MTA1 C: MCF-7 cells passed through the filter and attached to the lower side of the filter (400×) before silencing MTA1. D: MCF-7 cells passed through the filter and attached to the lower side of the filter (400×) after silencing MTA1.
Mentions: The effects of inhibiting MTA1 gene on invasion of breast cancer cells were evaluated by Boyden chamber migration assay. The invasion index before silencing MTA1 in MDA-MB-231 and MCF-7 cells were 76.3 ± 2.4%, 25.6 ± 1.9%, respectively, the difference was obvious(P < 0.05). After silencing MTA1 gene in MDA-MB-231 cells, the invasion index was 27.2 ± 2.1%, compared to before transfection, the statistics difference was obvious(P < 0.05). But in MCF-7 cells, invasion index was 23.3 ± 1.6% after silencing MTA1, compared to blank control, it's no statistics difference(P > 0.05). The invasion index in MDA-MB-231 and MCF-7 cells treated with empty vector were 73.2 ± 2.0%, 23.1 ± 2.1%, compared to blank control, its' no statistics difference(P > 0.05), respectively. (Figure 5)

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