Abstract
BackgroundMTA1(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.MethodsShRNA 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.ResultsDown-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).ConclusionsShRNA 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.
Highlights
Metastasis associated antigen 1 (MTA1)(metastasis associated-1) is a tumor metastasis associated candidate gene and overexpression in many human tumors, including breast cancer
The construction of pGenesil-1/MTA1 short hairpin RNA (shRNA) expression plasmid 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 indicated that, more MDA-MB-231 cells were blocked in G0/G1 stage after inhibiting MTA1 gene by pGenesil-1/MTA1 shRNA
Summary
MTA1(metastasis associated-1) is a tumor metastasis associated candidate gene and overexpression in many human tumors, including breast cancer. 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. Metastasis associated antigen 1 (MTA1) is a tumor metastasis associated candidate gene, it was originally identified by differential screening of a cDNA library from highly metastatic and non-metastatic rat mammary adenocarcinoma cell lines[3,4]. Overexpression of MTA1 plays an important role in tumorigenesis and tumor aggressiveness, especially tumor invasiveness and metastasis, including breast cancer[5]. The consequence of ER activation appears to be alterations in transcriptional activity and expression profiles of target genes. A number of genes, including cyclinD1, are regulated by ER alpha[7]
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