OBJECTIVETo evaluate the impact of a new CD44 variant on invasion of human breast cancer cell line MCF-7, and its possible mechanisms.

METHODSThe full length cDNA encoding CD44v17 was obtained from the total RNA isolated from the MCF-7/ADR cells by reverse transcript-polymerase chain reaction (RT-PCR) and subcloned into pMD19-T vector. The CD44v17 gene sequence and reading frame were confirmed by two restriction enzymes and nucleotide sequencing, and then inserted into the eukaryotic expression vector pcDNA3.1. The pcDNA3.1-CD44v17 was transfected into MCF-7 cells by Lipofectamine. The changes of MMP-2 and MMP-9 expression at gene and protein levels were detected by RT-PCR and gelatin zymography, respectively. The number of the cells through the artificial matrix membrane in every group was counted to compare the change of the invasive ability regulated by CD44 variant. The ERK and p-ERK were investigated by Western blotting to approach the molecular mechanisms of MMP-2 and MMP-9 expression regulated by CD44 variant.

RESULTSThe new gene sequence was successfully cloned into recombinant vector pcDNA3.1 and identified by the two restriction enzymes. It was confirmed that the reconstructed plasmid contained the sequence of CD44 gene variant which was composed of 1 to 4 exons, 16 to 17 exons, and 1 to 205 bases of 18 exons. The new gene sequence was sent to NCBI for publication and obtained the registered number FJ216964. The up-regulated levels of the CD44 gene mRNA and protein were respectively detected by RT-PCR and flow cytometry in MCF-7 cells transfected with pcDNA3.1-CD44v17. The invasiveness of the cells and the activity of MMP-2 and MMP-9 were clearly activated by hyaluronic acid (HA) treatment and blocked by CD44 neutralizing antibody. Pretreated MCF-7/CD44v17 cells with the neutralizing antibody against CD44 and the inhibitor of MAPKs signaling pathway strongly block the expression of p-ERK.

CONCLUSIONA new CD44 gene variant has been found in adriamycin-resistant human breast cancer MCF-7/ADR cells. The expression vector pcDNA3.1-CD44v17 has been cloned and constructed successfully. HA can be integrated with CD44 variant and then regulates the expression of MMP-2 and MMP-9, which increases the invasion ability of MCF-7 cells through the Ras/MAPK signaling pathway.