Experimental Study On Expression Of A New CD44 Variant In Multidrug Resistance Breast Cancer Cells And The Role Of The CD44 Variant To Human Breast Cancer Cells

Objective:To clone CD44 gene from multidrug resistant human mammary carcinoma cells(MCF-7/Adr)and construct its eukaryotic expression vector which lays the groundwork for the experiments with multidrug resistance,invasion and proliferation. Secondly,to analyze the relationship between CD44v expression and invasion of breast cancer cell line MCF-7,and to explore possible molecular mechanisms of MMP-2 and MMP-9 regulated by CD44 vatiant.Finally,to study the relationship between CD44v expression and the proliferation of MCF-7/CD44v cells.Methods:①The expression of CD44 gene and protein in the MCF-7 and MCF-7/Adr cells was detected by RT-PCR and flow cytometry.②The full length cDNA encoding CD44 was obtained by RT-PCR from the total RNA isolated from the MCF-7/Adr cells.In order to get CD44 gene contained the site of the two restriction enzymes EcoRⅠand KpnⅠ,the primer contained the site of the two restriction enzymes was used for polymerase chain reaction(PCR).Treated the PCR product with 1%Agarose DNA gel electrophoresis,then reclaimed and purified the CD44 gene according to the directions.③After purification,the cDNA fragment was cloned into the pMD19-T simple vector.The recombinant plasmid pMD19-T-CD44v was transformed into E.coli DH5αfor amplification,and then digested with two restriction enzyme EcoRⅠand KpnⅠafter being extracted with Plasmid Mini Preparation Kit.The nucleotides sequencing of the CD44 gene was confirmed by DNA sequencing.④The eukaryotic expression vector pcDNA3.1 and the recombinant plasmid pMD19-T-CD44v were digested with EcoRⅠand KpnⅠ,respectively,then connected by T4 DNA ligase and amplificated in DH5α.The eukaryotic expression vector pcDNA3.1-CD44v was identified by the two restriction enzyme and nucleotides sequencing.⑤The recombinant vector pcDNA3.1-CD44v was transfected into MCF-7 cells by Lipofectamine,and the expression changes of CD44 gene and protein were detected by Semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) and flow cytometry at 48 hour later,respectively.⑥After incubation with HA for 24 hour,all the cells in each group were collected for the detection of flow cytometry and RT-PCR.The conditioned medium were collected,clarified by centrifugation,and subjected to gelatin zymography.⑦The number of the cells through the artificial matrix membrane in every group was counted to compare the change of the invasion capability among the expermental groups.⑧Molecular mechanism of MMP-2 and MMP-9 expression regulated by CD44 vatiant was detected by western blotting.⑨.The number of the cells was counted by microscope to detect diverse proliferation among the experiment groups.Results:①.CD44 gene variant expressed in MCF-7/Adr cells,but not in MCF-7 cells.②The CD44 gene contained the site of the two restriction enzymes EcoRⅠand KpnⅠwas introduced into pMD19-T simple vector.After the identification and sequencing,the reconstructed plasmid was confirmed containing the sequence of CD44 gene variant which contains 1 to 4 exons,16 to 17 exons,and 1 to 205 basees of 18 exons.The new gene sequence was sent to NCBI for publication,registered number is FJ216964.③The new gene sequence was successfully cloned into recombinant vector pcDNA3.1 and identified by the two restriction enzyme as well as sequencing,the sequences of recombinant plasmid pcDNA3.1-CD44v were the same as that of FJ216964.④After transfered recombinant vector pcDNA3.1-CD44v to MCF-7 48 hour, RT-PCR and flow cytometry detected the mRNA and protein level of CD44 gene variant were obviously up-regulated.The expression ratio of CD44 protein in MCF-7/CD44v cells is 70.0±2.5%.Cells after transfection were screened by the conditioned medium G418 to obtain positive cell clones.Stable G418-resistant clones were obtained 2 week later and the expanded cells were then used for subsequent studies.⑤Overexpression of CD44v by transfecting tumor cells with CD44v cDNA increases MMP-2,MMP-9 expression as well as invasion capability of MCF-7 cells(P<0.05);⑥Our studies also indicate the activation of MMP-2 and MMP-9 secretion could be blocked by CD44 blocking anti-body.Pretreated MCF-7/CD44v cells with the neutralizing antibody against CD44 could strongly block the invasion of MCF-7/CD44v cells.⑦Finally,it is important to note that CD44v could inhibit the proliferation of MCF-7/CD44v cells.Conclusion:①The expression vector pcDNA3.1-CD44v was constructed successfully and could be expressed in human mammary carcinoma cells.A new CD44 gene vatiant was found in MCF-7/Adr cells(GeneBank NO.FJ216964);②Our study may start a new approach to study the function of CD44 gene vatiant and provide a method of gene therapy.③CD44 vatiant could increase invasion capability of MCF-7 cells though influence expression of MMP-2 and MMP-9.④HA could integrate With CD44 vatiant and regulate expression of MMP-2 and MMP-9,which increased invasion capability of MCF-7 cells through Ras/MAPK signaling pathways.④Over expression of CD44 gene vatiant could inhibit proliferation of MCF-7/CD44v cells.