| Objectives To investigate the effect of different expression levels of Rab25 gene on biological functions in breast cancer cells.To investigate the effect of Rab25 gene on ER and PR expression in breast cancer cell lines.To investigate the effect of different expression levels of Rab25 and Her-2/neu gene on biological functions in breast cancer cells.Methods Eukaryotic expression plasmid pDNR-Dual-Rab25 was constructed.Expression of Rab25 was up-regulated and down-regulated by transfection and RNA interference in breast cancer cell lines MCF-7, MDA-MB-231(MM231),SK-BR-3,respectively.The transfection rate of Rab25 mRNA and siRNA was measured by flow cytometry.The relative expression rate of Rab25 mRNA was measured by real-time quantitative PCR. Proliferation,colony forming,invasion ability of all the researched breast cancer cells were also detected.Breast cancer cell lines MCF-7,MDA-MB-231,SK-Br-3 with different expression level were constructed by transfection and RNA interference.ER,PR were measured before and after transfection,also after RNA interference by western blot.Eukaryotic expression plasmid pDNR-Dual-Rab25 and pDNR-Dual-Her-2/neu were constructed.Expression of Rab25 and Her-2/neu were up-regulated and down-regulated by transfection and RNA interference in breast cancer cell lines MCF-7,MM231,SK-BR-3,respectively.The transfection rate of Her-2/neu mRNA and siRNA was measured by flow cytometry.The protein expression of Her-2/neu was measured by Western blot.Proliferation, colony forming and invasion ability were also detected.Results Rab25 could be checked out only in MCF-7.After the recombinant eukaryotic plasmid pDNR-Dual-Rab25 was constructed successfully,determined by PCR and enzyme digestion analysis,The recombinant plasmids were transfected into all the researched breast cancer cell lines.Then by RNA interference,the expression of Rab25 was decreased in breast cancer cell lines.The tansfection rate of Rab25 mRNA and siRNA were detected,the results revealed there was significant difference(P＜0.01)about the tansfection rate of Rab25 mRNA and siRNA between experimental groups and control groups.The result of real-time PCR revealed there was significant difference(P＜0.01)about the relative expression quantity of Rab25 mRNA between experimental groups and control groups.So the Rab25 mRNA and siRNA were transfected successfully.In the researched breast cancer cell lines,the proliferation rate, colony forming rate,and invasion ability increased after the transfection of Rab25,while decreased after the interference of Rab25 gene.The difference was significant(P＜0.05)between experimental groups and control groups.Before Rab25 mRNA transfected,clear and corret ER,PR bands could be oberserved only in MCF-7,whereas they were expressed in all the three cell lines and the ER,PR band of MCF-7 got more evident after transfection. What's more,after RNA interference,the ER,PR protein expression reduced evidently,the ER,PR band of MCF-7 got lighter than the print before RNA interference.After the recombinant eukaryotic plasmid pDNR-Dual-Her-2/neu was constructed successfully,determined by PCR and enzyme digestion analysis,The recombinant plasmids were transfected into MCF-7.Then by RNA interference,the expression of Her-2/neu was decreased in breast cancer cell lines.The tansfection rate of Her-2/neu mRNA and siRNA were detected,the results revealed there was significant difference(P＜0.01)about the tansfection rate of Her-2/neu mRNA and siRNA between experimental groups and control groups.The Her-2/neu protein expression was detected by Western blot in all the researched breast cancer cell lines.The results revealed Her-2/neu was under-expressed in MCF-7 and over-expressed in MM231 and SK-BR-3 before Her-2/neu mRNA transfection,the Her-2/neu band of MCF-7 got more evident after transfection.After Her-2/neu RNA interference,the Her-2/neu protein expression in all the breast cancer cell lines of Her-2/neu over-expressed reduced evidently.So the Her-2/neu mRNA and siRNA were transfected successfully.In MCF-7 of Rab25 under-expressed,the proliferation rate,colony forming rate,invasion ability increased evidently with the transfection of Her-2/neu.In MCF-7 of Rab25 over-expressed,the proliferation rate,colony forming rate,invasion ability had no change with the transfection of Her-2/neu.In MM231 and SK-BR-3 of Her-2/neu over-expressed,the proliferation rate,colony forming rate,invasion ability increased evidently with the transfection of Rab25.In MCF-7,MM231 and SK-BR-3 of Rab25 and Her-2/neu over-expressed,the proliferation rate,colony forming rate,invasion ability had no change after Her-2/neu mRNA was shut down by RNA interference.Conclusions In MCF-7,MM231 and SK-BR-3,the proliferation rate,colony forming rate,and invasion ability increased with the transfection of Rab25,while decreased with the interference of Rab25 gene.Rab25 plays important roles in the growth,proliferation and invasion of breast cancer cells,which provides new ideas for the basical research and clinical application in breast cancer.The expression of ER,PR increased or decreased with Rab25 up-regulated and down-regulated in MCF-7,MDA-MB-231,SK-Br-3.There is Synchronous dependency between the expression of Rab25 gene and ER,PR in breast cancer cell lines.Expression of Rab25 may directly reflect the expression level of ER and PR,prompting new ideas and methods for the diagnosis and treatment of breast cancer.In breast cancer cell lines MCF-7,MM231,SK-BR-3,the proliferation rate, colony forming rate,invasion ability increased after the single gene transfection of Rab25.But expression of Her-2/neu can't change biological activity in breast cancer cell lines of Rab25 over-expressed.These prove Rab25 and Her-2/neu change biological activity of breast cancer cell lines by the different paths.The test provides new theoretical evidence for the basical research in breast cancer. |
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