| Background＆Obiective:Based on the GLOBOCAN statistics estimates,theincidence of breast cancer accounts for23%of total cancer cases and14%of thecancer deaths. Brast cancer cells invade lymphatic or blood vessels, leading toorgans’ metastases such as liver, lung,brain, bone, and so on, which is the majorcause of death in breast cancer patients. In recent years, many scientists found thatEMT play an important role in tumor dissemination and spread. EMT is amorphogenetic process in which cells undergo a developmental switch from polarizedepithelial phenotype to highly motile mesenchymal phenotype. It is characteristiclyshowed that cell adhesion force reduced,the ability of migration and invasionincreased; the molecular basis of EMT was showed that downregulation of epithelialphenotype markers(e.g:E-cadherin,CK18,etc), upregulation of mesenchymalphenotype markers(e.g:Vimentin,α-SMA,etc). Metadherin, also known as astrocyteelevated gene-1,has been proved to be a proto-oncogene in many types of humanmalignancy. It plays a key role in many malignant biological behavior, includingproliferation, apoptosis, migration, invasion and chemotherapy drug resistance andso on.The study of MTDH in EMT is rarely reported in breast cancer. In the presentstudy, with the assistant of RNA interference method for silencing MTDH inMDA-MB-231cells, in order to making sure it’s function and mechanism in theinvasion and metastasis.Methods:1.RNA interference plasmid that can express shRNA targeting MTDHor shRNA-negative plasmid that does not match any known human coding mRNAwas designed, constructed and named MTDH-shRNA and MTDH-shRNA-neg, which were transiently transfected into MDA-MB-231cells using LipofectamineTM2000. After48hours, the cell morphology and successful transient transfection ofrecombinant plasmid into MDA-MB-231cells was confirmed.2.The mRNA andprotein level of MTDH was determined by RT-PCR and Western blot.3.Theimmigration and invasion potential was examined by Wound-healing assay andTranswell chamber invasion assay.4.The mRNA and protein level of E-cadherin andα-SMA were determined by RT-PCR and Western blot.Results:1.The transfection efficiencies of the four plasmids and cellmorphology were measured using fluorescence microscope at48hours aftertransfection. The results showed that transfection rate of plasmids was70%. Thetypical phenotypic change showed: Blank control group: Cells clusters growth, lossof cell-cell contact, spindle-like morpology that were typical of fibroblasts. Negativecontrol group: Most of cells showed spindle-like morpology, a few of cells showedcobblestone-like appearance. Experimental group: Cells dispersion growth, showedcobblestone-like morpology, which are typical of the epithelial phenotype.2.Theresults showed that: Compared with MDA-MB-231or MTDH-shRNA-neg, themRNA and protein level of MTDH were abate in MTDH-shRNA, with statisticalsignificance. Compared with MDA-MB-231, the MTDH mRNA level wasdown-regulated by41.2%, the MTDH protein level was down-regulated by40.3%.3.The results of Wound-healing assay and Transwell chamber invasion assay showed:The immigration and invasion potential of cells was decreased after downregulationMTDH in MTDH-shRNA. Compared with MDA-MB-231, the migration of cellsdecreased by44%, the invasion of cells decreased by35%in MTDH-shRNA.4.Compared with MDA-MB-231or MTDH-shRNA-neg, the mRNA and protein levelof α-SMA was reduced and E-candherin were increased in MTDH-shRNA, withstatistical significance.Conclusion:Downregulation of MTDH increased E–candherin expression andreduced α-SMA expression, which inhibited EMT in MDA-MB-231cells. Thisknockdown significantly suppressed migration and invasion in MDA-MB-231cells. |
