MIRNA-199A-5P Inhibits The Invasion Of MDA-MB-231 Cells Via Regulating ERK5 Through SP1

Objective: miRNA,a kind of highly conservative endogenous non-coding single small RNAs,promote the degradation of target m RNA or inhibit its protein translation mainly through specific recognition for target m RNA 3'UTR,playing a role of negative regulation on the expression of genes.In recent years,a large number of studies have shown that miRNA in different tumors could be used as oncogenes or suppressor genes involved in regulating the invasion and metastasis of tumor cells.It was verified that in a wide variety of tumor tissues and cells the expression of miR-199a-5p was lower,including invasive breast cancer.miR-199a-5p could also promote tumor occurrence and progress via the MAPK signaling pathways.ERK5 was found to be the most late protein kinase of all MAPK family members and significantly enhanced the migration and invasion capacity of breast cancer cells.In this experiment,miR-199a-5p mimic was transient transfected into MDA-MB-231 cells.the molecular mechanism of invasion and the relationship between miR-199a-5p and ERK5 in breast cancer cells were discussed by using transwell invasion experiment,cell immunofluorescence and western blot methods,which could provide the basis on targeted miR-199a-5p for breast cancer therapy.Methods:(1)MDA-MB-231 cells were cultivated.The method of Lipofectamine2000 was used to transfect miR-199a-5p mimic transiently into cells when the density of cells was 80%,miR-199a-5p mock as a control group.Transfect Digoxin labeled short chains of si RNA into MDA-MB-231 cells to test exogenous RNA.Transwell invasion assay was applied to detect the invasion ability of the cells in miR-199a-5p mimic/MDA-MB-231 and miR-199a-5p mock/MDA-MB-231 groups.(2)Cell immunofluorescence and western blot were put into use to test the expressions of E-cadherin and vimentin in the cells of miR-199a-5p mimic/MDA-MB-231 and miR-199a-5p mock/MDA-MB-231.The ERK5,p ERK5,EGF and SP1 expressions were also investigated with Western blot.Further,using the LNA-si RNA inhibition of miR-199a-5p,the expressions of ERK5,p ERK5,EGF and SP1 were detected in miR-199a-5p LNA-si RNA and LNA-scramble si RNA cells.(3)Bioinformatics software on line was applicated and the result diaplayed that there is a region-201 /-207(GGGCGG)located upstream of ERK5 promoter which contains SP1 binding sites.The chromatin immunoprecipitation was adopted to detect the relationship between ERK5 and SP1.Results:(1)After Digoxin labeled short chains of siRNA transfected into cells 4hours,MDA-MB-231 cells showed large amounts of exogenous RNA and lasted for three days,which indicated that transfection was successful.Transwell invasion results: MDA-MB-231 cells with overexpressing miR-199a-5p,the number of invasive cells of the miR-199a-5p mimic/MDA-MB-231 group was decreased significantly compared with miR-199a-5p mock/MDA-MB-231 group(P<0.05).(2)Cell immunofluorescence and western blot results: in miR-199a-5p mimic cells,the E-cadherin expression was increased dramatically in the membrane while vimentin expression was significantly reduced in the cytoplasm compared with the miR-199a-5p mock group.At the same time,western blot test also showed that the expression of E-cadherinprotein was higher and the vimentin expression was lower markedly in the miR-199a-5p mimic cells than in the miR-199a-5p mock cells(P<0.05).(3)Western blot results displayed that in miR-199a-5p overexpressing cells,compared with miR-199a-5p mock cells,the expressions of ERK5,p ERK5,EGF and SP1 were dramatically decreased(P<0.05).However,with the treatment of application of LNA-si RNA to silence miR-199a-5p,ERK5,p ERK5,EGF and SP1 expressions in miR-199a-5p LNA-si RNA cells were obviously elevated compared with LNA-scramble si RNA cells(P<0.05).(4)Chromatin immunoprecipitation showed that MDA-MB-231 cells treated with antibody of SP1 appeared specific DNA banding,but without corresponding strip in the cells with IgG antibody treatment.Conclusion:(1)miR-199a-5p regulates the expressions of EMT related marker E-cadherin and vimentin,and inhibits the invasion of breast cancer MDA-MB-231 cells,which provides a reference basis for considering miR-199a-5p as a targeted treatment of breast cancer.(2)miR-199a-5p plays a role of inhibition of MDA-MB-231 cell invasion by regulating the expression of EGF and SP1 to cut down expression of ERK5 and suppress its phosphorylation signaling pathways.