| OBJECTIVE: To investigate the effects of targeted regulation of BDNF by micro RNA-204(miR-204)on proliferation,apoptosis,invasion and metastasis of breast cancer cells,and to explore the role of PI3K/ Akt signaling pathway.Methods: 1.The expression of miR-204 in normal breast epithelial cell line(MCF-10A)and two kinds of breast cancer cell lines(MCF-7 and MDA-MB-231)was detected by q RT-PCR,and the next transfected cells were screened according to the results.2,through the MCF-7 in breast cancer cells transfection miR-204 before to express slow virus construction of miR-204 expression cell line(miR-204-mimics),at the same time to build the corresponding negative Control group(miR-NC)and blank Control group(Control),the CCK-8,flow cytometry,Transwell invasion and migration experiment testing groups of breast cancer cell proliferation,apoptosis,migration,and attacking changes;3.The targeting relationship between miR-204 and BDNF was verified by dual luciferase reporter gene assay;4.The m RNA expression level of BDNF in breast cancer cells of each group was detected by q RT-PCR,and the protein expression levels of BDNF,p-PI3 K,PI3K,p-Akt and Akt in breast cancer cells of each group were detected by Western blot.5.According to the results of previous studies,breast cancer cell lines transfected with miR-204 overexpression by BDNF overexpressing plasmid were constructed,breast cancer cell lines simultaneously overexpressed with miR-204 and BDNF(miR-204+BDNF)and the corresponding negative control group(miR-204+NC)were constructed,and the biological function changes of breast cancer cells in each group were detected by the same method.Results:1.The relative expression level of miR-204 in MCF-10A(2.500±0.116)was significantly higher than that of MCF-7(0.993±0.004)and MDA-MB-231(0.826±0.080)(P=0.000 < 0.001).2.(1)CCK-8 method: compared with miR-NC and Control groups,the OD value of miR-204-mimics at 24 h,48h and 72 h decreased to(0.856±0.048)(P=0.002 < 0.01),(1.273±0.045)(P=0.000 < 0.001)and(1.690±0.058)(P=0.000 < 0.001),respectively.(2)Transwell invasion:Compared with miR-NC and Control groups,the cell invasion number of miR-204-mimics decreased to(65.333±7.024)(P=0.000 < 0.001);(3)Transwell migration method: Compared with miR-NC and Control groups,the cell migration number of miR-204-mimics decreased to(58.000±21.666)(P=0.000 < 0.001);(4)Flow cytometry: Compared with miR-NC and Control groups,the total apoptosis rate of miR-204-mimics cells was increased to(13.460±0.583)%(P=0.000 < 0.001).3.Dual luciferase reporter assay: Compared with miR-NC and BDNF 3,UTR WT(wild type)co-transfection group,miR-204-mimics and BDNF 3,UTR WT(wild type)co-transfection group decreased to(5.588±0.067)(P=0.000 < 0.001),the relative luciferase activity of miR-NC and BDNF 3,UTR MUT(mutant)co-transfected group(7.946±0.089),there was no significant in the relative luciferase activity of miR-204-mimics and BDNF 3,UTR MUT(mutants)co-transfected group(8.027±0.114)(P=0.388>0.05).4.(1)q RT-PCR: Compared with miR-NC and Control groups,the relative level of BDNFm RNA of miR-204-mimics decreased to(0.071±0.002)(P=0.005 < 0.01);(2)WB: Compared with miR-NC and Control groups,the expression levels of BDNF,p-PI3 K and p-Akt in miR-204-mimics showed a significantly decreased trend,the protein expression level of Bax showed an obvious upward trend.5.(1)CCK-8 method: compared with miR-204+NC group,the OD value of miR-204+BDNF at 24,48 and 72 h was increased to(0.947±0.067)(P=0.026 < 0.05),(1.419±0.073)(P=0.003 < 0.01)and(2.145±0.070)(P=0.000 < 0.001),respectively.(2)Transwell invasion method: Compared with miR-204+NC group,the number of cell invasion in miR-204+BDNF group was increased to 121.333±4.163(P=0.031 <0.05).(3)Transwell migration method: Compared with the miR-204+NC group,the number of cell migration in the miR-204+BDNF group increased to(154.667±7.506)(P=0.000 < 0.001).(4)Flow cytometry:Compared with the miR-204+NC group,the total cell apoptosis rate in the miR-204+BDNF group decreased to(8.853±0.262)%(P=0.000 <0.001).Conclusion:By down-regulating the experssion level of BDNF,miR-204 mimics may inhibit the activity of its downstream PI3K/AKT pathway and increase the expression level of pro-apoptotic protein Bax,thereby inbibiting the proliferation,migration and invasion ability of breast cancer cells and promoting their apoptotic ablility. |
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