MicroRNA-29a-3p Activates The P70s6k/S6 Signaling Pathway By Binding And Upregulating TRAF2 To Promote Breast Cancer Cells Proliferation,Migration And Invasion

Objective: To explore the expression of micro RNA-29a-3p in breast cancer cell lines,and to clarify the effects of miR-29a-3p on TRAF2 and p70s6k/S6 signaling pathways,as well as on the proliferation,migration and invasion of breast cancer cells.Methods: Firstly,the expression levels of miR-29a-3p in breast cancer cell lines MCF-7,T47 D,MDA-MB-231,BT-549,BT-474 and normal breast cell lines MCF-10 A were detected by real-time polymerase chain reaction RT-PCR.MCF-7 cells and MDA-MB-231 cells with higher expression of miR-29a-3p were selected for subsequent experiments.The effects of miR-29a-3p on the proliferation,migration and invasion ability of MCF-7cells and MDA-MB-231 cells were investigated by MTT assay,colony formation assay and transwell assay,and the protein expressions of ROCK,mmp2 and p21 were further detected by Western Blot.Subsequently,after transfection of miR-29a-3p mimic NC,miR-29a-3p mimic,miR-29a-3p inhibitor NC and miR-29a-3p inhibitor in MCF-7 and MDAMB-231 breast cancer cell lines.Under the premise of ensuring transfection efficiency,the expression levels of key proteins in the p70s6k/S6 signaling pathway were detected.After addition of specific inhibitor of p70s6k in the transfection of miR-29a-3p mimic,the expression levels of phosphorylated p70s6k and S6 were detected.The expression levels of TRAF2 m RNA and protein were detected by q RT-PCR and Western Blot respectively,and the combination of miR-29a-3p and TRAF2 was verified by the luciferase reporter gene assay using database analysis and prediction.Finally,after co-transfection with siTRAF2 and miR-29a-3p mimic,the expression levels of phosphorylated p70s6k and S6 were detected by Western Blot assay.Results: RT-PCR results showed that the expression of miR-29a-3p was significantly increased in breast cancer cell lines such as MCF-7 cells,MDA-MB-231 cells and BT-549 cells compared with human normal breast cancer cells(MCF-10A).Western Blot analysis showed that the proliferation,migration and invasion ability of MCF-7 cells and MDAMB-231 cells transfected with miR-29a-3p mimic were significantly increased,while the proliferation,migration and invasion ability of MCF-7 cells and MDA-MB-231 cells transfected with miR-29a-3p inhibitor were significantly decreased.MTT method and colony formation experiment results further verified that transfection of miR-29a-3p mimic promoted the cells proliferation.Transwell experiment results further verified that transfection of miR-29a-3p mimic improved the cells migration and invasion ability.The opposite results were found in transfection of miR-29a-3p inhibitor group.In addition,Western Blot results showed that after transfection of miR-29a-3p mimic in MCF-7 cells and MDA-MB-231 cells,the phosphorylation levels of p70s6k and S6 were significantly increased,but the total protein levels of p70s6k and S6 were not significantly changed.However,after transfection of miR-29a-3p inhibitor,the phosphorylation levels showed an opposite trend,but the total protein levels were still not significantly changed.After the specific inhibitor of p70s6k was added,the expression level of phosphorylated S6 was significantly inhibited.In addition,luciferase reporter gene assay verified that miR-29a-3p could bind to TRAF2.q RT-PCR and Western Blot results proved that miR-29a-3p had a regulatory effect on TRAF2,and miR-29a-3p was positively correlated with TRAF2 m RNA and protein expression levels.Finally,after co-transfection with si-TRAF2 and miR-29a-3p mimic,the down-regulation effect of si-TRAF2 on phosphorylated p70s6k and S6 was significantly reversed.Conclusion: In summary,we can conclude that miR-29a-3p promotes the proliferation,migration and invasion of breast cancer cells by binding and upregulating TRAF2 to activate the p70s6k/S6 signaling pathway.