MiR-519d-5p Modulates The Sensitivity Of Breast Cancer To Chemotherapy By Forming A Negative Feedback Loop With RELA

Purpose: To investigate the effect of miR-519d-5p on the sensitivity to chemotherapeutic agents of breast cancer and its functional mechanism.Methods:(1)RT-PCR was used to detect the expression of miR-519d-5p in breast cancer tissues and paracancer tissues,breast cancer cell lines and non-neoplastic cell line.(2)MTT assay was used to determine the effect of miR-519d-5p on the sensitivity to chemotherapeutic agents of breast cancer.(3)Western Blotting and RT-PCR were used to detect the expression of RELA in breast cancer cell lines and non-neoplastic cell line.(4)Bioinformatics tools were used to predict the target gene of miR-519d-5p,and the predicting result was verified by Dual luciferase reporter gene assay.(5)RT-PCR and Western Blotting were used to detect the effect of miR-519d-5p on the transcription and protein levels of RELA.(6)Bioinformatic prediction tool Jaspar was used to predict the combination of RELA and miR-519d-5p promoter,and the predicting result was verified by Ch IP and DNA pull down.(7)RT-PCR was used to detect the effect of RELA on miR-519d-5p transcription.(8)Scratch wound healing assay was used to detect the migration effect of miR-519d-5p and/or RELA on breast cancer cells.(9)MCF-7 cell subcutaneous xenotransplanted tumor was established to examine the cell proliferation and drug sensitivity effect of miR-519d-5p on breast cancer in vivo.Results:(1)miR-519d-5p was low-expressed in breast cancer tissues compared with paracancer tissues.Similarly,miR-519d-5p was low-expressed in breast cancer cells compared with non-neoplastic cells,which is contrary to the expression of RELA.(2)miR-519d-5p overexpression sensitized breast cancer cells to chemotherapeutic agents in vitro.(3)The result of bioinformatics tools and Dual luciferase reporter gene experiment indicated that RELA was one of the target genes of miR-519d-5p.(4)The results of Western Blotting and RT-PCR showed that miR-519d-5p negatively regulated the transcription and translation level of RELA.(5)Bioinformatic prediction tool Jaspar,Ch IP and DNA pull down analysis suggested that RELA may directly bind to the miR-519d-5p promoter.(6)RT-PCR analysis showed that RELA negatively regulated the transcription level of miR-519d-5p.(7)The result of Scratch wound healing assay showed that miR-519d-5p regulates the migration of breast cancer cells by forming a negative feedback loop with RELA.(8)miR-519d-5p inhibited the growth of MCF-7 tumor and sensitized MCF-7 tumor to Dox(Doxorubicine)in vivo.Conclusion: miR-519d-5p played a pivotal role in regulating the sensitivity to chemotherapeutic agents by forming a negative feedback loop with RELA.These findings provide a new therapeutic strategy for the combinational use of miR-519d-5p and chemotherapeutic agents to overcome chemoresistance.