The Study On The Effect Of MiR-23b-3p/GnRHR/DUSP1 Axis In Promoting Proliferation And Metastasis In Triple-Negative Breast Cancer And Its Underlying Mechanism

Objective:The microRNA miR-23b-3p,gonadotropin-releasing hormone receptor(GnRHR),and dual specificity phosphatase-1(DUSP1)are involved in various biological processes such as tumor cell proliferation and metastasis.However,their expression patterns,biological roles,and molecular regulatory mechanisms in triple-negative breast cancers(TNBC)are still unclear.In this paper,we focused on the biological effects of miR-23b-3p,GnRHR,and DUSP1 on the proliferation and metastasis of TNBC and their molecular mechanisms.Methods:(1)The expression level of miR-23b-3p in MDA-MB-231,MDA-MB-468,MCF-7 and MCF-10A was detected by the real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).The miRNA mimics and inhibitor of miR-23b-3p were constructed and transfected with in vitro cultured TNBC cells MDA-MB-231 and MDAMB-468,respectively.Then we used CCK-8 assay and clone formation assay to detect the proliferation ability of cancer cells,flow cytometry to detect apoptosis and cell cycle of cancer cells,scratch method to detect the migration ability of cancer cells,and Transwell assay to detect the invasion ability of cancer cells,respectively.(2)MDA-MB-231 cells were cultured in vitro and transfected with GnRHR plasmid to overexpress GnRHR and GnRHR siRNA to knock down GnRHR,respectively.CCK-8,scratch method,and Transwell were used to detect the effect of GnRHR on cell proliferation and metastasis,and to observe whether GnRHR could reverse the miR-23b-3p effect.(3)A miR-23b-3p stable low expression MDA-MB-231 cell line was constructed and used to construct a subcutaneous transplantation tumor model in nude mice to observe the effect of knockdown of miR-23b3p on tumor proliferation and changes in GnRHR expression in tumor tissues.The miR-23b3p and GnRHR expression data were obtained from the TCGA public database to analyse the relationship between the expression of both in breast cancer tissues and the relationship with prognosis.TNBC specimens operated at our centre were collected and GnRHR protein expression was detected using immunohistochemistry.(4)The interaction between miR23b-3p and GnRHR gene was detected by dual luciferase reporter gene analysis,and the effect of miR-23b-3p on GnRHR was detected by protein blotting(western blot,WB).MDA-MB-231 cells were cultured in vitro and transfected with GnRHR plasmid to overexpress GnRHR.Changes in mRNA expression profile were detected by RNAseq,and changes in p-AKT/AKT,p-ERK/ERK and MMP2 were detected by WB.Results:(1)The expression level of miR-23b-3p was higher in MDA-MB-231 and MDA-MB-468 cells than in MCF-7 and MCF-10A cells.Transfection of miR-23b-3p mimics in MDA-MB-231 and MDA-MB-468 cells cultured in vitro resulted in increased cell growth,proliferation,migration,and invasion,whereas transfection of miR-23b-3p inhibitor produced the opposite effect.(2)Transfection of GnRHR overexpression plasmid or siRNA in MDA-MB-231 cells cultured in vitro resulted in the overexpression of GnRHR inhibiting TNBC proliferation,metastasis,and reversing the effect of miR-23b-3p.(3)The miR-23b-3p low expression and wild-type MDA-MB-231 cells were used to construct subcutaneous implantation tumor models in nude mice,respectively.The low expression of miR-23b-3p resulted in slower tumor growth and higher GnRHR protein expression levels.The expression of miR-23b-3p and GnRHR in cancer tissues of TNBC patients was negatively correlated.High expression of miR-23b-3p in TNBC was associated with low disease-free survival(DFS)of patients,while low expression of GnRHR in TNBC was also associated with low DFS of TNBC patients.(4)MiR-23b-3p has the site that binds specifically to GnRHR,and they can bind directly through the site.And WB assay suggested that miR-23b-3p inhibited GnRHR protein expression.RNAseq assay revealed that DUSP1 expression was upregulated after GnRHR overexpression,and WB assay suggested that pAKT/AKT,p-ERK/ERK,and MMP2 were decreased after GnRHR overexpression.Conclusions:The high expression of miR-23b-3p in TNBC has a role in promoting TNBC proliferation and metastasis,and is associated with poor patient prognosis.The molecular mechanism may be that miR-23b-3p inhibits GnRHR expression,which in turn inhibits DUSP1 expression,so that ERK and AKT cannot be dephosphorylated and inactivated,and MMP2 expression is upregulated,ultimately promoting TNBC proliferation and metastasis.The miR-23b-3p/GnRHR/DUSPl axis may be a novel potential therapeutic target for TNBC.