The Mechanism Of LINC01189-miR-586-ZEB1 Feedback Loop In Regulation Of Breast Cancer Progression Through Wnt/β-catenin Signaling

Background The Global Cancer Statistics for 2020 reported that breast cancer as the most common diagnosed cancer in women had overtaken lung cancer,with a 2.3 million new cases of breast cancer(11.7%)in the worldwide last year.Statistical results of the 2020 Cancer Analysis of American Cancer Society showed that the long-term decline of breast cancer mortality had stopped,which taked more attention to the treatment of breast cancer.Nc RNA played an important role in the progression of breast cancer by regulating proliferation,differentiation,invasion and metastasis of breast cancer.Although some studies had shown that mi R-586 was an oncogene and the down-regulation of mi R-586 could inhibit proliferation and invasion of breast cancer in vitro,the role of mi R-586 in breast cancer had not been studied.Objectives The study aims to elucidate the role of mi R-586 in proliferation and metastasis of the breast cancer and the molecular mechanism in regulating the Wnt/β-catenin signaling pathway.Methods Firstly,the expression of mi R-586 in breast cancer cells and normal cells,breast cancer tissues and normal breast tissues were detected by RT-q PCR and the expression of mi R-586 on the prognosis of breast cancer patients was analyzed by the KM-plotter survival curve.We constructed MDA-MB-231 cell lines with stable down-expression of mi R-586.The effects of mi R-586 on the proliferation were determined by MTT and clonal formation experiments.The effects of mi R-586 on the migration and invasion were determined by transwell assays and Wound Healing assays.Tumor formation experiments in vivo were performed by injecting 231-anti-586 and 231-control cells to mice.Then we investigated the expression of mi R-586 on pathway activity by dual luciferase reporting assay.We analyzed the nuclear localization of β-catenin by immunofluorescence and WB after nucleoplasma separation.Then,the expression of mi R-586 on the interaction between TCF4 and β-catenin was detected by Co-IP.Then we predicted the downstream targets of mi R-586 by Target Scan and we verified it by RT-q PCR assay,WB assay,dual luciferase reporting assay.Thirdly,we predicted that the LINC01189 could sponge with mi R-586.We verified the cell localization of LINC01189 by RT-q PCR after nucleoplasmic isolation and FISH assay.Then,we defined the regulatory relationship between them by RIP experiment and dual luciferase reporting assay.We further clarified the function of LINC01189 by MTT assay,clonal formation experiments,transwell assay,wound healing assays in vitro and tumor formation experiments in vivo,then we further explored the mechanism.Lastly,we further explored the regulatory relationship among ZEB1 and LINC01189,β-catenin/TCF4 and ZEB1 by RT-q PCR,Ch IP assay and dual luciferase reporting assay.Results The expression of mi R-586 was higher in breast cancer cells and breast cancer tissues with a poor prognosis and lower in normal breast cells and normal breast tissues with a better prognosis.Our studies had confirmed that silencing mi R-586 can inhibit the proliferation and metastasis of breast cancer in vitro and in vivo.The overexpression of mi R-586 significantly induced the activation of Wnt signaling pathways in MCF7 and SKBR3 cell and the overexpression of mi R-586 could increase the nuclear localization of β-catenin.Co-IP showed that overexpression of mi R-586 significantly enhanced the interaction between β-catenin and TCF4 in MCF7 and SKBR3 cell.The overexpression of mi R-586 can reduce the expression of SFRP1 and DKK2/3 which was signaling pathway inhibitors of Wnt/β-catenin.Dual luciferase reporting assay and WB assay further proved that SFRP1 and DKK2/3 were target genes of mi R-586 and mi R-586 could inhibit the expression of SFRP1 and DKK2/3.We predicted that LINC01189 could bind to mi R-586.Further analysis showed that LINC01189 had a lower expression in breast cancer cells.LINC01189 was mainly expressed in the cytoplasm and the results of dual luciferase report assay and RIP assay indicated that LINC01189 can sponge with mi R-586.IHC results showed that the expression of mi R-586 and LINC01189 was correlated negatively.The overexpression of LINC01189 can inhibit the proliferation,migration and invasion of breast cancer in MDA-MB-231 cells.LINC01189 could inhibit tumor growth and lung metastasis in vivo experiments.KM-plotter showed that the higher expression of LINC01189 had a good prognosis.LINC01189 could increase the expression level of E-cadherin and decrease the expression level of VIM.LINC01189 was down-regulated in MCF7 cells with the overexpression of ZEB1.Ch IP assay and dual luciferase report assay showed that ZEB1 could inhibit the expression of LINC01189.IHC assay showed a negative correlation between the expression of ZEB1 and LINC01189 and a positive correlation between β-catenin nuclear expression and ZEB1.Ch IP assay,RT-q PCR assay and dual luciferase reporting assay showed that TCF4 could bind to ZEB1 and β-catenin/TCF4 transactivates ZEB1 expression.Conclusion In the present study,we showed that mi R-586 was an oncogene by inducing proliferation and metastasis of breast cancer both in vitro and in vivo.mi R-586 induces Wnt/β-catenin activation by directly targeting SFRP1 and DKK2/3 which were the signaling antagonist of the Wnt/β-catenin.Moreover,we demonstrated that LINC01189 functions as a tumor suppressor and inhibits breast cancer progression by inhibiting an epithelial-mesenchymal transition-like phenotype by sponging mi R-586.In addition,β-catenin/TCF4 transactivates ZEB1,resulting in a transcriptional repression of LINC01189 expression.Our data discovered the LINC01189-mi R-586-ZEB1 feedback loop and provided a mechanism to explain the Wnt/β-catenin signaling in progression of breast cancer.