Calycosin Inhibits Growth Of ER-negative Breast Cancer Cells Via Down-regulation Of HOTAIR

Objective: By exploring the effects of calycosin on the proliferation and apoptosis of ER-negative breast cancer cell and clarifying the role of lnc RNA-HOTAIR in human breast cancer progression,we aimed to provide new anti-cancer target for phytoestrogens and offer preliminary evidence for diagnosis and targeted treatment of human breast cancer.Methods: Two ER-negative breast cancer cell lines,MDA-MB-231 and SK-BR-3,were cultured with different concentrations of calycosin(0,20,40 and 80μM)for 48 h.MTT,Hoechst33258 staining and flow cytometry assays were respectively used to detect the effect of calycosin on cell proliferation and apoptosis.The expression levels of poptosis-related factors and HOTAIR were evaluated by WB and RT-PCR assays.Next,the involvment of HOTAIR in calycosin induced anti-cancer effects was demon-strated by transient overexpression or knockdown of HOTAIR in MDA-MB-231 and SK-BR-3 cells,followed by MTT,Hoechst33258 staining,flow cytometry and RT-PCR assays.Furthermore,the relationship between HOTAIR and the HOTAIR-related RNA binding proteins,HuR and IGF2BP1,was forecasted by Starbase database and verified by RNA binding protein immunoprecipitation(RIP).Similarly,calycosinmediated regulation of HuR,IGF2BP1,Bcl2 and Bax expression by HOTAIR were investigated in ER-negatived breast cancer cells with overexpression and knockdown of HOTAIR.Results: In MDA-MB-231 and SK-BR-3 cells,calycosin significantly inhibited cell proliferation and promoted apoptosis in a dose-dependent manner,which was confirmed by MTT,Hoechst 33258 staining and flow cytometry assays(P<0.05 or P<0.01).Accordingly,RT-PCR and WB results showed that calycosin treatment decreased the expression levels of HOTAIR and the anti-apoptotic Bcl2 but increased the levels of pro-apoptotic Bax(P<0.05).As expected,the up-regulation of HOTAIR promoted proliferation and inhibited apoptosis of ER-negative breast cancer cells.More importantly,the inhibitory effects of calycosin on MDA-MB-231 and SK-BR-3cells were also partially impaired by HOTAIR overexpression,whereas the down-regulation of HOTAIR had the opposite effect(P<0.05).The Star Base database showed that HuR and IGF2BP1 were the potential downstream target of HOTAIR in MDA-MB-231 and SK-BR-3 breast cancer cells,which was identified by RIP assay(P<0.05 or P<0.01).Subsequently,it was found that HOTAIR up-regulation led to the reduced expression of Bax and the elevated expression levels of HuR,IGF2BP1 and Bcl2(P<0.05).Furthermore,HOTAIR overexpression enhanced the induction of Bax and the inhibition of HuR,IGF2BP1 and Bcl2 by calycosin(P<0.05).In contrast,knockdown of HOTAIR exerted the opposite effects on regulation of HuR,IGF2BP1 and poptosis-related factors,and blocked calycosin-induced anti-cancer activity(P<0.05).Conclusion: Calycosin could inhibit proliferation and induce apoptosis of ER-negative breast cancer cells by down-regulating the expression of IGF2BP1 and HuR via suppressing HOTAIR expression.