Inhibition Of DFOG On Ovarian Cancer Stem Cell-like Features Is Mediated By Upregulating MiR-199a-3p And Inhibiting Its Target CD44 Transcription

Purpose: To explore the mechanism by which 7-difluoromethoxyl-5,4’-di-n-octyl genistein(DFOG),a newly synthesized genistein analogue,inhibits the transcription of CD44 and the characteristics of ovarian cancer stem cells(OCSLCs)by upregulating the expression level of miR-199a-3p.Methods: Human ovarian cancer SKOV3 cells and A2780 cells and normal ovarian epithelial IOSE80 cell were adherent cultured in vitro,and then OCSLCs derived from SKOV3 or A2780 cell were obtained by the sphere formation assay.Then the expression levels of miR-199a-3p in SKOV3 cells or A2780 cells and their derived OCSLCs were analyzed by qRT-PCR.The features of OCSLCs in vitro were evaluated by detecting the sphere formation rate and by analyzing the expression levels of CD133,Bmi1 and Sox2 protein respectively using sphere formation assay and Western blot.The functional role of miR-199a-3p and CD44 in SKOV3 cells or OCSLCs was determined by transfection with miR-199a-3p mimic or miR-199a-3p inhibitor or CD44 small interfering RNA(CD44 siRNA),and then by evaluating the features of OCSLCs(including the sphere formation rate,CD133,Bmi1 and Sox2 protein expression level).A luciferase assay was applied to determine the interaction between miR-199a-3p and CD44 mRNA 3’UTR sequence.The cell vitalities of normal ovarian epithelial IOSE80 cells and ovarian cancer SKOV3 cells and A2780 cells treated with different concentrations of DFOG(1.0,5.0,10.0,20.0 μM)and genistein(GEN: 10.0,20.0,40.0,80.0 μM)were measured by CCK8 assay.The effects of DFOG on the expression levels of miR-199a-3p and its main target molecule CD44 protein,as well as the features of OCSLCs derived from ovarian cancer SKOV3 cells were systematically investigated.DFOG in combination with transfection of miR-199a-3p mimic or inhibitor or CD44 siRNA were carried out the cell loss-of function and acquisition-of function experiments based on the overexpression or knockdown of miR-199a-3p expression and knockdown of CD44 expression in SKOV3 cells or OCSLCs,respectively via comparison of the level of miR-199a-3p and CD44 expression as well as the characteristics of OCSLC.Finally,we established a nude mouse xenograft model which based on SKOV3-derived OCSLCs.The antitumor effect of DFOG in nude mice was evaluated by tumor size and tumor weight.The expression levels of miR-199a-3p and CD44 mRNA of tumor in mice bearing SKOV3-derived OCSLCs were detected by HE staining and qRTPCR to clarify the molecular mechanism underlying DFOG’s anti-tumor effect in vivo.Results: Compared with normal human ovarian epithelial cells,the expression of miR-199a-3p is lower in SKOV3 cells and derived OCSLCs.MiR-199a-3p mimic significantly increased the expression level of miR-199a-3p and down-regulated CD44 protein expression in OCSLCs,while knocking down the CD44 gene did not affect the expression of miR-199a-3p.Additionally,A luciferase assay demonstrated that CD44 mRNA 3’UTR sequence was directly targeted by miR-199a-3p in SKOV3-derived OCSLCs.MiR-199a-3p mimic or knockdown of CD44 gene can inhibit the self-renewal ability of OCSLCs and reduce the expression level of CD133,Bmi1 and Sox2 proteins.In turn,miR-199a-3p inhibitor significantly decreased the expression level of miR-199a-3p,up-regulated the expression of CD44 protein and promoted OCSLCs characteristics in SKOV3 cells.The results for drug treatments showed that DFOG and genistein up-regulated the expression of miR-199a-3p and inhibited the expression of CD44 protein in SKOV3-derived OCSLCs,in a concentrationdependent manner,while the efficacy of DFOG inhibited OCSLCs characteristics was better than genistein.Interestingly,miR-199a-3p mimic or CD44 siRNA could cooperate with DFOG to inhibit the OCSLCs characteristics when DFOG combined with miR-199a-3p mimic or CD44 siRNA,respectively.In addition,miR-199a-3p inhibitor attenuated the inhibition of DFOG on OCSLCs characteristics in SKOV3 cells.The results from a nude mouse xenograft model showed that miR-199a-3p mimic,CD44 siRN and DFOG respectively reduced the size,volume and weight of tumor in mice bearing SKOV3-derived OCSLCs.In addition,DFOG and miR-199 a treatment significantly reduced the expressions of CD44 mRNA in tumor tissue.Consistent with the result that CD44 siRNA did not affect the expression of miR-199a-3p in vitro,treatment of CD44 siRNA also did not affect the expression of miR-199a-3p of tumor in mice bearing SKOV3-derived OCSLCs in vivo.Conclusions:1.MiR-199a-3p targets for regulation of CD44 transcription in SKOV3-derived OCSLCs;the lower expression of miR-199a-3p promotes CD44 transcription and enhances OCSLCs characteristics in ovarian cancer.2.DFOG inhibits OCSLCs characteristics by inhibiting transcription of CD44 through up-regulating the expression of miR-199a-3p in ovarian cancer.