miR-141-3p、KLF9异常表达对前列腺癌细胞株药物敏感性和雄激素受体表达的影响及靶向关系OACSTPCD

Objective To observe the effects of microRNA-141-3p(miR-141-3p)and Krüppel-like factor 9(KLF9)on the drug sensitivity and androgen receptor(AR)expression of prostate cancer(PCa)cells,and to verify the targeting relationship between miR-141-3p and KLF9 in order to explore the regulatory effects and mechanism of miR-141-3p and KLF9 on the drug sensitivity of PCa cells.Methods Androgen-sensitive LNCaP cell line with AR expression was selected for this study.The cells were divided into low miR-141-3p expression group,normal miR-141-3p group,KLF9 overexpression group,and normal KLF9 expression group,respectively,which were transfected with the miR-141-3p-in-hibitor,inhibitor-negative control(NC)group,KLF9 overexpression plasmid,and empty plasmid,respectively,using li-posome transfection method.The cells were treated with different concentrations of bicalutamide(0,10,25,50,75,and 100 μmol/L)or docetaxel(0,0.2,0.5,1.0,2.5,and 5.0 nmol/L).After 48 h of culture,cell OD value was detected by CCK-8,and cell proliferation rate was calculated to evaluate drug sensitivity.The expression of AR and AR-V7 of LN-CaP cells was analyzed by qRT-PCR and Western blotting.TargetScan database was used to predict binding sites between miR-141-3p and KLF9.Then,the luciferase reporter gene experiment was used to verify the targeted regulatory relation-ship between miR-141-3p and KLF9.LNCaP cells were divided into groups A,B,C and D.Cells in the group A were transfected with wild-type KLF9 luciferase reporter gene plasmid(KLF9-WT)and inhibitor-NC;cells in the group B were transfected with KLF9-WT and miR-141-3p-inhibitor;cells in the group C were transfected with mutant KLF9 luciferase re-porter gene plasmid(KLF9-Mut)and inhibitor-NC;cells in the group D were transfected with KLF9-Mut and miR-141-3p-inhibitor.The relative luciferase activity of cells in each group was detected by dual luciferase reporter gene assay at 24 h after transfection.The inhibitory effect of miR-141-3p targetedly regulating KLF9 on the drug sensitivity of LNCaP cell was further examined by the rescue experiment.LNCaP cells were divided into groups a,b,c and d.Cells in the group a were transfected with si-KLF9 negative control(si-Ctrl)and inhibitor-NC;cells in the group b were transfected with si-Ctrl and miR-141-3p-inhibitor;cells in the group c were transfected with si-KLF9 and inhibitor-NC;cells in the group d were trans-fected with si-KLF9 and miR-141-3p-inhibitor.After transfection for 24 h,the cells were treated with 75 μmol/L bicaluta-mide or 2.5 nmol/L docetaxel for 48 h,respectively.The OD value of the cells was detected by CCK-8,and the cell prolif-eration rate was calculated.Results LNCaP cells were cultured for 48 h.When no drug was added,the cell prolifera-tion rate of the low miR-141-3p expression group was lower than that of the normal miR-141-3p expression group(P<0.05),and the cell proliferation rate of the KLF9 overexpression group was lower than that of the normal KLF9 expression group(P<0.05).With the increase of bicalutamide or docetaxel concentrations,the cell proliferation rates decreased(all P<0.05).Under the same drug concentration,the cell proliferation rate of the low miR-141-3p expression group was lower than that of the normal miR-141-3p expression group(P<0.05),and the cell proliferation rate of the KLF9 overexpression group was lower than that of the normal KLF9 expression group(P<0.05).The LNCaP cells were cultured for 48 hours.Compared with the normal miR-141-3p expression group without adding the drug,the relative expression levels of AR in-creased in the normal miR-141-3p expression groups added with 75 and 100 μmol/L bicalutamide(both P<0.05),and the relative expression levels of AR-V7 increased in the normal miR-141-3p groups added with 50,75,and 100 μmol/L bicalutamide(all P<0.05);the relative expression levels of AR and AR-V7 increased in the normal miR-141-3p groups added with 0.5,1.0,2.5,and 5.0 nmol/L docetaxel(all P<0.05).The relative expression levels of AR and AR-V7 in the low miR-141-3p expression group were lower than those in the normal miR-141-3p expression group added with the same drug concentration(both P<0.05),and the relative expression levels of AR and AR-V7 in the KLF9 overexpression group were lower than those in the normal KLF9 expression group added with the same drug concentration(both P<0.05).TargetScan database predicted the existence of binding sites between miR-141-3p and KLF9.Dual luciferase report experi-ment showed that the relative fluorescence activity was significantly higher in the group B than in the groups A,C and D(all P<0.05).Further rescue experiments showed that the cell proliferation rate of group d was significantly higher than that of the group b(P<0.05),and was significantly lower than that of the group c(P<0.05),but there was no significant difference between group d and group a(P>0.05).Conclusions Low expression of miR-141-3p or overexpression of KLF9 can enhance the drug sensitivity and inhibit the expression of AR and AR-V7 in LNCaP cells.MiR-141-3p and KLF9 have a targeted relationship.The inhibitory effect of miR-141-3p targeting KLF9 on drug sensitivity of LNCaP cells may be achieved by promoting the expression of AR-V7.