Effects of abnormal expression of miR-141-3p and KLF9 on drug sensitivity and androgen receptor expression of prostate cancer cells and the targeting relationship
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.
microRNA-141-3pKrüppel-like factor 9drug susceptibilitybicalutamidedocetaxelandrogen receptorandrogen receptor splice variant 7prostate cancer
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