雷公藤甲素通过miR-320bAR轴对前列腺癌细胞的抑制作用

Triptolide inhibits prostate cancer cells via miR-320b/AR axis

目的观察雷公藤甲素(triptolide,TPL)对前列腺癌细胞恶性生物学行为的作用,探究其作用机制。方法用实时荧光PCR(real-time fluorescence PCR,RT-PCR)检测LNCaP、PC-3、DU145前列腺癌细胞、RWPE-1人正常前列腺上皮细胞中miR-320b和雄激素受体(androgen receptor,AR)mRNA的表达水平。用不同剂量的TPL处理PC-3细胞,用MTT法检测细胞的增殖活力。用RT-PCR检测miR-320b和AR的表达水平。用6.25 nmol·L^(-1) TPL处理细胞,在TPL处理的基础上转染miR-320b NC/mimics,用Transwell和平板克隆形成实验检测细胞的侵袭和克隆能力。用Western blot检测AR及上皮间质转化(epithelial-mesenchymal transition,EMT)过程中E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)和波形蛋白(vimentin)的表达水平。用荧光素酶实验验证miR-320b与AR的靶向关系。结果与RWPE-1人正常前列腺上皮细胞比较,miR-320b和AR分别在前列腺癌细胞中呈低表达和高表达状态(P<0.01)。与Control组比较,TPL可上调PC-3细胞miR-320b的表达水平、下调AR的表达水平,抑制细胞的增殖、侵袭能力,抑制AR、N-cadherin和vimentin的表达、上调E-cadherin的表达水平(P<0.05)。与TPL组比较,miR-320b可提高TPL对PC-3细胞侵袭和增殖能力的抑制作用,抑制AR、N-cadherin和vimentin的表达、上调E-cadherin的表达(P<0.05)。荧光素酶实验显示,AR是miR-320b的潜在靶基因,miR-320b对AR存在显著调控作用(P<0.01)。结论TPL可能通过miR-320b/AR轴抑制前列腺癌细胞的恶性生物学行为。

Objective To observe the effect of triptolide(TPL)on the malignant biological behavior of prostate cancer cells and explore its mechanism.Methods Real-time fluorescence PCR(RT-PCR)was used to detect the expression levels of miR-320b and androgen receptor(AR)mRNA in LNCaP,PC-3,DU145 prostate cancer cells and RWPE-1 human normal prostate epithelial cells.Different doses of TPL were used to treat PC-3 cells,MTT method was used to detect cell proliferation,and RT-qPCR was used to detect the expression of miR-320b and AR.Cells were treated with 6.25 nmol·L^(-1) TPL,and then miR-320b NC/mimics were transfected on the basis of TPL treatment.Transwell and plate clone formation experiments were used to detect cell invasion and cloning ability.Western blot method was used to detect AR and E-cadherin,N-cadherin,vimentin expression levels during pithelial-mesenchymal transition(EMT)process.Luciferase experiment was used to verify the targeting relationship between miR-320b and AR.Results Compared with RWPE-1 human normal prostatic epithelial cells,miR-320b and AR showed low and high expression levels in prostate cancer cells(P<0.01).Compared with control group,TPL could up regulate the expression level of miR-320b,down regulate the expression level of AR,inhibit the proliferation,invasion and cloning ability of PC-3 cells,inhibit the expression levels of AR,N-cadherin and vimentin,and up regulate the expression level of E-cadherin(P<0.05).Compared with TPL group,miR-320b could enhance the inhibitory effect of TPL on the invasion and cloning ability of PC-3 cells,inhibit the expression of AR,N-cadherin and vimentin,and increase the expression of E-cadherin(P<0.05).Luciferase assay showed that AR was a potential target gene of miR-320b,and miR-320b had a significant regulatory effect on AR(P<0.01).Conclusion TPL may inhibit the malignant biological behavior of prostate cancer cells through miR-320b/AR axis.