piRNA在双酚A促进前列腺癌细胞侵袭和迁移中的作用机制研究

Mechanism of piRNA in bisphenol A-promoted invasion and migration of prostate cancer cells

目的研究PIWI蛋白相互作用RNA(piRNA)在双酚A(BPA)促进前列腺癌细胞侵袭和迁移中的作用机制。方法利用癌症基因组图谱(TCGA)数据,分析并筛选在前列腺癌组织中表达显著升高的piRNA。使用不同浓度BPA对前列腺癌PC-3细胞进行12、24和48 h染毒,结合CCK-8实验确定20%抑制浓度(IC20),并使用实时荧光定量PCR检测BPA染毒前后piRNA表达水平的改变。随后利用比较毒理基因组学数据库(CTD)筛选受BPA调控且与前列腺癌相关的靶基因,经双荧光素酶报告基因实验验证piRNA与靶基因的靶向调控关系,并通过Western blotting检测piRNA靶基因的表达水平,并进行细胞侵袭和迁移实验探究piRNA及其拮抗剂对PC-3细胞恶性表型的影响。结果160μmol/L BPA处理PC-3细胞piR-sno48表达水平升高幅度较大(P<0.05)。转染piR-sno48拮抗剂可导致内源性piR-sno48表达下降及其靶基因GSTP1表达水平升高(P<0.05),但是BPA染毒的细胞中GSTP1表达未见明显改变(P>0.05)。双荧光素酶报告基因实验结果显示,piR-sno48与GSTP1的3′-UTR形成反向互补序列继而靶向抑制GSTP1表达。Transwell实验结果显示,BPA刺激促进了前列腺癌细胞侵袭和迁移(P<0.01),而piR-sno48拮抗剂可显著抑制这种促进作用(P<0.01)。结论BPA可能通过上调piR-sno48表达和靶向抑制GSTP1表达促进前列腺癌细胞侵袭和迁移;通过干扰内源性piR-sno48表达抑制前列腺癌细胞恶性表型。

ObjectiveTo investigate the regulatory mechanisms of piwi-interacting RNA(piRNA)in bisphenol A(BPA)-induced prostate cancer cell invasion and migration.MethodsThe Cancer Genome Atlas(TCGA)data was used to analyze and screen for piRNAs with significantly increased expression in prostate cancer tissues.PC-3 cells were treated with different concentrations of BPA for 12,24,and 48 h,respectively,and the 20%inhibitory concentration(IC20)was measured using a CCK-8 assay.The expression levels of piRNAs before and after BPA treatment were determined by reverse transcription-quantitative PCR.Target genes regulated by BPA and associated with prostate cancer were screened in the Comparative Toxicogenomics Database(CTD).Dual-luciferase reporter gene assay was performed to verify the relationship between piRNA and target genes,and the expression change of the piRNA target gene was detected by Western blotting.Cell migration and invasion assays were used to determine the effects of piRNA on the malignant phenotype of prostate cancer cells.ResultsAfter treatment of PC-3 cells with 160μmol/L BPA,the expression of piR-sno48 was most significantly increased(P<0.05).Transfection of piR-sno48 antagomir resulted in decreased expression of endogenous piR-sno48 and a significant increase in the expression of its target gene GSTP1(P<0.05).However,the expression of GSTP1 did not change significantly in BPA-treated PC-3 cells after transfection with piR-sno48 antagomir(P>0.05).The dual-luciferase reporter gene confirmed that piR-sno48 inhibited the expression of GSTP1 by forming an inversely complementary sequence with the 3′-UTR of GSTP1.The Transwell assay results showed that treatment with BPA significantly increased the invasion and migration ability of prostate cancer cells(P<0.01),whereas piR-sno48 antagonists significantly inhibited the effects above(P<0.01).ConclusionBPA promotes the invasion and migration of prostate cancer cells by upregulating the expression of piR-sno48 and suppressing the expression of GSTP1.Interfering with the expression of endogenous piR-sno48 may inhibit the malignant phenotype of prostate cancer cells caused by BPA.