环状RNA circ-MYBL2通过吸附miR-324-3p抑制前列腺癌细胞的增殖和迁移

Circular RNA circ-MYBL2 inhibits the proliferation and migration of prostate cancer cells by adsorbing miR-324-3p

目的探讨环状RNA circ-MYBL2在前列腺癌组织中的表达及影响前列腺癌发生和转移的分子机制。方法选取2017年2月—2021年4月荆门市第二人民医院泌尿外科收治的45例前列腺癌患者手术切除的前列腺癌组织和癌旁组织。采用实时荧光定量聚合酶链式反应(qRT-PCR)检测前列腺癌组织和癌旁组织中circ-MYBL2的表达差异、前列腺癌细胞系和永生化前列腺导管上皮细胞中circ-MYBL2的表达差异,筛选circ-MYBL2低表达细胞系,分别转染circ-MYBL2质粒(circ-MYBL2组)或阴性对照质粒(对照组)至前列腺癌细胞。qRT-PCR检测circ-MYBL2质粒转染效率。CCK-8法和细胞划痕实验分别检测circ-MYBL2对细胞增殖和迁移能力的影响。starBase v2.0软件分别预测circ-MYBL2作用的miRNA及miRNA的靶基因。用双荧光素酶报告基因实验验证circ-MYBL2和miRNA之间的调控关系。qRT-PCR分别检测circ-MYBL2对miRNA表达的影响及miRNA对靶基因mRNA表达的影响。Western blotting检测靶基因蛋白和Wnt/β-catenin信号通路蛋白表达。计量资料以均数±标准差(Mean±SD)表示,多样本均数间比较采用单因素方差分析,两样本均数间比较采用t检验。结果前列腺癌组织中,circ-MYBL2表达量显著低于癌旁组织,差异具有统计学意义(P<0.01)。前列腺癌细胞系中,circ-MYBL2表达量显著低于前列腺导管上皮细胞,DU-145细胞表达量最低,差异均具有统计学意义(P<0.01)。与对照组相比,circ-MYBL2组DU-145细胞circ-MYBL2表达量显著增加(P<0.01),circ-MYBL2能够降低DU-145细胞的增殖活性(P<0.05)和迁移能力(P<0.01)。circ-MYBL2作为海绵吸附miR-324-3p,miR-324-3p互补结合SUFU基因。circ-MYBL2抑制miR-324-3p的表达(P<0.01),SUFU基因表达增加(P<0.01),Wnt/β-catenin信号通路转导被抑制。结论circ-MYBL2通过吸附miR-324-3p促进SUFU基因的表达,抑制Wnt/β-catenin信号通路,进而降低前列腺癌细胞增殖活性和迁移能力。

Objective To explore the expression of circular RNA circ-MYBL2 in prostate cancer tissue and the molecular mechanism of its influence on the occurrence and metastasis of prostate cancer.Methods From February 2017 to April 2021,45 cases of prostate cancer tissues and paracancerous tissues from patients with prostate cancer in the Department of Urology,Jingmen No.2 People′s Hospital were selected.quantitative real-time fluorescence polymerase chain reaction(qRT-PCR)was used to detect the difference in expression of circ-MYBL2 in prostate cancer tissues and adjacent tissues,and the difference in expression of circ-MYBL2 in prostate cancer cell lines and immortalized prostate duct epithelial cells.Cell lines with low circ-MYBL2 expression were respectively transfected with circ-MYBL2 plasmid(circ-MYBL2 group)or negative control plasmid(control group).qRT-PCR was used to detect the transfection efficiency of circ-MYBL2 plasmid.CCK-8 method and cell scratch test were used to detect the effect of circ-MYBL2 on cell proliferation and migration.The starBase v2.0 software was used to predict the miRNA bound by circ-MYBL2 and the target gene of miRNA.The dual luciferase reporter gene experiment was used to verify the regulatory relationship between circ-MYBL2 and miRNA.qRT-PCR was used to detect the influence of circ-MYBL2 on miRNA expression and the influence of miRNA on target gene mRNA expression.Western blotting was used to detect the expression of target gene protein and Wnt/β-catenin signaling pathway proteins.The measurement data were expressed as mean±standard deviation(Mean±SD),the comparison between the means of multiple samples used one-way analysis of variance,and the comparison between the means of two samples used the t-test.Results The expression of circ-MYBL2 of DU-145 cells in prostate cancer tissue was significantly lower than that in adjacent tissues(P<0.01).The expression of circ-MYBL2 in prostate cancer cell lines was significantly lower than that of prostate ductal epithelial cells(P<0.01),and the expression of DU-145 cells was the lowest(P<0.01).Compared with the control group,the expression of circ-MYBL2 of DU-145 cells in the circ-MYBL2 group increased significantly(P<0.01),and circ-MYBL2 reduced the proliferation activity(P<0.05)and migration ability(P<0.01)of DU-145 cells.circ-MYBL2 acted as a sponge to adsorb miR-324-3p,and miR-324-3p complementarily bound to the suppressor of SUFU gene.circ-MYBL2 inhibited the expression of miR-324-3p(P<0.01),SUFU gene expression was increased(P<0.01),and Wnt/β-catenin signal pathway transduction was inhibited.Conclusion circ-MYBL2 promotes the expression of SUFU gene by adsorbing miR-324-3p,inhibits the Wnt/β-catenin signaling pathway,thereby reducing the proliferation activity and migration ability of prostate cancer cells.