目的 检测环状 RNA SR 相关 CTD 相关因子 11(CircRNA SR-related CTD associated factor 11,circSCAF 11)在胰腺癌(Pancreatic cancer,PC)中的表达,分析其对PC细胞增殖、迁移的影响及其潜在分子作用机制.方法 利用实时荧光定量聚合酶链反应(Quantitative reverse transcription-polymerase chain reaction,qRT-PCR)和蛋白免疫印迹实验检测 PC 细胞(PANC-1,AsPC-1,Capan-2,SW1990,BXPC-3)及人正常胰腺导管上皮细胞(HPDE6-C7)中circSCAF11 mRNA和蛋白表达水平;构建circSCAF 11干扰表达细胞系,通过细胞计数试剂盒-8(Cell counting kit-8,CCK-8)法、克隆斑点形成实验及Transwell实验检测下调circSCAF11对PC细胞增殖、克隆形成及迁移能力的影响;通过生物信息学网站预测circSCAF11的潜在微小RNA(Mi-croRNA,miRNA)分子靶标及其调控网络,采用双荧光素霉报告实验验证circSCAF11和miRNA-138-5p(miR-138-5p)和性别决定区相关高迁移率族盒蛋白4(Sex-determining region Y-related high mobility group box 4,SOX4)的靶向调控关系.构建过表达或干扰miR-138-5p细胞系及SOX4过表达细胞系,探究circSCAF11,miR-138-5p,SOX4间靶向调控对PC细胞克隆形成及迁移的影响.结果 PC细胞中circSCAF11 mRNA和蛋白表达均显著高于HPDE6-C7细胞(P＜0.05).PC组织中circSCAF11表达显著上调,主要定位在细胞质中.circSCAF11由SCAF11基因的外显子环化而成,成熟的circSCAF11大小为6787 bp.与NC组相比,下调circSCAF11组细胞增殖、克隆形成及迁移能力明显抑制(P＜0.05).miR-138-5p是circSCAF11的下游靶点,SOX4是 miR-138-5p 的直接靶标.circSCAF11 靶向负调控 miR-138-5p,miR-138-5p 靶向负调 SOX4,circSCAF11 正调控 SOX4.下调miR-138-5p或过表达SOX4可逆转干扰circSCAF11对细胞克隆形成及迁移的抑制作用.结论 PC细胞中circSCAF11显著高表达,干扰其表达能够抑制PC细胞的增殖、克隆形成及迁移;circSCAF11可能通过靶向调控miR-138-5p/SOX4信号轴来发挥作用影响PC细胞的生物学行为.
Down-regulation of circSCAF11 inhibited the mechanism of mir-138-5p/SOX4 signaling axis mediated proliferation pancreatic cancer cells
Objective objective To detect the expression of circRNA SR-related CTD associated factor 11(circSCAF11)in pancreatic cancer(PC),and analyze its effect on the proliferation and migration of PC cells and its potential molecular mechanism.Meth-ods Quantitative reverse transcription-polymerase chain reaction(qRT-PCR)and Western blot were used to detect PC cells(PANC-1,AsPC-1,PANC-1,ASPC-1).Capan-2,SW1990,BXPC-3)and human normal pancreatic ductal epithelial cells(HPDE6-C7)in mRNA and protein expression levels of circSCAF11.Cell lines with circSCAF11 interference were constructed,and the effects of circSCAF11 knockdown on PC cell proliferation,clonal formation and migration were detected by CCK-8 assay,clone spot formation assay and Transwell assay.Potential miRNA molecular targets and regulatory networks of circSCAF11 were predicted through bioinformatics websites,and circSCAF11 and miR-138-5p were verified by dual-luciferase assay.The targeted regulatory relationship between miR-138-5p and SOX4.Overexpression or interference of miR-138-5p cell lines and SOX4 over-expression cell lines were constructed to explore the effects of targeted regulation among circSCAF11,miR-138-5p and SOX4 on the formation and migration of PC cell clones.Results The mRNA and protein expressions of circSCAF11 in PC cells were signif-icantly higher than those in HPDE6-C7 cells(P＜0.05).The expression of circSCAF11 was significantly up-regulated in PC tissues,mainly located in the cytoplasm.circSCAF1 1 is cyclized from the exon of SCAF11 gene,and the mature circSCAF11 is 6787 bp in size.Compared with NC group,cell proliferation,clonal formation and migration were significantly inhibited in down-regulated circSCAF11 group(P＜0.05).MiR-138-5p is the downstream target of circSCAF11,and SOX4 is the direct target of miR-138-5p.circSCAF11 negatively regulates miR-138-5p,miR-138-5p negatively regulates SOX4,and circSCAF11 positively regulates SOX4.Downregulation of miR-138-5p or overexpression of SOX4 reversed the inhibition of circSCAF11 on cell clonal formation and migration.Conclusion circSCAF11 was significantly overexpressed in PC cells,and interference with its expression could in-hibit proliferation,clonal formation and migration of PC cells.circSCAF11 may play a role in influencing the biological behavior of PC cells through targeted regulation of miR-138-5P/SOX4 signaling axis.
Circular SR-related CTD associated factor 11Pancreatic cancermicroRNA-138-5pSex-determining region Y-related high mobility group box 4Proliferation
万方数据
维普
