环状RNA SAMD8调控miR-223-3p/RHOB表达抑制胰腺导管腺癌进程的分子机制

Molecular mechanism of regulating miR-223-3p/RHOB expression by circRNA SAMD8 to inhibit progression of pancreatic ductal adenocarcinoma

  • 摘要:
    目的 探讨环状RNA SAMD8(circ-SAMD8)在胰腺导管腺癌(PDAC)进展中的潜在机制。
    方法 基于Microarray数据(GSE79634)分析PDAC组织中circRNAs的表达谱。通过实时荧光定量聚合酶链反应(qRT-PCR)验证circ-SAMD8(hsa_circ_0006148)在PDAC组织和细胞(CFPAC-1和PANC-1)中的表达。采用生物信息学分析预测circ-SAMD8的靶微小RNA(miRNA)及其下游mRNA, 并采用双荧光素酶报告基因实验进行鉴定。采用MTT法和集落形成法检测PDAC细胞的增殖能力。采用免疫印迹法(Western blot)检测抗凋亡蛋白Bcl-2和促凋亡蛋白Bax的表达水平。采用流式细胞术检测凋亡细胞百分比。
    结果 circ-SAMD8在PDAC组织和细胞中的表达水平低于癌旁组织和正常细胞,差异有统计学意义(P < 0.05)。过表达circ-SAMD8能有效促进PDAC细胞凋亡,抑制PDAC细胞增殖。双荧光素酶报告基因实验显示, miR-223-3p是circ-SAMD8的一个潜在相互作用分子, miR-223-3p的下游mRNA靶点是RHOB。miR-223-3p在PDAC组织和细胞中异常过表达,并伴有RHOB低表达。在转染miR-223-3p模拟物或sh-circ-SAMD8的PDAC细胞中, RHOB表达显著降低,增殖能力增强,凋亡率降低。
    结论 circ-SAMD8通过海绵化miR-223-3p, 调控下游RHOB的表达,有效抑制PDAC的发生发展。

     

    Abstract:
    Objective To investigate the potential mechanism of circRNA SAMD8(circ-SAMD8) in development of pancreatic ductal adenocarcinoma (PDAC).
    Methods The expression profile of circRNAs in PDAC tissues was analyzed based on Microarray data (GSE79634). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the expression of circ-SAMD8 (hsa_circ_0006148) in PDAC tissues and cells (CFPAC-1 and PANC-1).The target microRNA (miRNA) of circ-SAMD8 and its downstream mRNA were predicted by bioinformatics analysis, and identified by double luciferase reporter gene assay. The proliferation ability of PDAC cells was detected by MTT assay and colony formation assay. The expression levels of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax were detected by Western blot. The percentage of apoptotic cells was detected by flow cytometry.
    Results The expression levels of circ-SAMD8 in PDAC tissues and cells were significantly lower than those in paracancer tissues and normal cells (P < 0.05). Overexpression of circ-SAMD8 could effectively promote apoptosis of PDAC cells and inhibited proliferation of PDAC cells.Dual luciferase reporter gene experiments showed that miR-223-3p was a potential interacting molecule of circ-SAMD8, and the downstream mRNA target of miR-223-3p was RHOB. The miR-223-3p was abnormally overexpressed in PDAC tissues and cells, accompanied by low RHOB expression. In PDAC cells transfected with miR-223-3p mimics or sh-circ-SAMD8, RHOB expression was significantly decreased, proliferation ability was enhanced, and apoptosis rate was decreased.
    Conclusion Circ-SAMD8 regulates the expression of RHOB downstream by sponging miR-223-3p, and effectively inhibits the occurrence and development of PDAC.

     

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