circ-MTHFD1L促进胰腺导管腺癌进展的作用机制

Mechanism of circ-MTHFD1L promoting the progression of pancreatic ductal adenocarcinoma

目的 探讨circ-MTHFD1L在胰腺导管腺癌(PDAC)进展中的作用,并分析其与miR-217与E2F3的相互调控关系,为PDAC的标志物选择和靶向疗法提供依据。方法 利用Arraystar Human circRNAs微阵列分析法分析20例PDAC组织和癌旁组织中差异表达的circRNAs。通过qRT-PCR检测circ-MTHFD1L在PDAC组织样本和癌旁组织样本中的表达水平。TCGA数据库的数据用于分析PDAC患者的总体生存数据。采用MTT细胞增殖实验、流式细胞术凋亡实验、Transwell迁移侵袭实验研究不同转染组的细胞增殖、凋亡和侵袭。在体内裸鼠的肿瘤形成试验中研究肿瘤发展的速度。此外,采用双荧光素酶报告基因检测和蛋白质印迹检测证明了circ-MTHFD1L与miR-217和E2F3之间的靶向关系。结果 通过微阵列数据分析筛选circ-MTHFD1L在肿瘤组织中的差异最为显著作为进一步研究的目标分子。与癌旁组织相比,circ-MTHFD1L在PDAC组织中的表达水平显著升高(P<0.05)。当circ-MTHFD1L被敲低时,PDAC细胞的增殖、迁移和侵袭能力将被抑制,细胞凋亡率显著升高且差异均具有统计学意义(P<0.05)。同时,在胰腺癌细胞PANC-1中,过表达miR-217或沉默circ-MTHFD1L,E2F3蛋白表达水平降低(P<0.05),而且通过双荧光素酶报告基因实验验证了CIRC-MTHFD1L与miR-217和E2F3之间的靶向关系。结论 Circ-MTHFD1L可通过抑制miR-217表达水平并增强E2F3蛋白表达水平促进PDAC细胞的进展。

objective To explore the role of circ-MTHFD1L in the progression of pancreatic ductal adenocarcinoma(PDAC), analyze its relationship with miR-217 and E2F3, and provide a basis for PDAC marker selection and targeted therapy. Methods Arraystar Human circRNAs microarray analysis method was used to analyze the differentially expressed circRNAs in PDAC tissues and adjacent tissues. The expression level of circMTHFD1L in PDAC tissue samples and adjacent tissue samples was detected by qRT-PCR. The overall survival data of PDAC patients were analyzed with data from TCGA database. MTT cell proliferation experiment, flow cytometry apoptosis experiment, Transwell migration and invasion experiment were employed to study cell proliferation, apoptosis and invasion in different transfection groups. The rate of tumor development was studied in an in vivo tumor formation test in nude mice. In addition, dual luciferase reporter gene detection and Western blotting analysis were conducted to prove the targeting relationship between circ-MTHFD1L, miR-217 and E2F3. Results circ-MTHFD1L was screened as the target molecule for further research through Microarray data analysis since the difference of circ-mthfd1l in tumor tissues was the most significant. Compared with adjacent tissues, the expression level of circ-MTHFD1L in PDAC tissues was significantly increased(P<0.05). When circMTHFD1L was knocked down, the proliferation, migration and invasion ability of PDAC cells were inhibited, and the apoptosis rate increased significantly and the difference was statistically significant(P<0.05). At the same time, the expression level of E2F3 protein was significantly reduced in pancreatic cancer cells PANC-1 by overexpressing miR-217 or silencing circ-MTHFD1L, and the dual luciferase reporter gene experiment verified the targeting relationship of circ-MTHFD1L and miR-217, as well as miR-217 and E2F3. Conclusion Circ-MTHFD1L can promote the progression of PDAC cells by inhibiting the expression level of miR-217 and enhancing the expression level of E2F3 protein.