环状人网状蛋白4调节miR-582-5p/神经纤毛蛋白1轴对结直肠癌细胞恶性生物学行为的影响

Effect of CircRTN4 on the Malignant Biological Behavior of Colorectal Cancer Cells by Regulating the miR-582-5p/NRP1 Axis

目的探讨环状人网状蛋白4(CircRTN4)调节miR-582-5p/神经纤毛蛋白1(NRP1)轴对结直肠癌(CRC)细胞恶性生物学行为的影响。方法选取2019年1月至2023年1月河南科技大学第一附属医院收治的CRC患者50例,通过实时荧光定量PCR(qRT-PCR)、蛋白质免疫印迹法分别检测患者的癌组织、癌旁组织、人正常结肠黏膜上皮细胞和CRC细胞中CircRTN4、miR-582-5p mRNA及NRP1的蛋白表达;将SW480细胞分别转染si-RTN4、miR-582-5p inhibitor及相应对照,qRT-PCR检测转染效率;通过平板克隆实验、Transwell小室和流式细胞仪等研究CircRTN4对CRC细胞的影响;蛋白质免疫印迹法检测增殖细胞核抗原(PCNA)、基质金属蛋白酶-2(MMP-2)、Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)及NRP1蛋白表达;双荧光素酶报告基因实验检测miR-582-5p与RTN4和NRP1的作用;构建肺癌裸鼠模型,分析敲低RTN4对肿瘤质量、体积及移植瘤中miR-582-5p、NRP1、Ki-67蛋白表达的影响。结果在CRC组织和细胞系中,RTN4、NRP1表达升高,miR-582-5p表达降低(P<0.05);敲低RTN4,降低SW480细胞迁移、增殖与侵袭,下调PCNA、Bcl-2、MMP-2、NRP1表达,促进miR-582-5p、Bax表达及细胞凋亡(P<0.05);miR-582-5p作为RTN4靶点,下调miR-582-5p可减弱RTN4敲低对SW480细胞恶性行为的抑制作用(P<0.05);体内实验显示,抑制RTN4可降低移植瘤质量和体积,降低Ki-67、NRP1表达水平,升高miR-582-5p表达(P<0.05)。结论在CRC组织和细胞系中,CircRTN4高表达,敲低CircRTN4可能通过调节miR-582-5p/NRP1轴抑制CRC细胞的恶性行为。

Objective To investigate the effect of circular reticulon protein 4(CircRTN4)on the malignant biological behavior of colorectal cancer(CRC)cells by regulating the miR-582-5p/neuropilin 1(NRP1)axis.Methods A total of 50 CRC patients admitted to the First Affiliated Hospital of Henan University of Science and Technology from January 2019 to January 2023 were selected.The expression levels of CircRTN4,miR-582-5p mRNA,and NRP1 protein in cancer tissues,adjacent tissues,normal colon mucosal epithelial cells,and CRC cells were detected by real-time fluorescence quantitative PCR(qRT-PCR)and Western blot.SW480 cells were transfected with si-RTN4,miR-582-5p inhibitor and corresponding control,respectively.qRT-PCR was applied to detect the transfection efficiency.The effect of CircRTN4 on CRC cells was studied by plate cloning experiment,Transwell chamber and flow cytometry.The expressions of proliferating cell nuclear antigen(PCNA),matrix metalloproteinase-2(MMP-2),Bcl-2 associated X protein(Bax),anti apoptotic factor B cell lymphocyte tumor 2(Bcl-2),and NRP1 protein in cells were detected by Western blot.The relationship between miR-582-5p and RTN4,miR-582-5p and NRP1 were detected by dual luciferase reporter gene assay.A nude mouse model of lung cancer was constructed to analyze the effects of RTN4 knockdown on tumor mass,volume and expression of miR-582-5p,NRP1 and Ki-67 in transplanted tumors.Results In CRC tissues and cell lines,the expression of RTN4 mRNA and NRP1 protein increased,while the mRNA expression of miR-582-5p decreased(P<0.05).Knocking down the expression of RTN4 significantly inhibited the proliferation,migration,and invasion of SW480 cells,reduced the expression of PCNA,Bcl-2,MMP-2,and NRP1 proteins,promoted the expression of miR-582-5p and Bax,and cell apoptosis(P<0.05).Down-regulation of miR-582-5p,a target of RTN4,could attenuate the inhibitory effect of RTN4 knockdown on the malignant behavior of SW480 cells(P<0.05).In vivo experiments showed that inhibition of RTN4 expression could significantly reduce the mass and volume of transplanted tumors,decrease the expression of Ki-67 and NRP1,and increase the expression of miR-582-5p(P<0.05).Conclusion CircRTN4 is highly expressed in CRC tissues and cell lines.Knockdown of CircRTN4 may inhibit the malignant behavior of CRC cells by regulating the miR-582-5p/NRP1 axis.