Circ_0072088靶向miR⁃377促进肝癌细胞进展的机制研究

Circ_0072088 targeting miR⁃377 promotes the progression of hepatocellular carcinoma

目的研究环状RNA 0072088(circ_0072088)在肝癌细胞及异种移植瘤模型中的生物学功能,并探讨其参与调控肝细胞癌(HCC)发生进展的关键机制。方法培养人正常肝细胞(THLE⁃2)和HCC细胞系(Huh⁃7、Hep G2、Hep 3B),采用RT⁃qPCR方法检测circ_0072088、miR⁃377和含三方基序23基因(TRIM23)mRNA表达水平;Western blot检测TRIM23蛋白表达水平。取Hep G2细胞分为sh⁃NC组、sh⁃circ_0072088组、mimic NC组、miR⁃377 mimic组,Huh⁃7细胞分为mimic NC组、miR⁃377 mimic组和circ_0072088+miR⁃377 mimic组。分别采用CCK8法、克隆形成试验测定各组细胞的增殖能力变化情况;Transwell实验检测迁移能力变化情况;根据circ_0072088与miR⁃377的结合位点设计并合成了circ_0072088⁃WT和circ_0072088⁃MT双荧光素酶报告质粒,并分别与miR⁃377 mimic或mimic NC共转染Hep G2细胞后使用双荧光素酶报告试剂盒测量荧光素酶活性;RNA结合蛋白免疫沉淀(RIP)测定Ago2或IgG RIP复合物中circ_0072088和miR⁃377的富集水平。在异种移植瘤裸鼠模型中探讨circ_0072088的体内功能。结果circ_0072088在HCC中较瘤旁组织表达上调。与THLE⁃2细胞比较,HCC细胞系(Huh⁃7、Hep G2、Hep 3B)circ_0072088的mRNA表达水平升高,差异均有统计学意义(t=13.77、30.11、33.27,P均<0.001)。与sh⁃NC组比较,Hep G2转染sh⁃circ_0072088后细胞中circ_0072088 mRNA表达降低,差异有统计学意义(t=23.31,P均<0.001),细胞增殖活力(48、72 h)、克隆形成能力和迁移能力降低,差异均有统计学意义(t=11.78、8.42、12.64,P均<0.01),TRIM23蛋白表达水平明显降低。RIP检测结果显示,相对于lgG,circ_0072088(t=60.59)和miR⁃377(t=35.68)在Ago2免疫沉淀中富集,差异均有统计学意义(P均<0.001)。双荧光素酶检测结果显示,与转染mimic⁃NC组比较,转染miR⁃377 mimic组含有circ_0072088⁃WT质粒细胞的荧光素酶活性降低,差异有统计学意义(t=21.88,P<0.001)。RT⁃qPCR及Western blot结果显示,与mimic NC组相比,miR⁃377 mimic组中TRIM23 mRNA(t=8.45,P<0.001)和蛋白表达水平明显降低。在异种移植瘤裸鼠模型中,与sh⁃NC组相比,sh⁃circ_0072088组移植的肿瘤体积减小,肿瘤组织中circ_0072088和TRIM23 mRNA表达降低,而miR⁃377 mRNA表达升高,差异有统计学意义(t=8.63、5.56、5.52、11.97,P均<0.001),TRIM23的蛋白水平下降。结论circ_0072088通过结合miR⁃377,间接上调TRIM23的表达,从而促进HCC细胞的增殖和迁移。

Objective To investigate the biological functions of circ_0072088 in hepatocellular carcinoma(HCC)cells and HCC xenograft mouse models,and explore its key mechanisms involved in regulating the occurrence and development of HCC.Methods Human normal liver cell and HCC cell lines were cultured.The mRNA expression levels of circ_0072088,miR⁃377 and tripartite motif containing 23 gene(TRIM23)were detected by RT⁃qPCR and the protein expression level of TRIM23 was detected by Western blot.Hep G2 cells were divided into sh⁃NC group,sh⁃circ_0072088 group,mimic NC group,miR⁃377 mimic group.Huh⁃7 cells were divided into mimic NC group,miR⁃377 mimic group,and circ_0072088+miR⁃377 mimic group.CCK8 method and colony⁃formation assay(CFA)were used to determine the proliferation ability of cells in each group.Transwell experiment was used to detect the change of migration ability.According to the binding sites of circ_0072088 and miR⁃377,the circ_0072088⁃WT and circ_0072088⁃MT dual luciferase reporter plasmids were designed and synthesized.Luciferase activity was measured after cotransfection of circ_0072088⁃WT and circ_0072088⁃MT with miR⁃377 mimic or mimic NC by using a dual luciferase reporter kit.Enrichment levels of circ_0072088 and miR⁃377 in the Ago 2 or IgG RNA binding protein immunoprecipitation(RIP)complexes were determined by RIP.Xenograft tumor nude mouse model was used to explore the in vivo function of circ_0072088.Results The circ_0072088 was upregulated in HCC compared with paratumoral tissues.Compared with THLE⁃2 cell,the mRNA expression level of circ_0072088 in HCC cell line(Huh⁃7,Hep G2,Hep 3B)increased,and the difference were statistically significant(t=13.77,30.11,33.27;all P<0.001).After Hep G2 was transfected with sh⁃circ_0072088,compared with sh⁃NC group,the mRNA expression of circ_0072088 decreased,the difference was statistically significant(t=23.31,P<0.001);cell proliferation viability(48,72 h),colony formation ability and migration ability significantly decreased,the differences were statistically significant(t=11.78,8.42,12.64;all P<0.01),and TRIM23 protein expression level reduced obviously.The RIP assay showed that circ_0072088(t=60.59)and miR⁃377(t=35.68)were enriched in Ago 2 immunoprecipitates relative to lgG(P<0.001).The dual luciferase assay showed that the luciferase activity of cells transfected with miR⁃377 mimic containing circ_0072088⁃WT plasmid was decreased compared with that of the mimic⁃NC transfection group,and the difference was statistically significant(t=21.88,P<0.001).RT⁃qPCR and Western blot results showed that compared with the mimic NC group,the mRNA(t=8.45,P<0.001)and protein expression levels of TRIM23 in the miR⁃377 mimic group were significantly decreased.In the xenograft tumor nude mouse model,compared with the sh⁃NC group,the transplanted tumor volume of the sh⁃circ_0072088 group was reduced,the mRNA expression of circ_0072088 and TRIM23 in the tumor tissue was decreased,while the mRNA expression of miR⁃377 was increased;the differences were statistically significant(t=8.63,5.56,5.52,11.97;all P<0.001),and the protein level of TRIM23 was decreased.Conclusion Circ_0072088 indirectly upregulated the expression of TRIM23 by binding to miR⁃377,thereby promoting the proliferation and migration of HCC cells.