Circ_0072088 targeting miR-377 promotes the progression of hepatocellular carcinoma
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.
NETL
NSTL
万方数据
