MiR-218-5p Suppresses Cell Proliferation And Migration By Targeting ZMIZ2 In Non-small Cell Lung Cancer

Background and Objectives:Lung cancer is the most usual malignant tumors worldwide,and its morbidity and mortality are the highest among all tumors.Lung cancer can be divided into small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC),of which NSCLC accounts for up to 85%.With the emergence and continuous improvement of targeted therapy,the clinical treatment methods for tumor have been enriched and refined.However,the 5-year survival rate and prognosis of NSCLC patients have not been significantly improved,which is mainly due to the malignant proliferation and metastasis of tumor.MicroRNA can be widely involved in the occurrence and development of tumors by negatively regulating the expression of target genes and is an important molecular target for the diagnosis,treatment and prognosis of tumors.Studies have shown that ZMIZ2(zinc finger MIZ-type containing 2)is highly expressed in a variety of malignancies and is closely related to the proliferation and invasion of cancer cells,but its role in NSCLC and its regulatory mechanism have not been reported.The purpose of this study was to investigate the effects of miR-218-5p-mediated abnormal expression of ZMIZ2 on the proliferation and migration of NSCLC cells and its regulatory mechanism,which is expected to provide a new theoretical basis for the clinical diagnosis and treatment of NSCLC.Methods:(1)We detected the expression levels of miR-218-5p and ZMIZ2 mRNA in NSCLC clinical samples by real-time PCR.The relationship between ZMIZ2 expression and NSCLC patients' age,gender and metastasis was further analyzed.(2)The potential binding relationship between miR-218-5p and ZMIZ2 gene was predicted by bioinformatics database(TargetScan),the dual-fluorescence reporter recombinant plasmid was constructed,and the targeting effect of miR-218-5p on ZMIZ2 was verified by the dual luciferase reporter assay in NSCLC cells.(3)MiR-218-5p mimics were transiently transfected into A549 and SPC-A1 cell lines,and its overexpression efficiency was detected by real-time PCR,and then mRNA and protein expression levels of ZMIZ2 were detected by real-time PCR and western blot.(4)Transwell,CCK-8 and flow cytometry assays were used to detect the effects of the overexpression of miR-218-5p on the migration,invasion,proliferation and cell cycle of NSCLC cells.(5)NSCLC cell lines with ZMIZ2 overexpression were constructed by lentivirus vectors,and the overexpression effect of ZMIZ2 was detected by real-time PCR and western blot.The effects of ZMIZ2 overexpression on NSCLC cell migration,invasion,cellular proliferation and cell cycle were detected by the assays of transwell,CCK-8 and flow cytometry,respectively.(6)Transfection of ZMIZ2-targeting siRNAs(si-ZMIZ2-1 and si-ZMIZ2-2)were carried out to silence ZMIZ2 expression in A549 and SPC-A1 cells.Knockdown effect of ZMIZ2 was detected by real-time PCR and western blot.The effects of ZMIZ2 interference on migration,invasion,proliferation and cell cycle of NSCLC cells were detected by transwell,CCK-8 and flow cytometry assays,respectively.(7)MiR-218-5p mimics were transiently transfected into ZMIZ2-overexpressed NSCLC cell lines,and the effects of miR-218-5p on migration,invasion,proliferation and cell cycle of ZMIZ2-overexpressed NSCLC cells were detected by transwell,CCK-8 and flow cytometry assays.(8)MiR-218-5p mimics were transiently transfected into A549 and SPC-A1 cell lines,real-time PCR was used to detect C/EBP? and IL-6 mRNA expression levels,and western blot was performed to detect ZMIZ2,C/EBP?,p-C/EBP? and IL-6 expression levels.Result:(1)In 59 clinical NSCLC samples,compared with paracancer tissues,the expression level of miR-218-5p mRNA in cancer tissues was significantly down-regulated(p<0.05),while the expression level of ZMIZ2 mRNA was significantly up-regulated(p<0.01).(2)TargetScan database prediction suggested that the 617-624 sequence in the 3'UTR region of ZMIZ2 gene was complementary to miR-218-5p targeting.The dual luciferase reporter assay further confirmed the targeting effect of miR-218-5p on ZMIZ2.(3)In NSCLC cell lines,overexpression of miR-218-5p significantly decreased ZMIZ2 mRNA and protein expression levels,and significantly inhibited cell migration,invasion,proliferation,and cell cycle arrest.(4)NSCLC cell lines with ZMIZ2 overexpression were successfully constructed and screened out,which significantly enhanced the cell migration,invasion,proliferation and cell cycle.However,si-ZMIZ2 effectively weakened the expression levels of ZMIZ2 and significantly inhibited the cell migration,invasion,proliferation and cell cycle.(5)The results showed that ZMIZ2 could significantly restore the inhibitory effects of miR-218-5p on the migration,invasion and proliferation of NSCLC cells,as well as the inhibitory effect of miR-218-5p on the cell cycle of NSCLC cells.(6)MiR-218-5p inhibited the expression levels of ZMIZ2,C/EBP?,p-C/EBP?and IL-6.Conclusion:ZMIZ2 plays a role in promoting NSCLC cells proliferation and migration.By targeting ZMIZ2,miR-218-5p inhibits the proliferation and migration of NSCLC cells.The possible mechanism is that miR-218-5p negatively regulates ZMIZ2/C/EBP?/IL-6 axis to exert tumor suppressor function.