The CircRNA CircP4HB Promotes NSCLC Aggressiveness And Metastasis By Sponging MiR-133a-5p

Objective:To explore the effects of circP4HB on the invasion and migration of NSCLC and the underlying molecular mechanism.Contents:1.The correlative study of circP4HB with NSCLC clinical characteristics.(1)To evaluate the level of circP4HB expression in bronchial epithelial cells.(2)To examine the the expression of circP4HB in carcinoma tissue and adjacent normal tissue of NSCLC.(3)To analyse the correlation of circP4HB with NSCLC aggressiveness and metastasis.2.The examination of circP4HB effects on NSCLC aggressiveness and metastasis.(1)To examine the effects of circP4HB on NSCLC cell invasiveness and metastasis in vitro.(2)To evaluate the effects of circP4HB on NSCLC metastasis in vivo.3.The investigations of the molecular mechanism underlying circP4HB effects on NSCLC.(1)To examine the sponging action of circP4HB on miR-133a-5p(2)To evaluate the effects of circP4HB on the expression of vimentin regulated by miR-133a-5p in NSCLC cells.Results:1.qPCR assay revealed that the expression of circP4HB in carcinoma tissues was significantly increased compared to the adjacent normal tissues(P <0.01).Similarly,CircP4HB level in NSCLC carcinoma tissues accompanied by lymph node invasion was notably higher than the ones without node invasion.Coincidentally,the expression of circP4HB in bronchial epithelial cell line BEAS-2B was significantly attenuated compared to NSCLC cell lines H23、H1755、H522.These results indicate that CircP4HB might be correlated with the pathogenesis of NSCLC.2.Wound healing assay and Transwell assay revealed that Knocking down the expression of circP4HB in NSCLC cells H23 and H1755 notably suppressed the ability of cells invasion and migration.However,the overexpression of circP4HB in NSCLC cells H522 could result in cells migrating faster and being more invasive.H23 cells were stably transfected with shcircP4HB as well as sh Ctrl and then injected into mice tail veins.The examination of lung tumor burden in mice showed that the transfection of shcircP4HB in H522 cells significantly reduced the tumor burden relative to control.Conversely,by injecting H23 cells that had undergone stable transfection for circP4HB overexpression into mice tail veins,lung tumor burden was greater than the control group.These results indicate that circP4HB may be involved in the invasion and migration of NSCLC.3.H23 cells were transfected with AGO2-FLAG followed by RIP technology assay using anti-FLAG antibody,and the results showed that obvious expression of circP4HB was evaluated by IP fraction,indicating the combination of circP4HB with AGO2 and the ability of circP4HB to sponge miRNA.The luciferase reporter assay showed that H23 cultures underwent co-transfection with Luc-circP4HB and miR-133a-5p mimic had a decrease in signal intensity compared with the control group.However,H23 cultures underwent co-transfection with Luc-circ P4HB-mutant and miR-133a-5p mimic had no obvious decrease in signal intensity compared with the control group.The pull-down experiment using circP4HB probe showed that circP4HB in H23 cells could bind with miR-133a-5p to form a complex,which was further confirmed by the pull-down assay showing that the level of circP4HB in the complex of H23-circ P4HB-KD was down-regulated,and the content of miR-133a-5p also decreased.4.Vimentin transcript levels were markedly decreased in H23 cells transfected with miR-133a-5p mimics in comparison to the control group.We next used q PCR、Western blot assayed protein levels of vimentin,which demonstrated that circ P4HB-KD cells expressed less vimentin in comparison to cultures that underwent transfection with scrambled control sh RNA.Whereas circ P4HB-OE cultures expressed more vimentin in comparison to cultures that underwent transfection with empty control plasmid.Vimentin protein levels were rescued in circ P4HB-KD cells that had underwent transfection with miR-133a-5p inhibitor.These findings indicate that circ P4HB-KD can promote vimentin expression in NSCLC cells through sponging miR-133a-5p.5.The wound healing assay,transwell assay and tumor xenograft experiments all demonstrated that the knock-down of circP4HB in H23 cells could notably reduce the migratory and invasive capacity of the cells,which was restored by the transfection of H23 cells with miR-133a-5p inhibitor.In addition,the overexpression of circP4HB in H522 cells could induce an increase in the migratory and invasive capacity of the cells,which was rescued by the transfection of H522 cells with a miR-133a-5p mimic.These findings indicate that CircP4HB can promote NSCLC aggressiveness and metastasis through affecting miR-133a-5p.Conclusion:CircP4HB can promote NSCLC aggressiveness and metastasis,which was probably mediated by miR-133a-5p/vimentin pathway.