Effects Of CircHIPK3 On The Proliferation,Migration And Invasion Of Neuroblastoma Cells By Regulating The MiR-653-5p/RSF1 Axis

Objective:Neuroblastoma(NB)is a common malignant solid tumor in children,known as the "king of childhood malignant tumors".Refractory and recurrence are still the biggest challenges in the treatment of NB.Circular homeodomain interacting protein kinase 3(circHIPK3)plays an important role in the progression of many tumors,but its role in NB remains unclear.This study aimed to explore the expression and mechanism of circHIPK3 in neuroblastoma.Methods:Circular RNAs with differential expression in NB tissues were screened by nextgeneration sequencing.Real-time quantitative PCR(RT-q PCR)was used to verify the expression of circHIPK3,miR-653-5p and RSF1 in NB tissues and human NB cell lines SH-SY5 Y,SK-N-SH,SK-N-BE(2),IMR-32 and HEK-293.SH-SY5 Y and SK-N-SH were transfected with small interfering RNAs si-NC and si-circHIPK3 or lentiviral knockdown vectors sh-NC and sh-circHIPK3.CCK-8 and plate clone formation experiments were used to verify the effects of circHIPK3 on the proliferation of SH-SY5 Y and SK-N-SH.The ability of circHIPK3 to migrate and invade SH-SY5 Y and SK-N-SH was detected by scratch assay and Transwell assay.Western blot was used to detect the expression of epithelial-mesenchymal transition(EMT)-related proteins in NB cell lines SH-SY5 Y and SK-N-SH.The effect of circHIPK3 on NB in vivo was investigated through subcutaneous tumorigenesis experiments in nude mice.Bioinformatics methods were used to predict the downstream regulatory targets of circHIPK3,and luciferase reporter gene assay was used to prove the target regulatory relationship between the two.The miR-653-5p inhibitor was transfected into NB cell lines with stable knockdown of circHIPK3,cck-8 and Transwell assay were used to explore the association between miR-653-5p and proliferation,migration and invasion of NB cells.Results:CircHIPK3 and RSF1 were highly expressed in NB cells and tissues,and miR-653-5p was lowly expressed in NB cells and tissues.When sh-circHIPK3 was transfected into SHSY5 Y and SK-N-SH cells,the expression of circHIPK3 decreased.The results of functional experiments such as CCK-8,colony formation assay,scratch assay and Transwell assay showed that when circHIPK3 was silenced,the proliferation,migration and invasion of NB cells were inhibited.Western blot results displayed that when circHIPK3 expression was inhibited,the expression of N-cadherin and Vimentin were declined,while the expression of E-cadherin was increased.The results of subcutaneous tumorigenesis experiments in nude mice showed that silencing the expression of circHIPK3 could inhibit the growth of NB in vivo.The results of luciferase reporter gene experiments confirmed that there are targeted regulatory sites between circHIPK3 and miR-653-5p,and between miR-653-5p and RSF1.The results of cell function recovery experiments confirmed that circHIPK3 affects the malignant phenotype of NB cells by regulating the miR-653-5p/RSF1 axis.Conclusion:CircHIPK3 is obviously expressed in NB cells and tissues.Knocking down circHIPK3 can suppress the proliferation,migration,invasion ability and EMT of NB cells,as well as restrain the growth of NB in vivo.Further studies found that circHIPK3 affects NB cells by regulating the miR-653-5p/RSF1 axis proliferation,migration and invasion.Significance:Silencing the expression of circHIPK3 can inhibit the malignant phenotype of NB cells and the growth of NB in vivo.Therefore,circHIPK3 may be a new point for the treatment of neuroblastoma,providing a new idea for the development of anti-neuroblastoma drugs.