The Role Of CircHIPK3 On The Biological Behavior Of Neuroblastoma:a Mechanism Study

Background and ObjectivesNeuroblastoma（NB）,originates from adrenal medulla or the sympathetic nervous system,is one of the most common extra-cranial solid tumors in early childhood.The biological features,clinical characteristics and prognosis vary by age,tumor location,histopathological differentiation and genomic alterations.A possibility of spontaneous regression or cellular maturation may happen on several cases.However,parts of patients suffer from systemic metastasis at the time of diagnosis,and even develop life-threatening progression.The 5-year overall survival rate of these patients is less than 40%.The main treatment methods for NB include surgery,radiotherapy and high dose chemotherapy.Due to the heterogeneity of NB,there is lacking effective prognostic factors in clinical.Circular RNAs（circRNAs）are highly conserved and stable covalently closed non-coding RNA,which play important roles in functions of cell biology such as protein synthesis,gene expression,post-translational modification and et al.CircHIPK3（hsa_circ₀000284）is originated from the second exon of the HIPK3 gene,and abundantly expressed in the liver,brain,and lung.Mounting evidence displayed that circHIPK3 may function as a "microRNAs sponge",modulate the expression of targeted genes,and then regulate cell survival,proliferation,and invasion.But the latest study also showed that circHIPK3 modulates autophagy via AMP-activated protein kinase（AMPK）and mammalian target of rapamycin（mTOR）signaling and regulates cell proliferation.The aim of this study was to observe the function of circHIPK3 for cell proliferation,invasion and apoptosis in neuroblastoma,and try to disclose intrinsic synergistic molecular mechanism.Methods and materialsBioinformatic approaches were used to understand the molecular-characteristics of circHIPK3,and observe the expression of circHIPK3 in different cancer types.The possible mechanism of circHIPK3 was also predicted.Human neuroblastoma SK-N-AS cells,purchased from institute of Neuroscience,Soochow university（Suzhou,China）,human neuroblastoma SH-SY5Y cells,purchased from Shanghai Institute of Biological Science（Shanghai,China）,were maintained in DME/F-12 medium,with a 10%FBS,penicillin/streptomycin（1:100）,and incubated at 37℃ in a humidified air atmosphere containing 5%CO2.After establishing circHIPK3-shRNA stably transfected tumor cells（SK-N-AS and SH-SY5Y）,we performed the Clonogenicity assay and the EDU fluorescence assay to test the proliferation of cancer cells.Flow cytometry was used to test the cell cycle.Transwell migration assay and cell wound healing assay were performed to test the effect of downregulating circHIPK3 on cancer cells migration and invasion.Cell apoptosis was analyzed by Annexin V-PE assay and TUNEL assay.Western blot detected total and phosphorylated AMPKα,ACC,AKT,mTOR signaling pathway changings,and the expression of EGFR and PDGFRα.In order to validate the function of circHIPK3 in NB in vivo,nude model was established using SH-SY5Y and circHIPK3-shRNA stably transfected SH-SY5Y cells.Mice survival rate and tumor size were recorded.Statistical analysis:Each experiment was repeated a minimum of three times,with similar results obtained each time.In each experiment,a minimum of three wells/dishes were used.Quantitative data were presented as mean± standard deviation（SD）.Statistic was analyzed by one-way ANOVA followed by a Scheffe’ and Tukey Test using SPSS 16.0 software（SPSS Inc.,Chicago,IL,USA）.Significance was chosen asp<0.05.Results1.Bioinformatic Analysis of circHIPK3Bioinformatic Analysis indicated that the expression of circHIPK3 in different cancer types is different,which is associated with the disease stage and prognosis of cancer.We inferred that circHIPK3 was abnormally expressed in NB and may affect biological tumor behavior and prognosis from regulating signaling pathway.Therefore,we choose to study the function of circHIPK3 in NB.2.CircHIPK3-shRNA knockdown inhibited cellular proliferation,migration and invasion in NBthe clonogenicity assay and the EDU fluorescence assay were performed,and results showed that both cell lines（SK-N-AS and SH-SY5Y）proliferation were significantly inhibited after circHIPK3-shRNA knockdown.Cell cycle progression,examined by FACS assay,were blocked.Results from transwell migration assay and cell wound healing assay also revealed that cell migration and invasion were inhibited by downregulating circHIPK3.Taken together,these results demonstrated that circHIPK3-shRNA knockdown inhibited cellular proliferation,migration and invasion in NB.3.CircHIPK3-shRNA knockdown induced apoptosis in NBThe Annexin V-PE assay and TUNEL assay were performed to test cell apoptosis,and results showed that circHIPK3-shRNA knockdown induced apoptosis in SK-N-AS and SH-SY5Y cells.Based on these data,we suggest that downregulation of circHIPK3 induced apoptosis in NB.4.CircHIPK3-shRNA knockdown contributed to activation of AMPK and AKT/m TOR inactivationWestern blot results demonstrated that,in SK-N-AS cells and SH-SY5Y cells,circHIPK3-shRNA knockdown activated AMPK pathway with increased expressions of p-AMPKα（Thr 172）and p-ACC（Ser 79）.Meanwhile,AKT/mTOR signaling pathway was inactivated.circHIPK3-shRNA knockdown in both cell lines also inhibited platelet-derived growth factor receptor α（PDGFRα）/epidermal growth factor（EGFR）expressions.We considered that activation of AMPK and AKT/mTOR inactivation may be the main mechanism of circHIPK3-shRNA knockdown inhibiting proliferation and inducing apoptosis.Conclusions● circHIPK3-shRNA knockdown inhibited cellular proliferation,migration and invasion in NB.● circHIPK3-shRNA knockdown induced apoptosis in NB.● Downregulation of circHIPK3 modulates cell proliferation and apoptosis through activating of AMPK.● Activation of AMPK by circHIPK3-shRNA knockdown contributed to AKT/mTOR inactivation and EGFR/PDGFR degradation.● The results above combing with bioinformatic analysis indicated that circHIPK3 is an important regulator for NB and may be a potential therapy target.