The Expression Of PLK4 In Neuroblastoma And Its Underlying Mechanism

Neuroblastoma(NB)is the most common malignant tumor in infancy and the most common extracranial solid tumor in childhood.Owing to improvements of diagnosis and treatment,the survival rate of patients with low-risk and intermiderate-risk NB can reach up to 80-90%.But the progression-free survival rate(PFS)of high-risk NBs is still less than 40% because of the heterogeneity,complexity and refractory of NB.Great changes have taken place on targeted-therapy because of its specific advantages in killing tumor cells in development of anti-tumor drugs,but the application on treatment of pediatric oncology is limited.The pathogenesis of NB is still not completely clear,therefore it is of great significance to further explore the pathogenesis of NB and discovery new therapeutic targets for NB to improve the survival rate of NBs.PLK4,one of the Polo-like kinase family members,is an important regulator of centriole replication.Abnormal expression of PLK4 was found in liver cancer,colorectal cancer,pancreatic cancer,prostate cancer and so on,but the mechanism remained to be elucidated.The purpose of this study is to detect the expression level of PLK4 in NB tissues and whether there are relationships between PLK4 expression and clinical pathological features as well as prognosis of NBs.Then further experiments in vitro and in vivo were made to explore the mechanism of PLK4 on the occurrence and development of NB.Methods: 1.Tissue experiments: analysis of PLK4 expression level in NB tissuesCollect relevant biological information about PLK4 and explore the relationship between PLK4 expression level and survival rate of NBs using the chip.Detect the protein and mRNA expression level of PLK4 in 7 pairs of NB tumor tissues and paired normal tissues by western blot and RT-PCR technology.2.To verify the relationship between PLK4 expression and clinicopathological features as well as prognosis of patients with NBImmunohistochemical method was used to detect the expression of PLK4 in 85 patients with NB and the correlations between PLK4 expression and clinicopathological features and prognosis of NBs were analyzed by SPSS 22.0 software.3.Experiments in vitro: the impact of PLK4 on the biological behavior of NB cells(1)Construct NB cell lines with shcontrol and shPLK4 by lentivirus;(2)Using cell function experiments such as tablet cloning,MTT,apoptosis,transwell and scratch assays for exploration of the effects of PLK4 on proliferation,invasion movement and migration of NB cells;(3)Western blot and Immunofluorescence technique were used to detect the changes in the expression levels of epithelial-mesenchymal transition as well as transcription factors related to EMT between shPLK4 and shcontrol NB cells.The PI3K/Akt pathway was further inspected if it played an important role in the EMT of PLK4-mediated NB cells.4.Experiments in vivoShPLK4 and shcontrol NB cells were transplanted in nude mice.Tumor size and weight were regularly measured.Mice were sacrificed at 6 weeks.The actual measurement of tumor size,weight were observed after tumor removal.The expression levels of PLK4,proliferation,apoptosis and EMT were detected in tumor tissues.Results:1.In the software of bioinformatics,the survival rate of NBs was significantly higher in patients with lower mRNA expression of PLK4 than that of patients with higher expression(P<0.001).PLK4 expression was negatively correlated with the prognosis of NB patients.Protein and mRNA expression levels detected in NB tumor tissues were higher than those of the the paires normal tissues.2.The expression level of PLK4 was closely related with primary site(c2=4.093,P=0.038),the serum level of LDH(c2=29.181,P<0.001),recurrence(c2=7.280,P=0.006)and clinical stage by INSS(c2=5.270,P=0.019).Moreover,high expression level of PLK4 had relationship with Ki-67 expression and it had significantly negative correlations with 3-year OS(c2=12.485,P<0.001)and PFS(c2=10.616,P=0.001).However,there was no significant relationship between PLK4 expression and age,gender,N-myc status and bone marrow metastasis(P>0.05).3.Compared with the shcontrol NB cells,the abilities of cell invasion and migration in shPLK4 NB cells decreased and apoptosis rate increased,which further influenced the proliferation rate.ShPLK4 NB cells showed elevated expression levels of E-Cadherin,while decreased expression of N-cadherin,vimentin and Slug.Meanwhile,shPLK4 NB cells showed decreased expression of p-Akt with stable expression of Akt.Similar results were obtained by using the Akt inhibitor,LY294002 to interfere with pCDH-PLK4 NB cells.4.The abilities of tumorgenesis,matastasis were statistically decreased in shPLK4 NB cells.Conclusions:1.The expression level of PLK4 in NB tissues was remarkably elevated,and high expression of PLK4 was negatively correlated with the survival rate of NBs,which suggesting that PLK4 is a potential tumor promotor of NB.2.PLK4 promoted the migration and invasion of NB cells and suppressed apoptosis.3.PLK4 regulated EMT through PI3K/Akt signaling pathway to promote the occurrence and development of NB.