Cell Cycle Dysregulation With Ovexpression Of KIF2C/MCAK Is A Critical Event In Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma(NPC)is a common malignant tumor of the head and neck,and has obvious geographical distribution characteristics.The epidemiological trend of the past ten years has shown that its incidence rate has gradually decreased,and the mortality rate has reduced significantly.However,its pathogenesis is not yet fully understood,and the further investigation is needed.In this study,we systematically analyzed the transcriptome data set of NPC using bioinformatics analysis methods and various molecular oncology research techniques,and conducted a preliminary systematic study on the function of a new candidate key gene KIF2C(Kinesin family member 2C,KIF2C)in NPC.(1)Screening and analysis of potentially key hub genes in NPC cellsFirstly,GEO database(Gene Expression Omnibus,http://www.ncbi.nlm.nih.gov/gds/)was retrieved to select the four NPC gene expression profile datasets with high quality(GSE12452,GSE53819,GSE13597 and GSE118719),independent processing and subsequent normalization of the original data of different datasets,focused on identifying the critical molecular networks and novel key hub genes implicated in NPC.A total of 170 common overlapping differentially expressed genes(DEGs)were found in four NPC datasets.The GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis of DEGs show that cell cycle dysregulation is a critical event implicated in NPC tumorigenesis.Protein-protein interactions(PPI)network analysis found a core network composed of 15 hub genes,in which the overexpressed KIF2 C was used as the central regulatory factor.Therefore,we decided to focus on investigating the functional implication of KIF2 C in NPC.(2)KIF2C is a key gene that regulates the growth and motility of NPC cellsThen the double stranded negative control short hairpin RNA(sh RNA)oligonucleotides and sh RNA targeting human KIF2 C m RNA,designated as NCsh and KIF2 Csh.The lentivirus particles were used to infect the HNE-1 and CNE-1 cells,and forty-eight hours later,cells were collected and subjected to subsequent analysis.Firstly,cell proliferation assay results showed that compared with NCsh cells,the cell proliferation rate of the KIF2 C silenced was significantly decreased,and the cell viability was significantly reduced.And FACS analysis showed that KIF2 C silencing resulted in a significant G2/M and S phase arrest,and caused an increase in the apoptotic population in NPC cells.Then the wound-healing assay and motility analysis indicated that KIF2 C silencing significantly inhibited the migration and motility of HNE-1 and CNE-1 cells.Finally,we analyzed the effect of KIF2 C knockdown on mitotic events in NPC cells by fluorescence microscopy.The results showed that KIF2 C knockdown caused a significant increase in binulceate and multinucleate cells in both HNE-1 and CNE-1 cells.Remarkably,KIF2 C knockdown also caused cells with mitotic defects in spindle structure and chromosome alignment.These results suggest that KIF2 C is an important gene involved in the regulation of mitosis and motility in NPC cells.(3)Molecular mechanism of KIF2 C regulating the growth and motility of NPC cellsTo further investigate the potential molecular mechanism behind KIF2C-related NPC tumorigenesis,the KIF2 C knockdowned cells and NCsh cells were collected,total RNA was extracted and subjected to RNA-seq analysis.The results showed that KIF2 C gene silencing resulted in the downregulation of 1558 genes and up-regulation of 1385 genes.Further GO,KEGG and GSEA analysis showed that differentially expressed genes were mainly concentrated in important signaling pathways such as cell proliferation and cell movement.Quantitative RT-PCR results showed that KIF2C gene silencing could induce down-regulation of CCNA1,E2F1 and NID1,and up-regulation of TGFB2 and PTEN genes.Moreover,Immunoblotting verified that KIF2 C knockdown reduced phosphorylation of AKT in HNE-1 cells,and decreased phosphorylated m TOR in CNE-1 cells.These results together indicate that KIF2 C might be an important regulator for multiple cancer-related signaling pathways in NPC cells.(4)KIF2C silencing enhances paclitaxel sensitivity in NPC cellsFinally,we sought to examine whether KIF2 C knockdown could enhance paclitaxel sensitivity in NPC cells as KIF2 C is a critical regulator of microtubule dynamics.The results showed that paclitaxel treatment could strongly inhibit cell growth and caused significant abnormal morphological changes in both HNE-1 and CNE-1 cells.Live cell growth monitoring and trypan blue exclusion assay revealed that KIF2 C knockdown cells with 10 n M paclitaxel treatment had a dramatic decrease in cell growth and a significant increase in dead cells especially in HNE-1 cells as compared with KIF2 C knockdown or paclitaxel treatment alone.Together these data suggest that KIF2 C knockdown can exacerbate the effect of paclitaxel to block mitotic progression and increase cell death.In summary,our present study systematically analyzed the NPC transcriptomic datasets,and identified cell cycle dysregulation as a critical event in NPC,and KIF2 C is a key hub gene that regulates the molecular network of malignant phenotypes in NPC cells.The results of functional experiments confirmed that KIF2 C is a key hub gene that regulates the growth and motility of NPC cells.Our results suggest that KIF2 C may be a new target for molecular diagnosis and targeted therapy of NPC.This study has positive theoretical and practical significance for further research and clarification of the role,molecular mechanism and targeted diagnosis and treatment value of KIF2 C in the occurrence and development of NPC and other malignant tumors.