The Functional And Mechanical Study Of VRK1 In The Initiation And Progression Of Esophageal Squamous Cell Carcinoma

BackgroundEsophageal carcinoma is the eighth most common malignant disease and ranks as the sixth leading cause of cancer-related death worldwide.Esophageal squamous cell carcinoma(ESCC),the predominant histological subtype of esophageal carcinoma,accounts for approximately 90%of newly diagnosed esophageal cancers in developing country.Although enormous progress has been made in the diagnosis and treatment of ESCC,the 5-year survival rate for ESCC patients is only 5%-40%.VRK1(vaccinia-related kinase 1)is one of the three members of the VRK family of Ser-Thr kinases that participate in cell division,transcriptional activation,DNA repair and histone modification.Up-regulation of VRK 1 has been observed in various human cancers,and a high level of VRK1 at the protein or RNA level is associated with a proliferative phenotype.Although previous research has reported that VRK1 expression levels are elevated in ESCC,the biological functions of VRK1 in ESCC are still unclear.In this study,we aim to investigate the biological functions of VRK1 in the ESCC and the underlying mechanism.The expression profile of VRK1 in ESCC was evaluated.We further explored the biological function and the relevant mechanism of VRK 1 in ESCC.ObjectiveThe aim of this study was designed to illustrate the expression profile of VRK1 in ESCC and its impact on the biological behavior of ESCC cells.We also tried to evaluate the biological functions of VRK1 in the initiation and progression of ESCC as well as the underlying mechanism.Our results will provide the experimental and theoretical basis for the diagnosis,prognostic evaluation and novel targeted therapy of ESCC.Materials and Methods1.Clinical samples and cell lines,2.RNA extraction and Real-time quantitative PCR,3.Western blot analysis,4.Immunohistochemistry,5.Cell culture,6.Vectors construction and transfection,7.Small interfering RNA,8.Luciferase assay,9.Apoptosis assay,10.Cell counting kit-8 assay,11.Migration and invasion assays,12.Colony formation assay,13.Wound-healing assay,14.Xenograft model,15.Statistical analysis.Result1.We examined gene profiling data for VRK1 expression in 41 paired fresh ESCC tissues using RT-PCR.Compared with the adjacent non-cancerous tissues(ANCTs),the tumor tissues presented significant VRK1 overexpression(P<0.01).VRK1 was differentially up-regulated in the ESCC cell lines compared with that in the normal Het-1a and HECC cells.The semiquantitative IHC analysis showed that high VRK1 expression was observed in 59.84%(79/132)of tumor tissues.Consistent with this observation,statistical analysis revealed that aberrant VRK1 protein levels were significantly correlated with the depth of invasion,lymphatic involvement and TNM stage.Furthermore,Kaplan-Meier and log-rank analyses indicated that patients with high levels of VRK1 expression had worse overall survival(OS)or disease-free survival(DFS)than patients with low levels of VRK1 expression.Ultimately,the univariate and multivariate Cox regression analyses demonstrated that VRK1 expression,along with T classification and N classification,were independent prognostic factors in ESCC.2.The ESCC cell lines were stably transfected with either negative control lentivirus or VRK1 silencing lentivirus.Compared with those transfected with negative control vectors,ESCC cells transfected with shRNA targeting VRK1 exhibited an increased proliferation,colony-forming ability,migration and invasion capacity as well as higher apoptotic rates.In contrast,ectopic expression of VRK1 in ESCC cells led to a significant increase in cell proliferation,colony formation,migration and invasion as well as lower apoptotic rates.3.Reconstitution of VRK1 expression in ESCC cells notably elevated cell survival relative to that of the control cells in response to cisplatin in a dose-and time-dependent manner.Conversely,knockdown of endogenous VRK1 markedly sensitized ESCC cells to cisplatin.Xenograft model showed that tumors with VRK1 overexpression plasmid presented increased chemoresistance to cisplatin,while tumors transfected with VRK1-shRNA exhibited a marked cytotoxic response to cisplatin.4.Western blotting showed that VRK1 up-regulated the proportion of phosphorylated c-Jun was in ESCC cells.Activated c-Jun displayed reduced sensitivity to cisplatin,increased proliferation and decreased apoptosis.Moreover,targeting c-Jun largely blocked VRK1-induced malignant behavior in ESCC cells.5.We found by western blotting and RT-PCR showed that down-regulation of c-Jun attenuated the expression of c-MYC in ESCC cells.Moreover,down-regulation of c-MYC was observed in ESCC cells with stable VRK1 knockdown.Furthermore,a dual luciferase reporter system showed that knockdown of c-Jun inhibited the luciferase activity of the c-MYC promoter in ESCC cells,indicating that c-Jun regulated the expression of c-MYC at the transcriptional level.In addition,silencing c-MYC strongly blocked c-Jun-enhanced cisplatin resistance in ESCC cells.6.In response to luteolin,a VRK1 inhibitor,ESCC cell proliferation was significantly reduced and apoptosis was significantly increased in a dose-dependent manner.Similar results were obtained from a colony formation assay.Additionally,luteolin also suppressed the invasive and migratory ability of ESCC cells.Luteolin inhibited c-Jun phosphorylation and c-MYC expression.Moreover,as analyzed by western blotting and CCK-8 assay,luteolin enhanced the cytotoxic effect of cisplatin on ESCC cells.In vivo assay showed that the tumor size in luteolin-treated group was significantly smaller than the NS-treated group.Additionally,luteolin administration rendered tumors more sensitive to cisplatin than NS administration.7.VRK1 expression was strongly correlated with c-MYC expression in 132 ESCC specimens(P<0.001).Kaplan-Meier analysis revealed that patients in the VRK1low/c-MYClow group exhibited significantly longer OS and DFS in comparison with the other three groups,while patients in the VRK1high/c-MYC high group carried the worst prognosis.ConclusionThis study clarified that VRK1 was up-regulated in ESCC and unveiled its correlation with clinicopathological characteristics.VRK1 was found to promote cell proliferation and migration as well as cisplatin(CDDP)resistance in ESCC.Furthermore,our results indicated that VRK1 up-regulates c-MYC through c-Jun activation and that this axis is responsible for VRK1-mediated CDDP resistance.We also examined the inhibitory effect of VRK1 using luteolin,a VRK1 inhibitor,in ESCC cells both in vitro and in vivo.These data provide the first evidence that VRK1 plays an essential role in the initiation and progression of ESCC and also indicate that VRK1 may serve as a therapeutic target for ESCC treatment.