Expression Of VNN1 Gene Associate With Preoperative Chemoradiotherapy Response And Prognosis Of Patient With Colorectal Cancer And The Mechanism Of VNN1 Regulate Cancer Cell Apoptosis

Part1:VNN1 overexpression is associated with poor response to preoperative chemoradiotherapy and adverse prognosis in patients with rectal cancersObjective:Colorectal cancer is prevalent worldwide and it is also the fourth most common cause of cancer mortality.For rectal cancer,neoadjuvant concurrent chemoradiotherapy(CCRT)followed by radical proctectomy is gold standard treatment for patients with stage Ⅱ/Ⅲ rectal cancer.By data mining a public dataset of rectal cancer transcriptome(GSE35452)from Gene Expression Omnibus,National Center of Biotechnology Information(GEO,NCBI),we identifed that VNN1 was the most signifcantly upregulated gene among those related to nitrogen compound metabolic process(G0:0006807).Therefore,we analyzed the clinicopathological correlation and prognostic impact of VNN1 protein(pantetheinase),which encoded by VNN1 gene.Methods:VNN1 immunostaining was performed in 172 rectal adenocarcinomas treated with preoperative CCRT followed by surgery,which were bisected into high-and low-expression subgroups.Furthermore,statistical analyses were performed to correlate the relationship between VNN1 immunoreactivity and clinicopathological features,as well as three survival indices:disease-specifc survival(DSS),local recurrence-free survival(LRFS)and metastasis-free survival(MeFS).Results:High VNN1 immunoexpression was signifcantly associated with advanced pre-treatment and post-treatment disease and poor response to CCRT(all p<.026).In addition,VNN1 overexpression was linked to adverse DSS,LRFS and MeFS in univariate analysis and served as an independent prognosticator indicating worse DSS and LRFS in multivariate analysis(all p<.019).Conclusion:VNN1 may play a crucial role in rectal cancer progression and responsiveness to CCRT,and serve as a novel prognostic biomarker.Additional studies to clarify the molecular pathway are essential for developing potential VNN1-targeted therapies for rectal cancer.Part 2:Mechanism of VNN1 regulate apoptosis and proliferation of RKO cells after chemoradiotherapy Objective:This part of the study examined the apoptosis and proliferation of RKO cells,and verified the expression of the protein related with signaling pathways by Western-blot and RT-PCR analyses.To explore the mechanism of VNN1 gene regulating the apoptosis and proliferation of RKO cells after chemoradiotherapy.Methods:We cultured RKO cell strain in vitro,and construct post-chemoradiotherapy cell model.Then we up and down regulate the expression of VNN1 gene through virus transfection,and RKO cell transfected virus were randomly divided into 5 groups:normal control group(Control),VNN1 over-expression group(VNN1 Over),VNN1 inhibited expression group(VNN1 siRNA),VNN1 no-load group(VNN1 Null).All of the groups received chemotherapy and radiation therapy for 48 hours respectively,then we separately observed the cell morphology,and detected VNN1 transfection efficiency.The proliferation and apoptosis of RKO cells were analyzed in different groups,while we detected the protein related with signal pathway by Western-blot analyses.Results:The Western-blot and RT-PCR test results showed that the transfection efficiency of VNN1 was significantly increased in VNN1 Over group(p<0.05),while significantly decreased in VNN1 inhibited group compare to the control group(p<0.05).The number of cells were significantly decreased in the VNNldownregulated group than that in the control group(p<0.05),however significantly enhanced in VNN1 overexpressed group(p<0.05).downregulated group of VNN1 elevate apoptosis rate significantly(p<0.05),nevertheless,overexpression of VNN1 decreased it remarkably(p<0.05).The Akt and bcl-2 was significantly decreased in the VNN1 siRNA group(p<0.05),while the expression of Caspase-3 was significantly increased(p<0.05).In the VNN1 overexpression group,the Akt and bcl-2 were upregulated significantly(p<0.05),while the expression of Caspase-3 was significantly reduced(p<0.05).VEGF expression of the VNN1 inhibited group was significantly lower than the control and VNN1 no-load groups(p<0.05),while significantly increased in VNN1 overexpressed group(p<0.05).Conclusion:VNN1 may inhibiting the classic apoptotic pathway bcl-2/Bax/casp-3 by activate the expression of p-Akt,,inhibiting the apoptosis of RKO cells.It is also possible to promoting the proliferation of RKO cells by enhancing the expression of VEGF,so that affecting the apoptosis and proliferation of RKO cells.