The Role Of DEK Expression In The Proliferation Of Human Colorectal Adenocarcinoma Cell Line HCT116

BackgroundThe morbidity of colorectal cancer is gradually increased in recent years. Andpatients with concealed pathogenesis often lost the optimal opportunity of surgicaltherapy when colorectal cancer was diagnosed at advanced stage. Therefore, about40%of these patients have poor prognosis even after multimodality therapy (surgical therapy,radiotherapy and chemotherapy). Recent studies have demonstrated that DEK isoverexpressed in a variety of malignant tumors and is closely associated with tumordevelopment and chemoresistance. So it could be used as a new target for tumorbiotherapy. However, the role of DEK in colorectal cancer has rarely been reported. Inthis experiment, DEK expression levels of the human colorectal cancer cell line HCT116is altered by lentivirus infection, then the changes of biological characteristics that related to cell proliferation are observed. This will show the possibility to take DEK as apotential target of the biotherapy of colorectal cancer.ObjectiveshRNA vector and expression vector of DEK were constructed to package lentivirusthat could knock down or overexpress DEK. DEK levels in HCT116were changedthrough lentivirus infection. Then the changes of cell proliferation rate, colony formingability and cell cycle were observed. All these would provide the foundation of theoryand experiment to explore DEK as a new target of colorectal cancer therapy.Methods（1） Lentivirus production: constructed3shRNA vectors that target at DEKaccording to the instruction of the plasmid. Transfected HCT116cells with the3shRNA vectors respectively using LipofectamineTM2000and checked theknockdown efficiency by Western Blot. The most effective shRNA vector andits control were co-transfected with2lentivirus packaging plasmids respectivelyto harvest lentivirus that containing shRNA and relative control. ConstructedDEK expression vector. The DEK expression vector and its control wereco-transfected with2lentivirus packaging plasmids respectively to obtain DEKexpression lentivirus and relative control.（2） Infection: infected HCT116cells with lentivirus to gain stabilized DEKsilencing HCT116cell line, stabilized DEK overexpressed HCT116cell line,and their relative controls.（3） Changes of cell biological characteristics: MTT was performed on day1,2,3,4,5,6and7respectively to get the growth curve of DEK silencing HCT116cell line, DEK overexpressed HCT116cell line and their relative controls. Softagar colony formation assay was taken to detect the changes of colonyformation ability of HCT116cell after DEK knockdown or overexpression. Thecell cycle changes in DEK knocked down or overexpressed HCT116isdetermined by flow cytometer after PI staining.Results （1） Compaired with the DEK level in HCT116cells transfected with pLKO.1-TRCcontrol, all of the3shRNA vectors pLKO.1-DEK RNAi A, pLKO.1-DEK RNAiB and pLKO.1-DEK RNAi C could downregulate DEK expression in HCT116.Among them, pLKO.1-DEK RNAi A was the most effective one. pLKO.1-DEKRNAi A and pLKO.1-TRC control were packaged with2plasmids pMD2.G andpsPAX2respectively to produce lentivirus “DEK RNAi” and “RNAi CON”.After infection, DEK expression was stabilized knocked down in HCT116-DEKRNAi compired with HCT116-RNAi CON.（2） pLVX-AcGFP1-N1-DEK and pLVX-AcGFP1-N1were packaged with2plasmids pMD2.G and psPAX2respectively to produce lentivirus “DEK” and“CON”. After infection, DEK expression was stabilized upregulated inHCT116-DEK compaired with HCT116-CON.（3） MTT assay showed that the cell proliferation rate of HCT116-DEK RNAi waslower than HCT116-RNAi CON; the cell proliferation rate of HCT116-DEKwas higher than HCT116-CON.（4） Soft agar colony formation assay revealed that colony formation was decreasedin HCT116-DEK RNAi compare with HCT116-RNAi CON; was increased inHCT116-DEK in contrast with HCT116-CON.（5） The percentage of G0/G1phase in HCT116-DEK RNAi was increased comparewith HCT116-RNAi CON (74.9%vs63.3%), and S phase was decreased(15.5%vs22.2%), P<0.05; the percentage of G0/G1phase in HCT116-DEKwas decreased compare with HCT116-CON (55.9%vs65.8%), and S phase wasincreased (28.5%vs22.1%), P<0.05.Conclusion（1） DEK could influence HCT116cell proliferation rate: DEK knockdown coulddecrease HCT116cell proliferation while DEK overexpression could increaseHCT116cell proliferation. （2） DEK could affect HCT116cell colony formation ability: DEK knockdowncould inhibit HCT116cell colony formation while DEK overexpression couldpromote HCT116cell colony formation.（3） DEK could impact HCT116cell cycle: DEK knockdown could arrest HCT116cell in G0/G1phase while DEK overexpression could promote HCT116cell intoS phase.（4） DEK could be further explored as a potential target in colorectal cancer therapy.