The Role And Molecular Mechanisms Of CDCA3 In The Growth And Anti-apoptosis Of Colorectal Cancer

Backgroud and ObjectiveColorectal cancer(CRC)is one of the most common types of cancer worldwide.About 1.4 million people worldwide are diagnosed with CRC every year,and approximately 700 thousand patients died directly or indirectly of CRC.Overwhelming majority of early CRC patients can be cured by surgical operation.Unfortunately,the disease is usually diagnosed at an advanced stage,and so prognosis is poor.Therefore,it is of great importance to understand the molecular mechanism underlying colorectal tumorigenesis and progression and identify new markers and effective therapeutic strategies for colorectal cancer patients.Cell division cycle associated protein-3(CDCA3),also called Tome-1,is required for the process of mitosis and regulate cell cycle progression.CDCA3 functions as part of a SKP1-Cullin RING-F-box(SCF)ubiquitin ligase(E3)complex to degrade the endogenous cell cycle inhibitor Weel,and is a trigger of mitosis entry 1.CDCA3 are also regulated through the cell cycle by transcription,and protein degradation during the G1 phase of the cell cycle.Recent studies have reported that CDCA3 is frequently up-regulated in cancer,including lung cancer,prostate cancer and oral squamous cell cancer.And CDCA3 plays an vital role in the tumorigenesis and progression of cancer.However,little is known its role in colorectal cancer.Therefore,it is of great importance to understand the molecular mechanism underlying colorectal tumorigenesis and progression and identify new markers and effective therapeutic strategies for colorectal cancer patients.Methods1.The expression of CDCA3 in CRC tissues and cells(1)Real-time quantitative PCR(qRT-PCR)was employed to determine the expression of CDCA3 in 32 pair samples from fresh CRC biopsies and CRC cell lines SW480,SW620,HT29,LOVO,Caco-2,RKO,HCT116 and colorectal epithelium cell line FHC.(2)The relationship between CDCA3 expression and clinical parameters,including tumor size,differentiation,serosal invasion,lymph metastasis,and tumor-node-metastasis(TNM)stage.2.The role of CDCA3 in growth of CRC in vitro and in vivo(1)CDCA3 knockdown Lentivital vector was purchased from company and CDCA3 overexpression plasmid was constructed.The results from sequencing showed insertion sequence was correct and there was no mutation or loss.Then Lentiviral vectors were transfected to SW480 and SW620 for establishing stable CDCA3 k:nockdown cell lines.The plasmids were transfected to SW620 and Caco-2 for establing transient CDCA3 overexpression cell lines.The results were confirmed by using qRT-PCR and western blot.(2)The proliferation ability of celll lines with CDCA3 overexpression and knockdown were measured by CCK8,colony formation and flow cytometry.(3)SW620-pLV-shCDCA3,SW620-PLV-shNC were injected subcutaneously into the left or right flank of nude mice.After 20 days,we analyzed primary tumor growth.3.The influence of CDCA3 in regulating the NF-?B signaling pathway(1)GEO database and GSEA were used to analyse the enrichment of NF-?B pathway in CRC patients with high CDCA3 expression.(2)Dual Luciferase activity assay was used to detect the activity of NF-?B signaling.(3)Western blot was used to measure downstream NF-?B pathway related proteins.(4)Nuclear protein extraction assay and immunofluorescence were used to determine the nuclear accumulation of p65.4.The role of compound CDCA3 and TRAF2 in regulating the NF-?B signaling pathway(1)Confocal immunofluorescence and co-immunoprecipitation(COI?)were used to explore the interaction between CDCA3 and TRAF2(2)Western blot analysed that the relationship between CDCA3 and TRAF2 and the regulation of the NF-?B pathway.5.CDCA3 regulates the expression of the NF-kB-related gene Cyclin D1(1)QRT-PCR and Western blot were used to detect the expression of NF-KB-related gene Cyclin D1.(2)QRT-PCR and Western blot analysed the Cyclin D1 expression after treatment with Bay 11-7082(3)QRT-PCR analysed the relationship between CDCA3 and Cyclin D1 expression in 24 paired human colorectal cancer tissues and adjacent normal tissues.6.Statistical analysesStatistical analyses were performed using SPSS20.0 software.The differences between groups were tested by using a two-tailed Student's t-test.Data was presented as the mean ± SD of at least 3 independent experiments.One-Way ANOVA was used for multiple comparisons with SNK or LSD.Relationships between CDCA3 expression and clinicopathologic characteristics were determined by ?2 test.The linear relationship between the expression levels of CDCA3 and Cyclin D1 in CRC tissues was analyzed using Spearman's correlation coefficient.Differences were considered significant if p<0.05:*,p<0.05;**,p<0.01;***,p<0.001.Results1.CDCA3 overexpression in CRC associated with serosal invasion and TNM stage(1)CDCA3 is up-regulated in colorectal cancer tissues and cellsThe results of qRT-PCR showed CDCA3 expression was indeed higher in 27 of 32 cases of CRC specimens compared with those of the adjacent normal mucosa tissues(p<0.001).The expression levels of CDCA3 were also up-regulated in investigated CRC cell lines(SW620,SW480,HT29,HCT116)compared with normal colorectal epithelium cell FHC.(2)The relationship between CDCA3 expression and clinicopathological parameters correlation analysis showed that CDCA3 expression was positively associated with serosal invasion and tumor-node-metastasis(TNM)stage in CRC.Furthermore,there is no relationship between CDCA3 expression and age,gender,differentiation,tumor size,lymph metastasis.2.The role of CDCA3 in the development of CRC(1)The construction of the CDCA3 knockdown Lentuviral vectors and overexpression plasmid.The molecular cloning technologies were used to construct the CDCA3 knockdown Lentuviral vectors and overexpression plasmid.The results from sequencing showed insertion sequence was correct and there was no mutation or loss.Then,lentiviral vectors were transfected to SW480 and SW620 for establishing stable CDCA3 knockdown cell lines.QRT-PCR showed CDCA3 were significantly reduced both in SW480 and SW620 cells(P<0.001).The plasmids were transfected to SW620 and Caco-2 for establing transient CDCA3 overexpression cell lines.The results were confirmed by qRT-PCR and western blot(P<0.001).(2)The effects of CDCA3 knockdown and overexpression on proliferation in CRC cells.CCK8 proliferation assays revealed that knockdown of CDCA3 significantly decreased cell growth(P<0.001)and overexpression of CDCA3 increased cell growth(P<0.001).Similarly,colony formation capacity was suppressed after the downregulation of CDCA3(P<0.05).(3)Downregulation of CDCA3 induces cell cycle arrest and apoptosis the number of cells in the G1 or G2/M phase was assessed by flow cytometry,we found pLV-shCDCA3 cells increased at the G1 phase and induced G1 phase cell cycle arrest significantly compared with the pLV-shNC cells.The downregulation of CDCA3 induced a significant increase of early apoptosis in SW480 cells(1.3%± 1.0 to 5.4%± 0.8)and SW620 cells(13.5%± 2.4 to 25.2%±3.6).To better understand the mechanism that induces cell apoptosis mediated by sh-CDCA3,the expression levels of some of the cell apoptosis associated proteins including Caspase 3 and PRAP,were detected.The downregulation of CDCA3 increased the expression levels of cleaved-caspase3 and cleaved-PARP.No change in the total protein amounts of precursors of caspase3 and PARP was observed under any circumstance.(4)CDCA3 knockdown inhibits the growth of CRC in vivo.Xenograft model showed that mice in SW620/shCDCA3 group developed smaller tumors than those in SW620/shNC group(P<0.05),IHC assay confirmed that Ki-67 proliferation index in the SW620/shCDCA3-xenografted tumors was lower than that in SW620/shNC-xenografted tumors(P<0.05).3.CDCA3 is involved in regulating the NF-?B signaling pathway(1)The luciferase reporter assay was performed to detect the luciferase activity of the NF-?B.An increase of the NF-?B luciferase activity was observed upon upregulation of CDCA3 in the SW620,which was decreased when CDCA3 was inhibited.The results of western blot analyses indicated that the down-regulation of CDCA3 decreased the expression of P105/50,IKK?,IKK?,and p-P65 compared to control cells.On the other hand,the up-regulation of CDCA3 caused the opposite effect.(2)Knockdown of CDCA3 led to the decreased nuclear p-P65 expression in SW480 and SW620 cells.Induction of p65 nuclear translocation by CDCA3 was further confirmed by immunofluorescence in Caco-2 and SW620 cells.4.CDCA3 activates the NF-?B signaling pathway by interacting with TRAF2 in CRC(1)Immunofluorescence indicated the co-localization of CDCA3 and TRAF2 in the cytoplasm of Caco-2 and SW620 cells.Reciprocal coimmunoprecipitation assay showed that TRAF2 and CDCA3 pulled down each other reciprocally.(2)The down-regulation of CDCA3 evidently descreased the expression levels of TRAF2 as compared with their control cells.Conversely,the up-regulation of CDCA3 led to the opposite effects.Treatment with the si-TRAF2 could abrogate the increase of IKKa,IKK?,and p-P65 by CDCA3.5.Overexpression of CDCA3 up-regulates the NF-?B-related gene Cyclin D1(1)Both qRT-PCR and western blot assays confirmed that CDCA3 suppressed the expression of Cyclin D1 and P21.(2)Treatment with the inhibitor of NF-?B signaling pathway(Bay 11-7082)led to the suppression of P65 and Cyclin D1.(3)CDCA3 and Cyclin D1 expression have positive relationship in colorectal cancer.Conclusion1.CDCA3 is up-regulated in colorectal cancer2.Knockdown of CDCA3 inhibits CRC cell proliferation in vitro and tumor growth in vivo3.Downregulation of CDCA3 induces cell cycle arrest and apoptosis4.CDCA3 activates the NF-?B signaling pathway by interacting with TRAF2 in CRC5.Overexpression of CDCA3 up-regulates the NF-?B-related gene Cyclin D1...