| Objective Bladder cancer(BCa)is one of the most common malignant tumors of the urinary system.Cell division cycle associated 8(CDCA8)is a protein closely related to the cell division cycle.More and more studies have shown that CDCA8 is closely connected to the occurrence and development of multifarious malignant carcinoma.Our previous research results found that CDCA8 also played a certain role in the development of Bca,however,the specific mechanism has not been studied.Usually,AKT presents an over-activated state in tumors,and the main reason that over-activated AKT/GSK3β signaling pathway could play an important role in tumors is the phosphorylation of GSK3β.We also found that CDCA8 is strongly linked to the AKT/GSK3β signaling pathway in Bca through gene set enrichment analysis(GSEA).Based on this,we speculate that CDCA8 and AKT/GSK3β signaling pathway together play a significant role in the occurrence and development of bladder cancer.This project intends to study the specific mechanism of the CDCA8-AKT-GSK3β axis in Bca.Methods Immunohistochemistry(IHC),real-time quantitative PCR(RT-q PCR)and western blot were used to detect the expression of CDCA8 in Bca samples and adjacent tissues,then analyze and verify the relationship between CDCA8 and clinicopathological parameters of Bca patients(both tissue-chip external-data group and 36-patients internal-data group).To study the effect of CDCA8 on the biological behavior of Bca at cell level,MTT assay,cloning survival assay and flow cytometry analyses were utilized to evaluate the effects of knockdown and overexpression of CDCA8 on Bca cell proliferation,transwell migration assay was applied to evaluate the effects of knockdown and overexpression of CDCA8 on Bca cell migration abilities in vitro.Western blot,immunoprecipitation(IP)and cellular immunolocalization were used to evaluate the role of the AKT/GSK3β signaling pathway in this process.In addition,lentiviruses were used to construct stable CDCA8-knockdown Bca cell line,xenograft model and pulmonary metastasis model are applied to study the effects of CDCA8 on Bca proliferation and migration abilities in vivo.Results In tissue-chip external-data group,we found that CDCA8 was highly expressed in Bca tissues,which was positively correlated with tumor stage(P = 0.024)and tumor grade(P = 0.031)of Bca,and patients with low expression of CDCA8 had a much better survival than those patients with high expression of CDCA8(P <0.001).The univariate and multivariate analyses revealed that high CDCA8 expression was an independent risk factor of BCa survival(HR: 1.941;P = 0.049;95% CI,1.003–3.757).In 36-patients internal-data group,we also found CDCA8 was highly expressed in Bca tissues,which was positively correlated with tumor stage(P = 0.032)and tumor grade(P = 0.013)of Bca,however the prognosis in CDCA8 high expression patients and low expression patients had no significant difference.Another our results showed that knockdown CDCA8 expression could reduce the proliferation rate,colony formation capacity of Bca cells and block Bca cells in the G0/G1 phase.Conversely,overexpression of CDCA8 can increase the proliferation rate and colony formation capacity of Bca cells and obvious activated Bca cells in G0/G1 phase.In addition,knockdown of CDCA8 inhibited the AKT/GSK3β signaling pathway,and overexpression of CDCA8 activated the AKT/GSK3β signaling pathway,furthermore,we found that CDCA8 activated the AKT/GSK3β signaling pathway through interacting with AKT.We found that CDCA8 regulates the proliferation and migration of Bca cells through the AKT/GSK3β signaling pathway since the overexpression of AKT could significantly rescue the suppression of proliferation and migration in CDCA8-silencing BCa cells.Also,our results demonstrated that knockdown of CDCA8 could inhibit the proliferation and migration of Bca in vivo.Conclusions In summary,our results proved that CDCA8 could be an independent prognostic risk factor of Bca.CDCA8 could promote the proliferation and migration of Bca,and this process was mainly achieved by CDCA8 interacting with AKT to activate the AKT/GSK3β signaling pathway. |
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