Role And Mechanism Of KIF4A High-Expression In Promoting Bladder Cancer Invasion And Progression

【Research Background】Bladder urothelial carcinoma(BLCA)is a common urological system cancer in China.Overall,5%–10% of BLCA cases are diagnosed during the metastasis period,and after radical cystectomy,approximately 50% patients experience distant or local tumour relapse.Despite the development in integrative treatment and surgical techniques,nearly50%–70% of new cases have a relapse risk in the following 5-years.Therefore,it is necessary to search for novel molecular markers related to the progression and metastasis of bladder cancer,and predict which patients are more likely to progress to muscularinvasive bladder cancer,so as to find the development direction of molecular targeted therapy.KIF4A(Kinesin family member 4A)is a member of KIFs.Up to now,more and more studies have confirmed that KIFs plays an important role in a variety of human cancers,including bladder cancer.KIFs proteins are involved in cell division.Abnormal proteins can change the distribution of genetic material in cells and cause cell cycle to be out of control and eventually lead to tumor formation gradually through excessive chromosome agglutination,abnormal spindle formation,cell division defects,late bridge or aneuploid formation and mitotic block.In addition,KIFs play an important role in transporting organelles,m RNA complexes and regulating plasma membrane receptors.In recent years,several studies have confirmed its carcinogenic effect in tumor,but its specific role in bladder cancer has not been thoroughly studied,and its functional mechanism needs to be further clarified.【Research Objective】To explore and verify the expression level of KIF4 A in urinary tract carcinoma and normal adjacent urothelial epithelium;To study the relationship between the expression of KIF4 A and the prognosis and clinicopathological stage of bladder cancer patients,and to further clarify the role of KIF4 A in the occurrence,development,migration and invasion of bladder epithelial carcinoma.【Research Methods】1.Transcriptome sequencing was performed on 10 pairs of urinary tract carcinoma and adjacent tissues,and the expression of KIF4 A in urinary tract carcinoma was determined according to the transcriptome sequencing results,database and relevant literature research.2.To study the expression of KIF4 A protein in bladder urothelial carcinoma cells,and extract proteins and m RNA from patients’ bladder urothelial carcinoma tissues to study the expression level of KIF4 A.3.Immunohistochemical staining was performed on tissue chips of 232 bladder cancer patients collected from our hospital to further determine the expression of KIF4 A in bladder cancer patients.Patients were divided into high expression group and low expression group according to the expression level so as to analyze the relationship between KIF4 A expression and clinicopathological features and prognosis of patients.4.Lentivirus and si RNA were used to construct overexpression,stable knockdown and transient silencing cell lines,and the stable interfering cell lines were used for secondgeneration transcriptome sequencing for the next step of pathway change analysis;The effects of KIF4 A on the proliferation of bladder cancer cells were studied by cell colony formation and CCK-8 tests,the effects of KIF4 A on the invasion and migration of bladder cancer cells were studied by Transwell chamber invasion test and cell scar test,and the role of KIF4 A in the apoptosis of bladder cancer was studied by flow cytometry.Western blot was used to further explore the changes of proteins related to proliferation,invasion,migration and apoptosis in KIF4 A knockdown and overexpression cell lines.5.The relationship between KIF4 A and transcription factor YAP1 and ITGB1/PI3K/Akt pathway was explored through bioinformatics analysis,immunofluorescence staining and cytoplasmic separation analysis.【Research Results】1.Transcriptome sequencing results of 10 tissues indicated that KIF4 A was significantly overexpressed in uroepithelial carcinoma of bladder compared with adjacent tissues(P<0,01).Real-time quantitative fluorescence PCR(RT-QPCR)and western blot(WB)showed that the expression of KIF4 A in bladder cancer patients was significantly higher than that in adjacent normal epithelial tissues.2.Immunohistochemical results of 232 cases of urinary tract carcinoma showed that the expression level of KIF4 A in MIBC was significantly higher than that in NMIBC.The weighted immunohistochemical score was determined by formula :0 × % no staining + 1× % weak staining + 2 × % moderate staining + 3 × % strong staining;When the score was between 0 and 300,the cancer-specific staining score of nucleus and cytoplasm was calculated separately and combined.The total score was divided into high or low according to the median(≤240,low expression;> 240,high expression),it was found that high expression of KIF4 A was closely associated with poor clinicopathological grade,larger tumour size,and poor prognostic indicators of OS and CSS(P <0.05).3.Sv-huc and BIU-87,5637,J82,UM-UC-3,HT1376,T24 cell protein detection,The expression levels of MIBC cell lines(5637,J82,T24,UM-UC-3)and NMIBC cell lines(BIU-87)were higher than those of normal uroepithelial cell lines(SV-HUC).4.Constructing KIF4A-overexpressed 5637 cell line and silencing the expression of KIF4 A in 5637 cell line and T24 cell line with small interfering RNA.Colony formation and CCK-8 cell proliferation test showed that the proliferation ability of knockdown T24 and 5637 cells was significantly decreased compared with the control group(P <0.001).Scratch test showed that the migration ability of KIF4 A knockdown 5637 and T24 cell lines decreased significantly compared with the control group(P <0.001).Transwell cell invasion test showed that KIF4A-knockdown 5637 and T24 cell lines had significantly decreased invasion ability(P <0.001).On the contrary,KIF4 A overexpressed 5637 cell lines showed significantly increased proliferation ability(P <0.001),migration and invasion ability(P <0.001)and KIF4 A expression was correlated with cell EMT.Flow cytometry showed that KIF4 A knockdown promoted the apoptosis of bladder cancer cells.5.KIF4 A knockout cell lines were constructed,and the downstream pathways were identified by second generation transcriptome sequencing.KIF4 A was found to be related to the AKT pathways and affected the activation of downstream AKT and YAP pathways by affecting the expression of ITGB1 and ITGB3.6.By constructing YAP overexpression 5637 cell line,it was found that YAP1 could promote the transcriptional expression of KIF4 A,and the m RNA and protein levels of KIF4 A in 5637 cell line with TEAD1 knockdown decreased,suggesting that YAP1 and TEAD1 may play an important role in the transcriptional expression of KIF4 A.7.In the knockout KIF4 A T24 and 5637 cell lines,WB found that the level of YAP1 protein decreased compared with the control group,while in the KIF4A-overexpressed5637 cell lines,the phosphorylated YAP decreased significantly,but the YAP1 protein did not.Further nuclear cytoplasmic separation and WB experiments showed that after KIF4 A was knocked down,compared with the control group,the level of YAP1 in nuclear cytoplasm decreased,while p-YAP(Ser127)increased in nucleus.After KIF4 A was overexpressed,the level of YAP1 was significantly upregulated in nucleus,while p-YAP(Ser127)was significantly down-regulated in nucleus,but the changes in cytoplasm were not significant.In addition,immunofluorescence experiments showed that compared with the control group,After KIF4 A is knocked down,the expression of YAP1 in the outside of the nuclear core is significantly weakened,which suggests that KIF4 A plays an important role in inhibiting the phosphorylation and degradation of YAP1 in the nucleus,indicating that KIF4 A plays an important role in the stability of YAP1.【Research Conclusion】KIF4A expression was significantly increased in urinary tract epithelial tumors of bladder cancer,and its expression level could be used as a possible molecular marker of bladder cancer stage and grade.The high expression of KIF4 A is a prognostic risk factor for urothelial carcinoma of bladder.Decreased expression of KIF4 A could inhibit the migration,invasion and epithelial-mesenchymal transformation(EMT)of urinary bladder epithelial cancer cells.In bladder urothelial cell lines,high expression of KIF4 A leads to upregulation of ITGB1,activation of AKT pathway,and maintenance of stability of YAP1,which is a potential mechanism leading to invasion and progression of bladder cancer.