Study On The Function And Mechanism Of KLF5 And TNFRSF11a In Cervical Cancer

Objective To investigate the expression of Krüppel-like Factors 5(KLF5)and Tumor Necrosis Factor Receptor Superfamily 11a(TNFRSF11a)in cervical squamous cell carcinoma and their molecular and cellular mechanisms on cell proliferation,migration and invasion of cervical cancer.Methods The normal cervical and pathological cervical tissues of Han females who were treated in gynecological clinic and inpatient department of Tangshan City workers' hospital were collected as experimental study specimens.In strict accordance with the inclusion criteria and exclusion criteria,from September 2014 to March 2016,a total of 160 cases were collected.All cervical tissues for experiments were studyed by the laboratory researchers and pathologists to confirm the pathological type and grade,includes 47 cases of the control group,73 cases of cervical intraepithelial neoplasia(CIN)(27 cases of CIN ?,46 cases of CIN II-III)and 40 cases of cervical squamous cell carcinoma(CSCC).Microarray technology was used to detect the mRNA expression of Gene responsed to cytocine stimulus in 3 cases of cervical squamous cell carcinoma and 3 cases of control cervical tissues.The quantitative real-time polymerase chain reaction(q RT-PCR)was used to validate the result of microarray screening and to analyze the correlation between the mRNA expression of KLF5 and TNFRSF11 a in different pathological cervical tissues.Clinical analysis of the relationship between mRNA expression of TNFRSF11 a and clinicopathological parameters of cervical squamous cell carcinoma.The expression of KLF5 and TNFRSF11 a in different pathological cervical tissues were detected by double immunofluorescence staining.He La cells were transfected specific si RNA to knockdown the endogenous TNFRSF11 a and KLF5,and were infected with Ad-KLF5 to overexpress KLF5,respectively.After the different treatment conditions,q RT-PCR was used to detect the mRNA expression level in He La cells and the protein level was detected by Western blot.The activity of the cell proliferation,migration and invasion was detected using CCK-8 assay and Transwell assay.The regulatory effect of KLF5 on candidate target gene(TNFRSF11a)was determined by dual-luciferase reporter assay and Chromatin immunoprecipitation(Ch IP)was used to test the binding site.Results 1 The results of Microarray technology showed that the expression of KLF5,Npnt,Socs5,TNFRSF11 a,Cxcl13,Il6 st,Cntfr and Plcb1 were significantly higher in cervical squamous cell carcinoma tissues compared with control cervical tissues(P<0.05).2 The results of q RT-PCR showed that KLF5,Npnt,Socs5,TNFRSF11 a,Cxcl13 and Il6 st were significantly higher in cervical squamous cell carcinoma tissues compared with control cervical tissues(P<0.05).3 Double immunofluorescence staining showed that the protein expression of KLF5 and TNFRSF11 a were significantly higher in CIN ?,CIN ?-? and cervical squamous cell carcinoma tissues compared with control cervical tissues(P<0.05).4 The results of q RT-PCR showed that KLF5 and TNFRSF11 a were significantly higher in CIN ?,CIN ?-? and cervical squamous cell carcinoma tissues compared with control cervical tissues(P<0.05),and they were significantly higher in cervical squamous cell carcinoma tissues compared with CIN ?-? cervical tissues(P<0.05).5 Spearman method was used to test the relationship of KLF5 and TNFRSF11 a in different cervical tissues.In control group and CIN I group the mRNA expression of KLF5 and TNFRSF11 a had no significant positive correlation;in CIN II-III group their expression of mRNA were positively correlated(r=0.326,P=0.027),and in cervical cancer tissues their expression of mRNA were positively correlated(r=0.393,P=0.012).6 Overexpression of KLF5 can upregulate the level of TNFRSF11 a protein and mRNA,and knockdown of KLF5 can down regulate the protein and mRNA levels of TNFRSF11 a with or without TNF-?treatment(P<0.05).Luciferase assay and Ch IP assay showed that the TNFRSF11 a gene promoter was significantly activated by KLF5 in He La cells.7 The test of cells function shows that overexpression of KLF5 could improve the proliferation,migration and invasion of He La cells,knockdown of TNFRSF11 a could inhibit the proliferation,migration and invasion of He La cells,overexpression of KLF5 after knockdown of TNFRSF11 a could partly inhibit the proliferation,migration and invasion of He La cells(P<0.05).8 The analysis of clinical parameters shows the degree of pathological differentiation,depth of myometrial invasion,clinical stage and lymph node metastasis is significantly related to the expression of TNFRSF11 a in cervical squamous cell carcinoma(P<0.05).Conclusion 1 The coexpression of KLF5 and TNFRSF11 a can promote the occurrence and development of cervical squamous carcinoma.2 KLF5 can directly interact with TNFRSF11 a promoter in order to activate the transcription of TNFRSF11 a.3 KLF5 promotes cervical cancer proliferation,migration and invasion partly dependent on the expression of TNFRSF11 a.