Roles Of Agrin In HPVs-associated Human Cervical Carcinoma Cells

BackgroundHigh-risk human papillomavirus,especially HPV16 and HPV18,are the primary causes of cervical carcinoma.The products of E6 and E7 gene induce malignant transformation of infected cellsby interacting with the target in infected cells.Agrin is a kind of heparansulfate proteoglycan(HSPG),which are the components of extracellular matrix(ECM).Involving in viral initially binding,storage,invasion and transcriptional transactivation,HSPG plays an important role in the process of viral infection.cDNA microarray analysis found that Agrin have a various expression between normal cervical and cervical cancer.However deeply research reports about this factor has not been found and the role and mechanism of Agrin in HR-HPVs related cervical cancer has remained unclear.ObjectivesTaken cervical carcinoma Caski cells(HPV16-positive),SiHa cells(HPV16–positive),HeLa cells(HPV18-positive)and C33-A cells(HPVs-negative)as the research objects,research into the role and preliminary mechanisms of Agrin in HR-HPVs related cervical cancer initial and development to enrich the carcinogenic mechanisms of HR-HPVs.Methods1.Real-time quantitative polymerase chain reaction(qPCR)and Western blotting methods were used to detect the expressions of Agrin mRNA and protein in cervical carcinoma Caski cells(HPV16-positive),SiHa cells(HPV16-positive),HeLa cells(HPV18-positive)and C33-A cells(HPVs-negative).2.qPCR and Western blotting methods were used to detecte the expressions of ?-catenin mRNA and protein in cervical carcinoma Caski cells(HPV16-positive)and HeLa cells(HPV18-positive),C33-A cells(HPVs-negative).3.Using immunofluorescenceco-localization technology to validate the relationshipbetween Agrin and ?-catenin in HR–HPVsassociated human cervical carcinoma cells.4.The small interferingRNA(siRNA)sequences targeting Agrin mRNA and negative control(NC)siRNA were designed and constructed,named siRNA-Agrin and siRNA-NC.5.The siRNA-Agrin and siRNA-NC respectively transfered into Caski cells(HPV16-positive),HeLa cells(HPV18-positive)by liposome.The Caskicells and HeLa cells transfected siRNA-Agrin were named as Caski-siRNA-Agrin cells and HeLa-siRNA-Agrin cells,while the Caski cells and HeLa cells transfected siRNA-NC were named as Caski-siRNA-NC cells and HeLa-siRNA-NC cells.The silencing efficiency is detected by Western blotting and qPCR methods.6.The proliferation,migration and invasion of Caski cells and HeLa cells after silencing Agrin expression were detected by CCK 8 experiment,transwell test and invasion test,scratch wound healing assay.Results1.Western blotting and qPCR results indicated: compared with C33-A cells,the expressions of Agrin mRNA and protein in Caski cells,SiHa cells,and HeLa cells were significantly increased(P<0.01 or 0.05).The order of Agrin mRNA and protein expressions in the four kinds of cervical carcinoma cells was as follows: C33-A cells < SiHa cells < Caski cells and HeLa cells.2.Western blotting and qPCR results indicated: the expressions of ?-catenin mRNA and protein in Caski cells and HeLa cells are higher than those in C33-A cells(P<0.01).3.Immunofluorescence co-localization technology to validate the relationship between Agrin and ?-catenin in cervical carcinoma Caski cells,HeLa cells,C33-A cells,the co-localization in the cell membrane and cytoplasmic.4.Silencing effect of siRNA targeting Agrin mRNA on the expressions of Agrin in cervical carcinoma Caski cells and HeLa cells by Western blotting and qPCR methods.The results of Western blotting and qPCR displayed that: the expressions of Agrin mRNA and protein in Caski-siRNA-Agrin and HeLa-siRNA-Agrin cells were respectively lower than those in Caski-siRNA-NC and HeLa-siRNA-NC cells(P<0.01).Compared with Caski and HeLa cells,the expressions of Agrin mRNA and protein in Caski-siRNA-NC and HeLa-siRNA-NC cells were not significantly different(P>0.05).5.The results of CCK8 showed that compared with Caski-siRNA-NC and HeLa-siRNA-NC cells,Caski-siRNA-Agrin and HeLa-siRNA-Agrin cell growth rate slowed down(P<0.01).6.The results from transwell migration test and invasion test illustrated that the number of Caski-siRNA-Agrin and HeLa-siRNA-Agrin cells through basement membrane of chamber were significantly less than those of Caski-siRNA-NC and HeLa-siRNA-NC cells(P<0.01).7.The scratch wound healing assay detected that compared with Caski-siRNA-NCand HeLa-siRNA-NC cells,Caski-siRNA-Agrin and HeLa-siRNA-Agrin cell migration slowed significantly.Conclusions1.Agrin plays an important role in HPV16/18-induced cervical cancer,which may involve the role of ?-catenin.2.Agrin expression silencing can significantly inhibit proliferation,migration and invasion abilities of HR-HPVs associated cervical cancer cells.