Studies Of URI Promoting The Migration And Invasion Abilities Of Cervical Cancer Cells Via Regulation Of Vimentin Expression

Background Cervical cancer is one of the most common malignant tumor in female reproductive system.It is also a major disease threatening women’s health and life in developing countries.For cervical cancer patients,the survival rate and curative efficacy will be severly affected once the distal metastasis occurs.Therefore,the research has been continuously focused on the metastatic mechanism of cervical cancer.A RNA polymerase II subunit 5(RPB5)-mediated protein RMP,also known as an unconventional prefoldin RPB5 interactor URI,has been shown to possess characteristics of oncoprotein.Previous studies from our group have shown that URI promotes the migration of cervical cancer cells in vitro,but the potential mechanism has not been well elaborated yet.We speculate that it might be associated with a cytoskeleton protein,Vimentin.Here,based on our previous work,we further explore the effect and mechanism of URI on the migration and invasion capabilities by altering expression of URI and Vimentin in He La and C33 A cervical cancer cells.We will also investigate the the relationship between URI and Vimentin during cervical cancer cells migration and invasion.Objectives In this study,we have three specific aims(1)To characterize the role of URI in the migration and invasion of two cervical cancer cell lines He La and C33 A.(2)To determine whether the effect of URI on cervical cancer cell motility abilities is associated with the expression of Vimentin.(3)To explore the potential mechanism of URI regulation of Vimentin expression.Materials and Methods(1)Expression analysis of URI in two cervical cancer cell lines using q RT-PCR and western blot.Then using chemical transfection reagents to transfect URI specific si RNA,URI expression plasmid PCMV6-URI respectively in He La and C33 A cells,in order to establish cell lines with URI knocked-down or over-expressed.(2)Transwell assays were conducted to detect the migration and invasion abilities of cervical cancer cells when URI was forcely knocked-down or over-expressed.(3)q RT-PCR and western blot analyses were performed to detect the expression level of Vimentin in He La and C33 A cells in which URI was knocked-down or over-expressed respectively.(4)Cell motility abilities were examined in URI manipulated cervical cancer cells in which Vimentin expression was induced by TGF-β or knocked-down by Vimentin specific si RNA.(5)The upstream promoter sequence of Vimentin gene was cloned into the p GL3/Basic firefly luciferase vector.Luciferase report assay was conducted to determine the promoter activity of Vimentin regulated by URI via co-transfecting with URI expression plasmid into 293 T cells.(6)Chromatin immunoprecipitation(Ch IP)assay was performed to determine whether URI binds to the promoter area of Vimentin gene.Results(1)URI basal expression and transfection studies in two cervical cell lines.q RT-PCR and western blot test results showed that the expression level of URI in He La cells was higher than in C33 A cells.In He La cells transfected with URI specific si RNA,the m RNA and protein expression levels of URI was obviously lower than the control groups.While,in C33 A cells which transfected with URI expression plasmid PCMV6-URI,the m RNA and protein expression levels was obviously higher than the control groups.(2)URI manipulation affects cell migration and invasion abilities.Transwell migration assay results showed that in URI knock-down group,the number of He La cells migrated through the chamber was less than untreated groups.There are more amount of migrated C33 A cells in URI overexpression group than control groups.Transwell invasion assay results showed that in URI knock-down group,the number of He La cells invaded through the chamber decreased significantly compared with untreated groups.The number of invaded C33 A cells in URI overexpression group increased significantly compared with control groups.(3)URI manipulation affects Vimentin expression.q RT-PCR and western blot test results indicated that when URI was knocked down in He La cells,Vimentin m RNA and protein levels were both reduced,while in URI over-expressed C33 A cells,Vimentin m RNA and protein levels were both increased.(4)URI-mediated migration and invasion of cancer cells are associated with Vimentin.Transwell migration assay results showed that URI knockdown in He La cells supplied with 10ng/ml TGF-β increased the number of cells migrating through the chamber compared with control groups.URI overexpression in C33 A cells treated with Vimentin specific si RNA decreased the number of cells migrating through the chamber compared with untreated groups.Transwell invasion assay results showed that URI knockdown in He La cells supplied with 10ng/ml TGF-β significantly increased the number of cells invading through the chamber compared with control groups.URI overexpression in C33 A cells treated with Vimentin specific si RNA significantly decreased the number of cells invading through the chamber compared with untreated groups.(5)URI enhances the Vimentin promoter activity indirectly.Luciferase reporter assay in 293 T cells indicated that the luciferase activity in URI force-expressed group was increased about 1.57 fold than that of control group.However,Chromatin immunoprecipitation(Ch IP)assay results for the binding area at Vimentin promoter regions demonstrated that there is no statistical differences between URI protein and negative control by q PCR in He La and C33 A cancer cells.Conclusions(1)URI promotes migration and invasion of cervical cancer cells.(2)URI upregulates Vimentin m RNA and protein expression.(3)Vimentin plays a role in URI-enhanced motility capacity of cervical cancer cells(4)URI enhances the activity of Vimentin promoter,but there is no evidence that supports a direct interaction between URI and the Vimentin promoter.