Mechanisms Of HPV E7 Oncoprotein In Regulating G1 Checkpoints

High-risk human papillomavirus(HPV)infection is one of the important factors for the occurrence of cervical cancer.In female malignancies,the incidence of cervical cancer is ranked only second to breast cancer.Although vaccines for HPV have been applied in many countries and have been successfully marketed in China,the HPV vaccine is only targeted at some HPV subtypes and can only prevent HPV virus infection.The vaccines have no effect on patients who have been infected or have immunosuppressive diseases.There is no specific treatment for HPV currently because of the poor knowledge of pathogenesis of HPV.Therefore,it is still significant to continue to investigate the pathogenic mechanism of HPV for the purpose of effective prevention and treatment of cervical cancer and HPV-related cancers.Human papillomavirus belongs to the Papillomaviridae family.It is a small,non-enveloped virus which contains an 8 Kb long double-stranded circular DNA divided into three regions,Long Control Region(LCR),Early Region(E1,E2,E4,E5,E6,and E7),and Late Region(L1,L2).Among them,the products of E6 and E7 are key proteins of HPV-induced tumors.E6 and E7 could bind and degrad p53 and pRb which are two classic tumor suppressor proteins.In addition,E6 and E7 also interfere with multiple pathways of cells by interacting with various host proteins which are involved in genomic stability,cell adhesion,cell proliferation,apoptosis,cell cycle,DNA repair,metabolism,translation,and transcriptional signals.Cell cycle progression is regulated by cyclins and cyclin dependent kinases(Cdks),including G1,G2/M,spindles and post-mitotic checkpoints.The G1 checkpoint is a critical checkpoint for determining whether a cell enters S phase or proliferation phase.Under normal circumstances,cells will be arrested in the G1 phase when DNA damage happened.Cells will block damaged cells to replicate DNA,and allow the cell repair system to repair damaged DNA.HPV-16 E7 eliminates the G1 cell cycle checkpoint by degrading pRb,resulting in dissociation of the E2F transcription factor family members from pRb.Free E2F binds to the promoter regions of many genes whose products are involved in cell cycle regulation or DNA replication.Then cell proliferation is disordered and and in turn genomic instability and tumorigenesis is induced.Although a variety of E7 interacting proteins have been identified,there is the need to identify the unknown proteins in E7-mediated cell cycle regulation and transformation.There are many studies on the mechanism of E7 regulation in G1 checkpoints.However,the exact mechanisms are still not fully understood.The provious research of this lab screened out several genes involved in the cell cycle regulated by E7,such as WDHD1,Cdc6,CIP2A,etc.In addition,regulator of chromatin condensation 1(RCC1)and GCN5(General control non-depressible 5)were also screened out,but whether the two genes were involved in the G1 checkpoint regulated by E7 needs further validation.Based on this,this study investigated the functional mechanisms of RCC1 and GCN5,respectively.Part Ⅰ Roles and Mechanisms of RCC1 in HPV E7-mediated G1 Checkpoints RCC1 is a guanine nucleic acid exchange factor of Ran-GTPase.Studies have shown that RCC1 is a key cell cycle regulator and its loss of function leads to G1 arrest.But the molecular mechanism of this phenomenon is unknown In addition,there is little research on the relationship between RCC1 and cancer.Expecially,there is no research on the functional mechanism of RCC1 in cervical cancer.Analysis of the mass spectrometry results of HPV E6 and control cells revealed that RCC1 expression was elevated in E6-expressing cells.E6 and E7 are important oncogenic proteins of HPV.There is a certain similarity in function between E6 and E7.So E7 may also affect the expression of RCC1.This study was investigated the roles and functional mechanisms of RCC1 in E7-expressing cellls.Methods1.The expression level of RCC1 mRNA was analyzed from GEO database of NCBI.The expression levels of RCC1 protein in normal cervical tissues and cervical cancer tissues were detected by immunohistochemical(IHC)technique.2.Real-time quantitative PCR(RT-PCR)and immunoblot assays were used to detect the expression of c-Jun and RCC1 in RPE1 and NIKS cells,and RPE1 cells treated with Bleomycin.The mRNA and protein expression of c-Jun and RCC1 were detected with transfected siRNA specific to c-Jun.The binding of c-Jun to RCC1 promoter was determined by Chromatin immunoprecipitation(ChIP)assay.3.PI treatment detected the proportion of G1 phase,and BrdU treatment detected S phase ratio after RCC1 knockdown and RCC1 overexpression.4.RT-PCR and immunoblot assays were used to detect the expression of cell cycle-associated proteins after RCC1 knockdown.Cells were treated with cycloheximide(CHX)to detect the half-life of E2F1.Flow cytometry assay to examine the effect of RCC1 knocknown and overexpression E2F1 on the cell cycle.5.PI assay was used to examine the effect of Cdkl knockdown and overexpression on cell cycle.6.Immunoblot,PI and BrDU experiments were used to detect the effect of RCC1 knocknown on cell cycle related proteins and cell cycle.7.Immunoblot was used to analysis the effect of RCC1 knockdown on E7 protein and its half-life.Results1.High RCC1 expression correlates with cervical cancer.GEO database analysis showed that RCC1 mRNA was up-regulated in cervical cancer tissues and cell lines compared with normal cervical epithelium.Immunohistochemical staining analysis of RCC1 protein in normal cervical and cervical cancer tissues showed that although there was no significant difference in the score between normal cervical and cervical cancer tissues,the expression of nuclear RCC1 in cervical cancer tissues was higher than that in normal cervix tissues.2.RCC1 expression is upregulated by c-Jun in HPV E7-expressing cells.RT-PCR and immunoblot assays showed that RCC1 mRNA and protein levels were higher in E7-expressing NIKS and RPE1 cells as well as Bleomycin-treated RPE1-E7 cells.Significantly,the results of cell localization showed that the RCC1 protein was mainly localized in the nucleus,and the expression level in the E7-expressing cells was higher than that of the control RPE1-Vector cells.These results indicate that E7 up-regulates the expression of RCC1.RT-PCR and immunoblot results showed that c-Jun mRNA and protein levels were up-regulated in both E7-expressing NIKS and RPE1 cells and Bleomycin-treated RPE1-E7 cells.ChIP assay showed that c-Jun bound to chromatin at three sites within the RCC1 promoter and significantly more c-Jun bound to chromatin in E7-expressing cells than in vector control cells.RCC1 mRNA and protein levels were significantly downregulated following knockdown of c-Jun.3.RCC1 knockdown inhibits G1/S phase progression and DNA replication.Cell cycle assays showed that upon treatment with Bleomycin,fewer E7-expressing cells than vector control cells arrested in G1,indicating that HPV E7 abrogated the G1 checkpoint.Notably,knockdown of RCC1 led to a significant increase in the G1 peak in both E7-expressing and control RPE1 cells.Furthermore,RCC1 knockdown increased more number of E7-expressing cells arrested in G1 than control RPE1 cells.BrdU proliferation assay showed that knockdown of RCC1 led to a significant reduction in BrdU incorporation in both RPE1-E7 cells and RPE1-Vector cells,as well as Bleomycin-treated RPE1-E7 cells,indicating that knockdown of RCC1 affected DNA replication.Overexpression of RCC1 further validated the role of RCC1 in the G1/S phase,and the results showed that overexpression of RCC1 resulted in a decrease in G1 phase and an increase in S phase in PBS and Bleomycin treated cells.These results suggest an important role for RCC1 in G1 cell cycle control and S phase entry.4.RCC1 knockdown promotes E2F1 degradation and E2F1 overexpression rescues the RCC1 knockdown-mediated inhibition of the G1/S transition.Immunoblot detected the expression of genes involved in the G1 checkpoint,E2F1,Cdk1,Cdk2,and Cdk4.E2F1,Cdk1,and Cdk2 protein levels were considerably higher in E7-expressing cells and were markedly decreased upon RCC1 knockdown.These results suggest a role for E2F1,Cdk1,and Cdk2 in RCC1-mediated G1/S transition in E7-expressing cells.E2F1 mRNA levels stayed relatively constant after RCC1 knockdown,which suggests that RCC1 does not affect E2F1 at the transcriptional level.Cell localization analysis showed that RCC1 knockdown resulted in a decrease in nuclear E2F1 but not in cytoplasm.We speculate that knockdown of RCC 1 may lead to the degradation of E2F1.CHX-treated cells showed that the steady-state E2F1 levels dropped more quickly with RCC1 knockdown,indicating that E2F1 degradation was increased.Knockdown of endogenous RCC1 and transfection of exogenous expression plasmids showed that E2F1 protein levels recovered when cells were transfected with Flag-RCC1,indicating that RCC1 protected the stability of E2F1.Knockdown of RCC1 caused an accumulation of cells in G1 while overexpression of E2F1 partly bypassed G1 arrest in E7-expressing cells.5.Cdkl is involved in RCC 1-mediated G1 checkpoint.Cell cycle assay results showed that transfection of siRNAs targeting Cdkl led to more increase number of G1 phase in the of Bleomycin-treated E7-expressing cells.With overexpression ofCdkl,cells with RCC1 knockdown partially bypassed G1 arrest.Therefore,Cdkl indeed rescues the effect of RCC1 knockdown on G1 arrest.6.RCC1 knockdown induces G1 arrest in cervical cancer cells.The steady-state levels of RCClwere higher in HeLa and SiHa cells compared with RPE1-Vector cells.SiHa and HeLa cervical cancer cells were treated with siRCC1s and observed downregulation of E2F1,Cdk1,and Cdk2,led to an increase in the G1 peak and a significant reduction in BrdU incorporation.These results demonstrate an important role of RCC1 in G1/S cell cycle control in cervical cancer cells.7.RCC1 knockdown reduces E7 protein expression.Immunoblot detected the changes of E7 protein levels after RCC1 knockdown,and the results showed that E7 protein was decreased after RCC1 knockdown.CHX-treated cells showed that the steady-state levels ofE7 dropped more quickly with RCC1 knockdown.The same phenomenon was occured in SiHa cells.These suggest that RCC1 regulates the degradation of E7 protein.Conclusions1.RCC1 was regulated at the transcriptional level by c-Jun in HPV E7-expressing cells.2.RCC1 regulated G1/S cell cycle progression in an E2F1-dependent manner and Cdkl was involved in RCC1-mediated G1 checkpoint.3.RCC1 promoted G1/S cell cycle progression in cervical cancer cells.Significance and innovationThis study discovered that RCC1 expression was upregulated by c-Jun in HPV E7-expressing cells.The functional mechanism of RCC 1 regulating G1/S cell cycle progression through E2F1 was explored.RCC1 may be involved in HPV E7-mediated genomic instability,leading to the development of cancer.Part Ⅱ Roles and Mechanisms of GCN5 in HPV E7-mediated G1 CheckpointsGCN5 is the first identified transcription-related lysine acetyltransferase(KAT),which is an important catalytic component of transcriptionally regulated SAGA(Spt-Ada-GCN5-acetyltransferase)and ATAC(containing the ADA2A)complex.Although GCN5 is involved in the development of cancer,its role in cervical cancer is still unclear.Previous studies have shown that E2F1 can transcriptionally activate GCN5 expression and GCN5 overexpression can promote cell growth and G1/S phase transition.In addition,transcription factor E2F1 is highly expressed in E7-expressing cells.It is speculated that GCN5 may be highly expressed in E7-expressing cells and plays a role in E7-mediated cell cycle regulation.Therefore,this study also explored the relationship between GCN5 and HPV E7 and its function mechanism in HPV E7-expressing cells.Methods1.RT-PCR and immunoblot assays were used to detect the expression of GCN5 in RPE1 and NIKS cells.2.PI and BrdU assays were used to detect the effect of GCN5 knockdown on cell cycle.3.RT-PCR and immunoblot assays were used to detect the expression of cell cycle-associated proteins after GCN5 knockdown.Immunoblot and flow cytometry assays were used to detect the effects of E2F1 knockdown on cycle-associated proteins and cell cycle.PI assay was used to examine E2F1 overexpression on the cell cycle.4.Immunoblot assay was used to detect the acetylation level of histone H3K9 in deacetylase inhibitor Trichostatin A(TSA)treatment and GCN5 knockdown.ChIP assay was used to analysis of GCN5 binding to the E2F1 promoter and the binding level of acetylated H3K9 at the E2F1 promoter after GCN5 knockdown.5.Immunoblot assay was used to analysis of the c-Myc expression with TSA treatment and GCN5 knockdown.Immunoprecipitation(IP)was used to detect the acetylation of c-Myc protein with GCN5 overexpression and GCN5 knockdown.Immunoblot and PI assays were used to detect the effect of c-Myc knockdown on E2F1 protein expression and cell cycle.ChIP assay was used to detect the binding level of c-Myc to E2F1 promoter after GCN5 knockdown.The effect of mutated c-Myc(K323R)on its expression and its binding property to E2F1 promoter were explored.Results1.GCN5 expression is upregulated in HPV-16 E7 expressing cells.RT-PCR and immunoblot results showed that RCC1:mRNA and protein levels were significantly up-regulated in E7-expressing NIKS and RPE1 cells.Transient transfection of plasmids encoding HPV-16 E7 showed an increase in GCN5 protein levels.2.GCN5 knockdown causes G1 arrest and inhibites DNA synthesis in HPV-16 E7-expressing cells.Flow cytometry results showed that knockdown of GCN5 led to an increase in the G1 peak in E7-expressing cells.BrdU assay showed that knockdown of GCN5 by siRNAs led to a statistically significant reduction of BrdU incorporation in RPE1 cells as well as Bleomycin-treated RPE1-E7 cells.These results demonstrate an important role of GCN5 in the G1 cell cycle control and S phase entry in E7-expressing cells.3.E2F1-dependent regulation of Cdkl by GCN5 in E7-expressing cells.Immunoblot results showed that E2F1,Cdk1,Cyclin A and p53 were significantly up-regulated in E7-expressing cells and down-regulated after transfecting the cells with siRNAs against GCN5.RT-PCR results showed that the mRNA level of E2F1 was down-regulated after GCN5 knockdown.This indicates that GCN5 affects the expression of E2F1 at the transcriptional level.The steady-state levels of Cdkl and cyclin A proteins were decreased after transfected cells with siE2F1s.Flow cytometry results showed that knockdown of E2F1 led to an increase in the G1 peak and decrease in S phase in E7-expressing cells.It is indicated that GCN5 knockdown-induced G1 arrest and down-regulation of Cdk1 are dependent on E2F1.Knockdown GCN5 caused an accumulation of cells in G1 peak while overexpression of E2F1 partly abrogated G1 arrest caused by GCN5 knockdown in E7-expressing cells.These results indicate that GCN5 is involved in E7-mediated G1 regulation by affecting E2F1.4.GCN5 binds to the E2F1 promoter and increases histone H3 acetylation in E7-expressing cells.Immunoblot results showed that the acetylated H3K9 was higher in E7-expressing cells,especially after treatment with TSA.The acetylation level of H3K9 was decreased when cells were treated with siGCN5,indicating that GCN5 was responsible for the acetylation level of H3K9 in E7-expressing cells.ChIP assay results indicated that GCN5 could bind to the E2F1 promoter.The acetylation level of H3K9 binding to the promoter of E2F1 was significantly decreased with GCN5 knockdown.These results indicate that GCN5 binds to and increases the histone H3 acetylation of the E2F1 promoter in E7-expressing cells5.Acetylation of c-Myc by GCN5 contributes to its stability and ability to regulate E2F1 expression in E7-expressing cells.The effect of GCN5 on c-Myc expression was detected by immunoblot.The result showed that the protein level of c-Myc was decreased after GCN5 knockdown and increased with TSA treatment.IP experiments results showed that the acetylation level of c-Myc was higher in E7-expressing cells than that in the vector control cells and the acetylation level of c-Myc was increased with GCN5 knockdown.These results indicate that GCN5 increases c-Myc acetylation in E7-expressing cells.Immunoblot and flow cytometry assays was used to examine the potential role of c-Myc in regulating E2F1 and G1 checkpoints in E7-expressing cells.These results showed that knockdown of c-Myc,led to a decreased in E2F1 expression and an increase in the G1 peak,indicating a role of c-Myc in G1 checkpoint regulation in E7-expressing cells.ChIP assay results showed that c-Myc bond to the promoter of E2F1.The level of c-Myc binding to E2F1 promoters was significantly decreased when cells were treated with GCN5 siRNA,suggesting that acetylation of c-Myc by GCN5 affect the ability of c-Myc binding to E2F1 promoters.A c-Myc mutant having a mutation at K323(K323R,mimicking the deacetylation state;K323Q,mimicking the acetylation state)was constructed,and the effect of the acetylation of K323 residue of c-Myc on its stability was examined.Immunoblot results showed that the steady-state levels of K323R mutant of c-Myc were lower than that of the wild-type.ChIP assay results showed that the relative level of c-Myc binding to E2F1 promoters was decreased for K323R,indicating that c-Myc acetylation is important for its stability.Conclusions1.GCN5 expression was up-regulated in HPV-16 E7-expressing cells and regulated G1/S cell cycle progression in an E2F1-dependent manner.2.GCN5 bond to and increased histone H3 acetylation of the E2F1 promoter in E7-expressing cells.3.GCN5 could acetylate c-Myc,stabilize it,affect its level of binding to the E2F1 promoter,regulate E2F1 expression,and controll cell cycle progression.Significance and innovationThese results revealed a novel mechanism of E7 function whereby elevated GCN5 acetylate histones and c-Myc to regulate E2F1 expression and cell cycle progression.