A Study On The Role And Mechanism Of Human Papillomavirus 16E6/E7 Modulating Autophagy In Cervical Cancer Cells

Cervical cancer,caused by high-risk human pappilomaviurs(HPV)infection,still possesses a poor prognosis in advanced patients due to limited effectiveness of current therapeutics.Autophagy regulation is being expected as a novel therapeutics,but the role of HPV on autophagy in cervical cancer is little known.Here,we found that autophagy was inhibited in the initiation stage when HPV 16E6/E7 was knockdown in SiHa and CaSki cells,and activated as autophagosomes synthesis increasing and degradation accelerating when 16E6 or 16E7 forcibly over-expressed in HEK293 cells.Further,cell viability was suppressed when autophagy was inhibited,but enhanced when autophagy activated.RNA sequencing and bioinformatics analysis revealed that Atg9B and LAMP1 acted as candidate down-stream molecules in 16E6/E7 modulating autophagy among 586 differential expressed genes.Atg9B knockout by CRISPR Cas9 resulted in LC3-Ⅱ decline and LAMP1 knockdown by RNAi induced LC3-Ⅱaccumulation.Further,LC3-Ⅱ was accumulated when Atg9B were over-expressed and reduced when LAMP1 over-expressed in 16E6/E7 knockdown cells.Immuoprecipitation assay showed that 16E7 interacted with Atg9B and Dual-luciferase reporter system verified that 16E6 most likely bound to-1750nt to-2000nt in Atg9B and-1800nt to-2000nt in LAMP1 promoter.Our study suggests that HPV16 E6/E7 possesses the ability to maintain proper activated autophagy via accelerating autophagic flux at the initiate and degradation stage in cervical cancer cells,and Atg9B and LAMP1 may be used as potential therapeutic targets for blocking autophagy maintained by 16E6/E7.PartⅠ The Effect of 16E6/E7 on Autophagy in Cervical Cancer CellsObjective:To investigate the effect of HPV 16E6/E7 on autophagy and whether modulating autophagy affects cell proliferation or apoptosis by autophagy regulatory drugs or RNA interference in cervical cancer cell lines.Methods:Autophagy marker protein LC3-Ⅱ expression and number of LC3 dots was detected when knock-down 16E6/E7 in cervical cancer cells SiHa and CaSki,with or without exposure to autophagy inducer EBSS,autophagy blocker Baf A1 or CQ.Then mRFP-GFP-LC3 double-labeled adenovirus was introduced to monitoring autophagy flux by confocal laser scanning microscope.After that,HEK293 cells were transfected with 16E6 and 16E7 over-expression plasmids and the effect of 16E6 and 16E7 on autophagy was detected.Optimal concentrations of autophgay inducer rapamycin,inhibitor 3-MA and blocker CQ were selected for subsequently experiments.CCK-8 assay was used to detect cell proliferation while Annexin V combined with PI assay to cell early apoptosis after treated with autophagy regulatory drugs or RNA interference.Results:1.LC3-II expression was decreased and autophagosome formation was inhibited when knock-down 16E6/E7,with or without exposure to EBSS,Baf A1 or CQ.2.Autophagy flux was induced when over-expressing 16E6 or 16E7 in HEK293 cells.3.Cell proliferation was inhibited and early apoptosis was induced when autophagy was impaired after exposure to 3-MA,CQ or RNA interference.Conclusions:1.HPV 16E6/E7 may maintain autophagy properly activated by regulating autophagy at the initiate stage in cervical cancer cells.2.Autophagy blockage inhibits cervical cancer cell viability.Part Ⅱ Atg9B and LAMP1 screened as downstream signaling molecules in 16E6/E7 modulating autophagy by RNA sequencing and bioinformatics analysisObjective:To find out down-stream molecules involved in HPV 16E6/E7 modulating autophagy by RNA sequencing and bioinformatics analysis in cervical cancer cells.Methods:HPV 16E6/E7 was knockdown by RNA interference in SiHa cells.The tested samples(3 samples for each group)were prepared for RNA sequencing in a Hi-seq 2500 platform after interference efficacy was confirmed by RT-PCR.DEseq analysis was utilized to dig the differential expressed genes.KEGG pathway and GO analysis were utilized to understand the function and distribution of all the differential expressed genes,and autophagy pathway related genes were selected as candidate genes.mRNA and protein expression of candidate genes were confirmed in SiHa and CaSki cells by RT-PCR and WB,respectively.Then down-stream molecules were verified by their effect on autophagy and subcellular distribution.Results:1.Totally 586 differential expressed genes,including 193 up-expressed genes and 393 down-expressed genes,were screened by RNA sequencing.2.The top enriched three pathways were metabolism,cell cycle,and cancer signaling pathway and the top enriched six GO cellular components were organelle,intracellular organelle,organelle part,nucleus,intracellular organelle part,and intracellular non-membrane-bounded organelle.3.SPATA18(spermatogenesis associated 18),RGS19(regulator of G-protein signaling,RGS19),Atg9B(Autophagy related 9B),and LAMP 1(lysosomal associated membrane protein 1),which had been reported to participant directly or indirectly in autophagy,were selected as candidate genes.4.RT-PCR confirmed that SPATA18 and RGS 19 mRNA were up-regulated and Atg9B and LAMP1 mRNA were down-regulated in SiHa cells with Si-16E6E7.But,LAMP1 mRNA was down-regulated and other three mRNAs were up-regulated in Caski cells with Si-16E6E7.All four proteins were decreased in both SiHa and CaSki cells.5.Atg9B and LAMP1 were mainly detected in ME and decreased after 16E6E7 knock-down in both SiHa and CaSki cells.6.LC3-Ⅱ was accumulated when LAMP1 knockdown by RNAi and decreased when Atg9B knockout by CRISPR Cas9 using Atg9B-KO plasmid transfection.LC3-Ⅱwas accumulated when Atg9B were enforcedly over-expressed in HEK293.Conclusions:1.Atg9B and LAMP 1 were selected as down-stream signaling molecules in 16E6/E7 modulating autophagy verified by RT-PCR and WB.Part Ⅲ The mechanism of 16E6 and 16E7 in regulating Atg9B and LAMP1Objective:Verify the role of Atg9B and LAMP1 in HPV 16E6/E7 modulating autophagy and clarify the mechanism of 16E6 and 16E7 in regulating Atg9B and LAMP 1.Methods:Atg9B and/or LAMP1 were over expressed in 16E6/E7 low expression cervical cancer SiHa and CaSki cells to clarify the role of Atg9B and LAMP1 in 16E6E7 modulating autophagy.Then the distribution of Atg9B and LAMP1 were detected after over expressing 16E6 or 16E7 in HEK293 cells by confocal laser scanning microscope.Immunofluorescence assay was also used to confirm whether Atg9B colocalized with LAMP1.Immunoprecipitation assay was carried out between 16E6 or16E7 and Atg9B or LAMP1 to investigate the interactions between them.The sequences of promoter region for Atg9B and LAMP]genes were searched in the NCBI database and constructed into pGL3-Basic vector.Dual-luciferase reporter assay was utilized to detect whether 16E6 would bind to the promoter regions of Atg9B and LAMP I.The JASPAR database was used to predict the binding sites in Atg9B and LAMP1 promoter regions for 16E6.Truncated promoter regions were constructed into pGL3-Basic vector respectively to clarify the binding sites for 16E6.Results:1.LC3-Ⅱ was accumulated when Atg9B were over-expressed and reduced when LAMP1 over-expressed,compared to the control group,in 16E6/E7 low-expression cells.2.Distribution of LAMP1,but not Atg9B,was dispersed in HEK293 cell with over-expressed 16E6 or 16E7.3.Red fluorescence of Atg9B and green fluorescence of LAMP 1 were colocalized in HEK293 cells with over-expressed Atg9B and LAMP1.4.Immunoprecipitation analysis showed the interaction between 16E7 and Atg9B,but not LAMP 1.5.16E7,Atg9B,and LAMP1 plasmids were simultaneously transfected into HEK293 cells,laser confocal scanning microscopy showed that Atg9B was colocalized with LAMP1 in cells regardless of starvation induction or CQ exposure.6.Dual-luciferase reporter assay showed that 16E6 positively regulated the transcription of Atg9B or LAMP1.7.The binding sites for 16E6 in Atg9B or LAMP 1 gene promoter region was predicted in the JASPAR database,respectively,and-720nt to-710nt,-1085nt to-1075nt and-1857nt to-1860nt in Atg9B promoter and-700nt to-690nt,-1265nt to-1255nt and-1880nt to-1870nt in LAMP1 promoter were found to be most likely sites for 16E6 to bind to.8.The most likely binding sequences for 16E6 may be located in-1750nt to-2000nt in Atg9B promoter and-1800nt to-2000nt in LAMP1 promoter.Conclusions:1.16E7,but not 16E6,interacts with Atg9B and Atg9B interacts with LAMP1,consequently activating autophagy,and Atg9B and LAMP1 are involved in 16E7 modulating autophagy in cervical cancer cells.2.16E6 regulates Atg9B or LAMP1 expression positively at the transcriptional level via binding to-1880nt to-1870nt in LAMP1 promoter and-1857nt to-1860nt in Atg9B promoter,respectively.