The Experimental Study Of The Anti-HPV Activities Of Human Keratinocytes In Response To Hyperthermia And Heat Shock Proteins

Objective: Human papillomavirus(HPV)infection is a common clinical condition seen in the department of dermatology.As a contagious disease transmitted by direct contact,HPV infection is problematic to both health and life quality.Likewise,as a sexually transmitted disease,HPV infection affects people both socially and mentally.Therefore,cutting off HPV transmission and curing HPV infections have become one of the top tasks for dermatologists around the globe.Conventional therapies,such as cryotherapy,laszer ablation and photodynamic therapy,have all been critisized for painful process,high recurrence rate or high medical expenses.Recently,a brand-new treatment method known as local hyperthermia has been implemented clinically to treat cutaneous HPV infecions.With advantages such as painless process,low recurrence rate,rare contra-indications,multi-lesions targeting as well as high therapeutic efficacy,local hyperthermia has been proved to be an excellent alternative treatment against HPV infectious diseases.In order to obtain a more precise understanding of the overall mechanism underlying local hyperthermia therapy,this project was carried out from the perspectives of hyperthermal effects and heat shock proteins to provide molecular targets for intervening,improve efficacy,shorten course of treatment,as well as reduce recurrence rate,and finally broaden our outlook in the realm of treating HPV infectious diseases.Methods: Ha Ca T cell lines,foreskin tissues and condyloma acuminatum(CA)tissues were studied in this research.Foreskin tissues were obtained from patients underwent circumcisions,and CA tissues were obtained from patients underwent pathological examinations.All methods concerning human subjects were proved by the ethics committee of the First Hospital of China Medical University.In the research,an ex-vitro/vivo 44 ± 0.1 °C water bath was used to mimic local hyperthermia therapy.Protemic changes of cell lines and tissues were monitored with help of i TRAQ or SILAC labelled mass spectrometry.Expression levels of m RNA and proteins were detected with real-time q PCR and western blotting,respectively.Protein expression and positioning in tissues were investigated via immuno-histo-chemistry(IHC)staining,whereas those of cell lines were studied via immuno-flurocence(IF)staining.HPV-DNA in the tissues was seperated and detected with help of agarose-gel electrophoresis.For functional studies,apoptosis and cell cycle analysis were accomplished through flow-cytometry,whereas proliferation index was analyzed via MTS assays.For data processing,bioinformatical analysis of high-throughput data were performed with use of R 3.4.1 platform,whereas protein-protein interaction(PPI)network was created by searching STRING online database and mined with help of Cytoscape 3.5.1 software.Statistically,student’s t test or one-way anova with post-hoc comparisons(bonferroni-corrected)were usd to compare data concerning cell lines,whereas pairwise t test was used when comparing data concerning clinical tissues.Finally,data visuallizations were illustrated by using ggplot2 package in R 3.4.1 platform and Photoshop 7.0 software combined.Results: 1)630 differentially expressed proteins(DEPs)were detected in Ha Ca T cells stimulated by 44 °C hyperthermia(1.3 foldchange cutoff,p-value < 0.05),including 251up-regulated and 379 down-regulated.Up-regulated DEPs were concentrated into 4clusters with their functions involved in cytoplasmic motion,apoptosis,stress responses,energy metabolism,virus-host interaction and viral infection regulations.The down-regulated DEPs were clustered into 5 clusters,functioning in multple catabolism and metabolism processes,viral genome transcription and translation,ribosome biogenesis,ribonucleoprotein assembly,and cell cycle regulations,etc.PPI analysis came out with a network containing 591 nodes with 6167 edges,with 4 major interactive modules mined both from up-and down-regulated networks.10 hub-proteins were detected within the entire PPI network,which provided us more future directions.2)In CA tissues,44 °C hyperthermia generated 102 DEPs(1.2 foldchange cutoff,p-value <0.05)in total,with 37 up-regulated and 65 down-regulated.For validation of proteomic data,10 target proteins were correspondingly regulated on m RNA level detected by real-time q PCR,whereas 4 out of 5 target proteins were correctly regulated on protein level detected by western blotting.Clustering and enrichment analysis revealed that the up-regulated DEPs were more concentrated on antigen presenting and anti-viral reactions,whereas the down-regulated DEPs were more enriched into multiple catabolism,metabolism and keratinocyte differentiation.PPI scaning of the DEPs found a network containing 49 nodes with 68 edges,with two major interactive modules mined form it.13hub-proteins containing CETN2 were discovered from the entire network,which could be our future research targets.3)Heat shock protein DNAJA4 was induced by 44 °C hyperthermia in a temporally expressing pattern,with peak value reached 8 hours after hypethermia treatment and gradually recovered in 24 hours.DNAJA4 was majorly induced in the epidermis of skin tissues as revealed by IHC staining,whereas it was majorly induced in cytoplasms in Ha Ca T cells as revealed by IF staining,both with a baseline expression level.DNAJA4 deficiency led to the hyper-phosphorylation activation of P65,and promoted downstream gene transcriptions,an activity that was blocked by NF-kappa B signal pathway inhibitors PDTC and Helenalin.DNAJA4 deficiency lowered the resistance of Ha Ca T cells against 44 °C hyperthermia by induction of cell senescence,decrease of S phase proportion and inhibition of cellular proliferation,effects which were all or partially reversed by PDTC pre-treatment.4)44 °C hyperthermia induced heat shock protein HSPA6 expression in keratinocytes from all sources(cell line,foreskin and CA),which also followed a temporal profile with peak value reached at around 4 hours after hyperthermia treatment.HSPA6 had a very low basic expression level at normal conditions,and induced majorly in cytoplasms upon hyperthermia treatment.HSPA6 deficiency severely damaged the resistance capacity of Ha Ca T cells towards 44 °C hyperthermia by causing apoptosis and cell death in large scales,as well as cell cycle blockage and proliferation inhibition.Results from PCR array also showed that 44 °C hyperthermia boosted anti-viral responses in Ha Ca T cells,which could be bi-directionally regulated by HSPA6.5)44 °C hyperthermia caused a total of240 proteins(311 lysine sites)with alterations in lysine acetylation level(differentially acetylated proteins,DAPs)in Ha Ca T cells(1.5 foldchange cutoff,p-value < 0.05),150proteins(194 lysine sites)among them were up-regulated,whereas 90 proteins(117lysine sites)were down-regulated.There were 11 DAPs containing both up and down-regulated acetylated lysine sites.Motif analysis found 15 most enriched lysine acetylation motifs,containing GKK and others,with high scoring.Enrichment analysis revealed that up-regulated DAPs were more concentrated in viral-genome regulation and telomeres assembly,whereas down-regulated DAPs majorly participated in P53 signal pathway and histone-chromosomal interactions.Both up and down-regulated DAPs were enriched in processes involving RNA processing,transferring and metabolism.6)Under44 °C conditions,DNAJA4 deficiency caused minor influence to DAPs related to DNA regulations,r RNA processing and metabolism,as well as P53 signaling,whereas it caused major influence to DAPs concering apoptosis,viral-genome regulation and m RNA splicing.On the other hand,HSPA6 caused an overall major influence to DAPs regarding RNA splicing/processing/metabolism,DNA replication,viral transcription regulation and P53 signaling.Conclusions: 1)44 °C hyperthermia compromises cellular energy and material metabolisms,inhibit keratinocyte differentiation,whereas improve antigen presenting capacity and anti-viral activities in human keratinocytes,which may be the molecular mechanism of local hyperthermia against HPV infections.2)DNAJA4 deficiency promoted NF-kappa B signal pathway related growth arrest in Ha Ca T cells in response to44 °C hyperthermia.3)Heat shock protein HSPA6 is an important and potent protector,as well as a bi-directional anti-virus regulator for Ha Ca T cells in response to 44 °C hyperthermia.4)In Ha Ca T cells,44 °C hyperthermia tends to cause more acetylation alterations in proteins regarding anti-viral activities,whereas less acetylation alterations in proteins related to chromosome controls,and bi-directionally regulate the acetylation levels of molecules involving RNA processing and metabolism,which may be the key factors in the control of virus life cycle during infections.Besides,the acetylation of proteins in Ha Ca T cells in response to hyperthermia is diversely influenced by DNAJA4 and HSPA6,where HSPA6 shows more potential as compared to DNAJA4.