Effect And Mechanism Of Hyperthermia On SIRT7 And Succinylation In Human Keratinocytes

BackgroundHuman papillomavirus is one of the most common skin infectious viruses.As a contact infectious virus,it spreads widely and causes a variety of skin diseases,such as genital warts,flat warts,plantar warts,etc.,of which genital warts alone affect about 1%of the global population.Commonly used treatment options,such as liquid nitrogen freezing,have problems such as pain and high recurrence rate,and improving treatment has become an important clinical task that dermatologists need to solve urgently.Hyperthermia treatment(HT)is a method of treating diseases at the temperature beyond normal body temperature,and the treatment temperature range is generally 40-44 °C for different diseases,and the effect lasts 30-60 minutes.In recent years,infrared thermotherapy device has been successfully applied to the treatment of HPV infectious skin diseases in clinical practice,and has achieved good curative effects,increasing the clinical cure rate of viral warts by about 10 percentage points.Only one targeted wart was removed by hyperthermia,other warts in the non-irradiated area would also be cured.Our previous studies confirmed a significant increase in CD4+ and CD8+ T lymphocyte infiltration in HPV-infected tissues after hyperthermia therapy.In addition,hyperthermia promoted apoptosis of keratinocytes infected with HPV,reduced the transcriptional activity of human papillomavirus,and enhanced endogenous interferon expression by 4-6times.However,the observed efficacy of thermotherapy is about 60%,and further exploration of its molecular mechanisms is needed to improve cure rates and effective rates.SIRT7 is a class III histone deacetylase,belonging to the sirtuin family,which mainly involves in aging,apoptosis,metabolism and other biological processes.In recent years,it has been reported that it has the activity of desuccinylase.The previous experiments of the research group found that the overall succinylation level of protein in HaCaT cells after hyperthermia increased,while the expression of SIRT7 decreased.Therefore,the role of SIRT7 in this process needs to be studied in depth.Early proteins E6 and E7 play an important role in the pathogenesis of HPV,and the influence of E6 protein on the stability of p53 protein is particularly important.E6 protein induces p53 degradation through ubiquitin-mediated proteolysis,resulting in a significant decrease in p53 activity.Studies have reported that stimulation of cervical cancer cells at 42°C for 1 hour would lead to the degradation of E6 protein,prevent the formation of E6-p53 complexes,and promote apoptosis of HPV+ cells.Whether the hyperthermia treatment in human keratinocytes also affects the expression of p53 molecules still needs further study.Knocking down SIRT7 in both cells and mice enhanced the transcriptional activity of p53 for apoptosis and further activated the p53-mediated pro-apoptotic signaling pathway.However,the interaction between p53 and SIRT7 is controversial,and more research is needed to determine whether p53 is indeed a substrate for SIRT7.Histone modifications are dynamic and reversible,involved in processes such as the regulation of chromatin structure,protein-histone binding,and DNA unfolding.It plays an important regulatory role in all chromatin-based biological processes,including gene expression,transcription,and DNA damage repair.SIRT7 was found to have desuccinylase activity.It could reduce the level of H3K122 succinylation,thereby promoting the binding of histone H3 to DNA and enhancing DNA damage repair.The functions of SIRT7 and histone modification in keratinocytes with the stimulus of hyperthermia are still rarely reported.In this study,starting from the role of hyperthermia on SIRT7 molecules in keratinocytes,we tried to figure out the relationship between SIRT7 and p53,and explore whether SIRT7 could be used as a desuccinylase to regulate the level of succinylation at histone H3K122 and H3K79 sites,thereby affecting the biological effects of cell cycle,apoptosis,and proliferation.It could help to provide more ideas and clues for further illustrating the molecular mechanism of hyperthermia,finding more suitable treatment targets and improving the cure rate and effectiveness.MethodsIn this study,human immortalized keratinocytes HaCaT cell lines and genital wart tissues were used as the research objects.Among them,the genital wart tissues were derived from excess wart tissues excised by patients in the clinical outpatient clinic of dermatology.The disease tissue was determined by pathological diagnosis,with the consent of the patient himself,and the research protocol was approved by the Ethics Committee.In this study,the method of resuming the 37℃ culture after 30 min of water bath at 44℃ simulated the in vitro hyperthermia treatment protocol.1.After a 30 min bath at 44 ℃,the HaCaT cells were returned to the 37 ℃ incubator for future culture.For foreskin tissue and genital wart tissue,the tissue was divided into two,placed in a Petri dish containing an appropriate amount of medium,treated for 30 minutes under water baths at 37 ℃ and 44 ℃,respectively,and then returned to the 37 ℃ incubator for further incubation.2.Immunohistochemistry(IHC)was used to detect the effect of hyperthermia on SIRT7 expression in genital warts tissues.3.Western blot method was used to detect the changes of SIRT7 and other molecules at the protein level.4.Immunofluorescence(IF)was used to detect the localization of SIRT7 molecules in HaCaT cells before and after warming.5.And co-immunoprecipitation(Co-IP)was used to detect protein molecules interacting with SIRT7.6.Immunoprecipitation(IP)was used to detect the level of succinylation of related molecules.7.RNAi technology was used to construct SIRT7 gene defective HaCaT cells.Lentiviral was utilized as the vector to construct HaCaT cells that overexpressed SIRT7 protein,then an appropriate amount of puromycin was added to obtain stable transfected cell lines.8.A specific SIRT7 inhibitor,SIRT7 inhibitor 97491(Selleck,USA),was uesd to inhibit the deacetylase activity of SIRT7.It was dissolved in DMSO,and added to the medium at a concentration of 5 μM for 24 h.9.CCK8 experiment detected the effect of SIRT7 on cell proliferation.10.The ATP detection kit was uesd to detect the effect of SIRT7 on intracellular ATP content.11.SILAC-tagged protein profiles detected the effect of hyperthermia on the level of succinylation in HaCaT cells.12.To analyze and visualize protein interaction networks,the STRING online database and Cytoscape software(v3.8.0)was used.13.All other images were drawn in R(v3.6.1),and the package used was ggplot2.All image stitching was performed by Photoshop CC 2019 software.14.The data obtained were paired with t-test or ANOVA using SPSS 24.0 software or Graph Pad Prim9.5 software,and relevant charts were made using Graph Pad Prim 9.5 software.A p-value < 0.05 was considered statistically significant.Results1.Effects of hyperthermia on the expression of SIRT7 and p53 molecules in human normal keratinocytes and HPV-infected skin tissues.Stimulation of human keratinocytes,and genital wart tissues at 44℃ could induce the downregulation of SIRT7 molecule expression.In HaCaT cell lines,hyperthermia stimulation can lead to downregulation of p53 and downstream molecules p21.In HaCaT cells,SIRT7 overexpression could induce upregulation of p53 and p21 expression levels.The results of immunofluorescence and nucleoplasmic separation showed that SIRT7 molecules were mainly localized in the nucleus and also existed in the cytoplasm of HaCaT cells,and no obvious position change was found after hyperthermia treatment.2.Effects of hyperthermia on histone H3 protein expression and succinylation modification levels.In HaCaT cells,44°C hyperthermia could downregulate the expression of histone H3 at 2 h.Then the low expression was maintained at 4 h,and gradually recovered at8-12 h.Under hyperthermia stimulation,the succinylation level of H3K122 in HaCaT cells was upregulated,and the expression level of H3K79 succ did not change significantly.The expression of SIRT7 showed a negative correlation with the changes of H3K79 succ and H3K122 succ,such as the expression levels of H3K79 succ and H3K122 succ were upregulated after knocking down SIRT7,and vice versa.KAT2 A,as a succinyltransferase of H3K79 succ,was downregulated after warmth,while knock-down or overexpression of SIRT7 did not affect the expression of KAT2 A.It was suggested that warming simutaneously downregulated the expression of succinyltransferase KAT2 A and dessuccinylase SIRT7,resulting in no significant change in the expression level of H3K79 succ under warming.The methylation of H3K79 was downregulated after hyperthermia,while the expression of the known H3K79 mel methyltransferase DOT1 L was also downregulated with the stimulation of hyperthermia.However,the knock-down or overexpression of SIRT7 did not have a significant effect on the expression of DOT1 L.3.Changes in protein succinylation levels in HaCaT cells stimulated by hyperthermia at 44 ℃.A total of 969 succinylation sites located on 387 proteins were identified,of which944 sites of 375 proteins were differentially expressed after hyperthermia treatment.With a change threshold of 1.5-fold,p-value <0.05 as standard,119 proteins(197 lysine sites)were hyper-succinylated and 1 protein(1 lysine site)was down-succinylated.After analyzing the amino acid motifs of the succinylation modification sites,two main succinylation modification motifs were found,namely Ix K and Vx K.GO results showed that succinylated proteins were mainly involved in biological processes such as cell metabolism,immune response,and stress response,among which proteins localized in the mitochondria accounted for 47.29%.4.Effect of Stimulating HaCaT cells at 44 ℃ on the expression level of succinylation regulatory molecules and the succinylation level of target molecules.Stimulation of HaCaT cells at 44℃ induced downregulation of succinyltransferase KAT2 A expression at 2 h.The remaining succinyltransferases CPT1 A and desuccinylases like SIRT3 and SIRT5 were not significantly changed.The immunoprecipitation(IP)results showed that the succinylation level of ANXA2 and LACTB molecules were upregulated in the 44℃ warm-stimulated group compared with the control group,and the co-immunoprecipitation(Co-IP)results showed that ANXA2 interacted with SIRT7.5.Effects of SIRT7 molecule on proliferation and ATP content in HaCaT cells.HaCaT cells which was knocked down of SIRT7 had a lower level of cell proliferation than the normal ones,and the level of cell proliferation was upregulated after SIRT7 overexpression.Knocking down SIRT7 increases the amount of ATP in human keratinocytes.Conclusion1.Hyperthermia stimulation at 44℃ could downregulate the expression levels of SIRT7,p53,and p21 molecules in HPV-negative human keratinocytes and HPV-positive genital wart tissues.SIRT7 overexpression could induce upregulation of p53 and p21 expression levels.2.In HaCaT cells,hyperthermia stimulation at 44℃ could induce the upregulation of succinylation level at H3K122 site,and no significant change in succinylation level of H3K79.Transient downregulation of H3 protein was observed after hyperthermia.SIRT7 might play the role as a dessuccinylase to regulate the succinylation level of H3K122 and H3K79,thereby regulating downstream gene transcription and expression.3.In HaCaT cells,succinylated proteins tended to participate in immune response,metabolic regulation,and stress response.In this process,KAT2 A and SIRT7 were downregulated compared with other known succinylation regulators such as CPT1 A and SIRT5,indicating that these two molecules played an important role in the hyperthermia-induced changes of succinylation level.4.In HaCaT cells,44 ℃ hyperthermia upregulated the succinylation levels of ANXA2 and LACTB proteins,and SIRT7 might function as a desuccinylase of ANXA2.5.SIRT7 molecule promoted cell proliferation of HaCaT cell.