Effects Of Hyperthermia On DNAJA4,CLU And MAPK Signaling Pathway In Keratinocytes

IntroductionHyperthermia treatment(HT)is a method of treating diseases with temperature beyond normal body temperature.According to different diseases,the range of treatment temperature is generally 40-44?,lasting 30-60 minutes during application.In the past,this method was mostly used for adjuvant treatment of tumor patients.In recent years,our team has successfully applied the infrared thermotherapy instrument(patent-No: ZL 2007 2185403.3)with independent intellectual property rights to the treatment of human papillomavirus infectious skin diseases,such as verruca vulgaris,verruca plana,verruca plantaris,genital warts(also known as condyloma acuminatum),verrucous epidermal dysplasia and so on.The clinical cure rate of verruca was increased by about 10%,and the recurrence rate was very low.However,for some patients,if no therapeutic response is observed,there is often no curative effect at all.At present,the clinical curative effect observed is about 50%-60%.Therefore,it is still necessary to further explore the molecular mechanism of hyperthermia treatment,find effective in vitro intervention molecules,enhance the effect of hyperthermia treatment,shorten the course of treatment,and further improve the cure rate.Heat shock protein 40 family(hsp40s)is a group of acute phase reaction proteins which molecular weight is about 40 kDa.Its basic biological function is to assist the ATP hydrolase activity of HSP70 and play the role of molecular chaperone.The previous data of our research group showed that DNAJA4,a member of hsp40 s family,was highly expressed in normal human prepuce,condyloma acuminatum and human epidermal keratinocyte(HaCaT)during hyperthermia treatment at 44?.And we found that the activity of NF-? B pathway in DNAJA4 deficient HaCaT cells was increased significantly under hyperthermia treatment compared with that of wild-type HaCaT cells.And NF-? B activation could further up regulate the expression level of a series of antiviral cytokines,such as TNF-? and IL-1 ?,suggesting that DNAJA4 is closely related to hyperthermia induced anti HPV infection.In addition,proteomic analysis showed that hyperthermia treatment could also change the expression level of a series of other molecules in HaCaT cells,such as clusterin(CLU),DUSP1(MKP),HERPUD1,TRIB1,MYC,BCl3,etc.Real-time fluorescence quantitative PCR technology was used to verify the changes of mRNA expression of the above molecules under the hyperthermia treatment.Most of the PCR results were consistent with proteomics.Among them,the expression level of CLU seems to be affected by the knockout of DNAJA4.So,it becomes the focus of the next research.Cluster protein(CLU),also known as testosterone inhibitory prostate messenger-2(trpm-2)or sulfurized glycoprotein,is a heterodimer sulfated multifunctional glycoprotein with a molecular weight of 75-80 kDa,which is widely expressed in body fluids and tissues of various organisms.It is found that CLU can be divided into two types: secretory and intracellular.CLU has a variety of biological functions,including promoting cell aggregation,regulating reproduction,tissue repair,complement inhibition / regulation of immunity,lipid transport,sperm maturation and programmed cell death.The up regulation of CLU mRNA and protein expression has been found in many pathophysiological changes,such as inflammation,tumor,tissue repair and injury.Previous studies have found that hyperthermia can induce the expression of CLU in rabbit testicular cells,human prostate cancer,bladder cancer and epithelial cancer cells,all of which play an antiapoptosis role.However,the changes of CLU expression in skin and condyloma acuminatum tissues and the results of functional study have not been reported.Mitogen activated protein kinase(MAPK)signaling pathway is a kind of important Ser / Thr protein kinase family,which widely exists in eukaryotes.At present,based on sequence homology and functional differences,at least four MAPK signal transduction pathways have been identified:(1)extracellular signal regulated kinase(ERK1 / 2)pathway;(2)c-Jun N-terminal kinase(JNK),also known as activated protein kinase pathway;(3)p38 mitogen activated protein kinase(p38 mitogen activated Protein kinase(p38 MAPK)pathway;(4)extracellular regulated kinase 5(ERK5)pathway.In recent years,more and more attention has been paid to the role of MAPK signaling pathway in hyperthermia.It was found that hyperthermia can activate MAPK pathway in keratinocytes.Moreover,MAPK pathway also plays an important role in the process of virus replication,suggesting that hyperthermia treatment may play a role through this pathway.In addition,a large number of research results have confirmed that hyperthermia can also change the activity of MAPK signaling pathway in a variety of other cells,and further affect the phenotypic changes of cell proliferation,apoptosis,inflammatory factor release and so on.However,results of the effects of hyperthermia on MAPK pathway and the subsequent changes of cell fate are not completely consistent,which may be related to cell types,temperature,duration,detection time point and other factors.Previous studies have found that CLU can activate ERK pathway in pancreatic cancer cells and astrocytes,suggesting that hyperthermia may further affect ERK pathway through CLU to regulate cell proliferation,apoptosis and other phenotypic changes.To understand the role of DNAJA4 / CLU / MAPK signaling pathway in hyperthermia and the interaction between the molecules / pathways will be helpful to explain the molecular mechanism of the effect of hyperthermia therapy in clinical practice.And this may provide powerful clues for the follow-up basic research and the development of in vitro intervention drugs.Material and Methods 1.SubjectsThe cell line was selected as a human keratinocyte cell line(HaCaT)and purchased from Jiangsu Kaiji Biotechnology Co.,Ltd.Human foreskin and condyloma acuminata originated from patients diagnosed with condyloma acuminatum and excessively long foreskin from November 2017 to January 2020 in the Department of Dermatology and Urology Clinic of the First Hospital of China Medical University.With his or her informed consent,remaining or discarded human tissues after circumcision or pathological biopsy were used for ex vivo hyperthermia studies.The study was approved by the Ethics Committee of the First Affiliated Hospital of China Medical University and was in line with the Helsinki Declaration.2.Hyperthermia treatment programEx vivo hyperthermia: Cut fresh foreskin or condyloma acuminata into two parts,infiltrate the dermis side into the culture medium,and expose the epidermal side to the air.Place the bottom of the petri dish in a thermostatic water bath,adjust the temperature of the water bath.After treatment at 37? or 44? for 30 minutes,the dishes were returned to the incubator at 37?.Cell hyperthermia: After plated for 24 hours,the cells were treated with 37? or 44? in a water bath for 30 minutes.The method is the same as ex vivo hyperthermia.37? is the control group and 44? is the experimental group.Then,cells were collected at different time points after hyperthermia according to the needs of the experiment.3.Immunohistochemical detection of related proteins4?m paraffin sections were used to detect the expression of CLU and p-ERK by immunohistochemical staining(SP method).Paraffin tissue sections were dewaxed,antigen repaired,primary antibody,secondary antibody,developed,counter-stained,observed and photographed under a 20 x objective.4.Real-time quantitative PCR detection of CLU phase changes after hyperthermiaThe expression of CLU mRNA was detected by real-time quantitative PCR.Total RNA was extracted according to the instructions of Qiagen extraction reagents;cDNA was synthesized by reverse transcription using PrimeScript RT regagent Kit(Takara)kit;SYBR Premis Ex Taq(Takara)kit was used for real-time fluorescence quantitative PCR amplification,and double standard curve method was used for relative quantification Result analysis.5.Western blot detection of CLU and MAPK signal pathway expressionWestern blot was used to detect the changes of CLU and MAPK signal pathway expression levels before and after hyperthermia treatment.The protein was extracted from the cells or tissues according to the protein extraction method;BCA was used to detect the protein concentration;After incubation with respective primary and second antibodies,the bands were visualized with ECL Western Blotting Substrate.6.Establishment of CLU gene knockout cells using RNAi technology and lentivirus as vectorsThe RNAi technology was used to knock out the CLU gene in HaCaT cells using lentivirus as a vector.The knockout cells were verified by fluorescence microscope observation,PCR,and Western blot.7.Inhibition of ERK pathway activityThe phosphorylated ERK activity inhibitor PD98059(Sigma Aldrich,USA)was dissolved in DMSO and added to the culture medium to a final concentration of 5 or 10 ?M.The inhibitor negative group was also treated with an equal amount of DMSO.PD98059 or DMSO treatment was performed 2 hours before hyperthermia.8.Effect of CLU and ERK pathways on cell cycle and apoptosis by flow cytometryHaCaT cells after lentiviral transfection or inhibitor treatment were seeded in six-well plates,and then subjected to hyperthermia treatment at 44? for 30 minutes.After 24 hours,the APC Annexin V apoptosis kit and PI reagent were used to detect the changes in apoptosis and the distribution of the cycles separately.9.statistical analysisSPSS 16.0 and GraphPad Prism 5 software were used for statistical analysis and chart drawing.Comparison between groups was performed using t test and analysis of variance.Differences were considered statistically significant when p <0.05.Results 1.Effect of hyperthermia on the expression of CLU molecules and MAPK signaling pathway in human and HPV-infected keratinocytesIn human keratinocytes,foreskin,and CA tissues,hyperthermia at 44? can induce the expression of CLU molecules to be up-regulated.In human keratinocytes,hyperthermia at 44? has no effect on the overall expression of JNK,ERK,and p-38,but can induce the up-regulation of JNK and ERK phosphorylation and the down-regulation of p-38 phosphorylation.In CA tissues,hyperthermia at 44? can induce upregulation of ERK phosphorylation.2.Mutual regulatory relationship between hyperthermia-induced upregulation of DNAJA4,CLU molecules and p-ERK in HaCaT cellsIn human keratinocytes,the up-regulated DNAJA4 can inhibit the expression of CLU and p-ERK under hyperthermia at 44?.The expression of p-ERK was inhibited by the upregulated CLU under hyperthermia at 44?.DNAJA4 and CLU double gene knockout can further induce the expression of p-ERK under hyperthermia at 44?.3.Effects of hyperthermia-induced CLU and p-ERK on proliferation and apoptosis of HaCaT cellsIn human keratinocytes,p-ERK,which expression was up-regulated by hyperthermia at 44?,exerted a cytoprotective effect through maintaining the cell cycle.Hyperthermiainduced CLU exerted a cytoprotective effect through inhibiting apoptosis.CLU knockout could enhance the expression of hyperthermia-induced p-ERK,which played a cytoprotective role through maintaining the cell cycle.Conclusion 1.Hyperthermia at 44? can up regulate the expression of DNAJA4,CLU and p-ERK in human normal keratinocytes and HPV infected CA tissues.2.In HaCaT cells,the upregulated CLU and p-ERK by hyperthermia can play a protective role by inhibiting apoptosis and maintaining cell cycle,respectively.3.In HaCa T cells,DNAJA4,CLU and p-ERK have mutual regulatory relationship.Inhibition of 1-2 protective factors can further up regulate the expression of other protective factors,suggesting that there is a regulatory relationship between the three,which may be the protective feedback regulation mechanism of cells under stress.4.The above results suggest that if we can inhibit these protective molecules or pathways as much as possible in the process of hyperthermia treatment,it is possible to achieve faster and stronger therapeutic effect.