Effect Of Heat Shock Protein DnaJA4 Induced By Hyperthermia On Migration Of Human Epithelial Cells

Background and ObjectiveHyperthermia treatment(HT)is a method of heating tissue to abnormal temperature(usually at 42-45℃)and sustaining for a period of time.Currently,HT is used to treat malignant tumors and human papillomavirus(HPV)infectious diseases.Previous studies have shown that HT can enhance local antigen-specific immune reaction and treat HPV infection in skin and cervix,improve the sensitivity of cancer tissues to radiotherapy and chemotherapy.HPV has high host specificity.So far,no natural host other than human has been found.HPV mainly infects human epithelial cells,such as skin,vulva,cervix,nasopharyngeal mucosa,etc,and causes various diseases,such as condyloma acuminatum,verruca vulgaris,cervical cancer,head-neck squamous cell carcinoma and so on.Among them,high-risk HPV(including HPV 16/18)infection is the main risk factor of cervical cancer and penile cancer.Our previous studies found that the expression of heat shock protein DnaJA4 was significantly up regulated after HT on human normal foreskin,condyloma acuminatum,immortalized human keratinocytes(HaCaT)and HPV16/18 positive human cervical cancer epithelial cells(Ca Ski).DnaJA4 knockout(KO)could enhance the activation of NF kappa B pathway and ERK pathway induced by HT.Heat shock proteins(HSPs)are usually expressed in intracellular or extracellular environment by cold and heat stimulation,microbial infection,ultraviolet radiation and other factors.HSPs can be divided into small molecule HSPs,HSP40 s,HSP60s,HSP70 s,HSP90s and HSP110 s according to their molecular weight.DnaJA4 is a member of HSP40 s and mainly functions at participating in protein folding and metabolism as a molecular chaperone of HSP70 s.Only DnaJA4,Dna JB1 and Dna JB4 among HSP40 s were significantly up regulated under HT.The role of DnaJA4 in the treatment of malignant tumors and HPV infectious skin diseases is worth further investigation.This study aims to clarify the effects of DnaJA4 on the function of human normal epithelial cells and HPV high copy epithelial cells.We aim to provide some evidence for the further application of HT.MethodsIn this study,HaCaT cells and Ca Ski cells were treated with 44℃ water bath,and the cell model of gene specific overexpression(OE)or knockdown(KD)was constructed by lentivirus transfection.To observe the effects of DnaJA4 KD under HT on the functions of HaCaT cells and Ca Ski cells.The proteomic changes of HaCaT cells after DnaJA4 KO were analyzed by i TRAQ labeled liquid chromatography-mass spectrometry.The effects of HT and DnaJA4 KD on cell proliferation were measured by CCK-8.The effects of DnaJA4 KD on cell migration and its regulatory mechanism were observed by wound healing assay and western blot.The m RNA levels of DnaJA4 and other molecules were detected by real-time fluorescence quantitative PCR.The expression of DnaJA4,MYH9,MMP-9,PKM and cell migration related proteins were detected by western blot.The protein-protein interaction between DnaJA4 and PKM was detected by co-immunoprecipitation.Statistical methods: paired t-test or analysis of variance were used for comparison between groups.The data were expressed by(?)±SEM.When the P value was less than 0.05,the difference was statistically significant.Graphpad prism 9.0 is used for statistical analysis and chart making.Explanation of statistical test in this study: * P value < 0.05,** P value < 0.01,*** P value < 0.001,**** P value < 0.0001,ns means no statistical significance.Results(1)In the differential proteomic analysis between DnaJA4 KO and wild-type HaCaT cells,5344 proteins were identified,of which 4573 proteins had quantitative information.A total of 388 differentially expressed proteins(DEPs)were screened,of which 178 were significantly up regulated and 210 were significantly down regulated.After GO annotation of these differentially expressed proteins,it was found mainly distributed in cell process,metabolic process,biological regulation and so on.DEPs mainly localized at protein complex,intracellular anatomical complex and so on.In molecular function,it is mainly reflected in binding,catalytic molecules,translation regulation,transport regulation,structural molecular activity.These differentially expressed proteins were mainly located in cytoplasm,cell membrane,nucleus,mitochondria and intercellular.The GO and KEGG enrichment analysis showed that the DEPs were mainly concentrated in pyruvate metabolism,lipid metabolism,glycolysis,binding activity,cytoskeleton,cell migration and adhesion.(2)DnaJA4 KO/KD increased the migration of HaCaT cells,decreased the migration of Ca Ski cells,while HT increased the migration of HaCaT cells,but had no significant effect on the migration of Ca Ski cells.DnaJA4 KO/KD up regulated the expression of MYH9 and MMP-9 protein in HaCaT cells and down regulated the expression of MYH9 protein in Ca Ski cells.Both HT and DnaJA4 OE down regulated the expression of MMP-9 protein in HaCaT cells.No significant change on the proliferation of HaCaT cells and Ca Ski cells under HT and DnaJA4 KD.(3)HSP90α inhibitor CH5138303 decreased the migration of HaCaT cells and Ca Ski cells,increased the expression of DnaJA4 and down regulated the expression of MYH9 in HaCaT cells.(4)After applying of CH5138303,DnaJA4 KD improved the migration and MYH9 expression of HaCaT cells,comparing to WT.Co-treatment of CH5138303 and HT decreased the expression of MYH9,HPV16 E6 in Ca Ski cells.(5)PKM transcription,PKM1 and PKM2 protein expression and PKM2 phosphorylation had no difference under the treatment of HT and DnaJA4 KO,in HaCaT cells.The transcription and protein expression of DnaJA4 increased under treatment of HT,while PKM1 and PKM2 protein expression and PKM2 phosphorylation showed no significant changes,in Ca Ski cells.Co-immunoprecipitation showed no interaction between DnaJA4 and PKM1/2 both in HaCaT cells and in Ca Ski cells.Conclusions(1)Proteomic studies showed that compared with the WT,the DEPs of HaCaT cells after DnaJA4 KO were mainly concentrated in molecular binding,metabolism,cell migration,cytoskeleton and so on.The functions of up regulated proteins are mainly to promote cell growth,mitosis,cell migration and cytoskeleton reorganization.(2)There is an interaction between DnaJA4 and MYH9 in HaCaT cells under hyperthermia.The deficiency of DnaJA4 can enhance the migration of HaCaT cells stimulated by hyperthermia and inhibit the migration of Ca Ski cells.(3)Overexpression of DnaJA4 participates in the process driven by HSP90α inhibitor,which inhibits the migration of HaCaT cells and Ca Ski cells.Hyperthermia combined with HSP90α inhibitor significantly down regulated the expression of HPV16 E6/E7 and MYH9 in Ca Ski cells.(4)HT could up regulate the expression of DnaJA4 in HaCa T cells and CaSki cells.DnaJA4 induced by hyperthermia had no significant effect on PKM1/PKM2.There was no direct interaction between DnaJA4 and PKM1/PKM2 in HaCaT cells and Ca Ski cells.