The Effect Of Hyperthermia On The Migration And Apoptosis Of Caski Cells By Regulating The Expression Of EDN1/2 By YAP Nuclear Translocation

Objective: The Human Papillomavirus(HPV)infection can lead to a variety of warts and neoplasms,including cutaneous and mucous warts such as acuminatum,plantar warts,plantar warts,and cervical,vulvar and penile cancers.Therefore,how to effectively block the transmission of HPV virus and treat HPV infectious diseases has become an important clinical task of dermatology.Hyperthermia(HT)refers to the treatment by heating the tissue temperature to 42-45℃ for 30-60 minutes at a time.It is reported that it is often used in the treatment of malignant tumors and human papillomavirus(HPV)infectious diseases.Previous studies have found that keratinocyte apoptosis occurs in skin tissues under warm intervention,and the apoptosis rate in HPV-positive tissues is significantly higher than that in normal skin tissues.Therefore,a high throughput PCR array was used to detect signal molecules related to apoptosis in human condyloma acuminatum.It was found that the m RNA expressions of most signaling molecules in Hippo,TGF-β,BMPs,Wnt,which are closely related to cell proliferation and apoptosis,were changed under the effect of warm heat.However,the m RNA expression of YAP,the link molecule of these signaling pathways,did not change significantly.As a key molecule in Hippo,TGF-β,BMPs and Wnt signaling pathways,whether YAP plays a key role in the treatment of malignant tumors and HPV infectious skin diseases with warm heat is worth further exploration.The purpose of this study was to clarify the effect of YAP on the function of human epithelial cells with high copy of HPV during thermotherapy,and to provide theoretical reference for further application of thermotherapy.Methods: In this study,HPV high copy human cervical carcinoma epithelial cells(Caski)were selected as the study object.In this study,44(±0.1)℃ water bath in vitro was used to simulate clinical thermotherapy.m RNA expression levels were examined by RT-PCR.Protein expression levels were examined by Western Blot.The protein interaction was measured by Co-IP.The protein localization in cells were examined by IF staining.DNA nucleic acid assay was performed using agarose gel electrophoresis for isolation and identification.For cell functional tests,cell apoptosis was detected by flow cytometry,cell proliferation activity was measured by CCK-8 cell proliferation assay,and cell migration was measured by Transwell migration assay.For statistical analysis,all spatial transcriptome data were calculated and analyzed using R language(version 3.4.1)platform.Independent sample t test or variance analysis were used to judge the data from cell lines,and the test level was P<0.05.Graph Pad Prism 9.0 software were used for the rest of the images and statistical analysis in this study,* represents P value <0.05,** represents P value < 0.01,ns indicates no statistical significance.All images were spliced using Photoshop.Results: 1)In m RNA and protein levels,YAP expression in Caski cells was higher than that in Ha Cat cells.To evaluate YAP expression in tissues,IHC results were obtained from HPA and analyzed,showing weak YAP staining in normal epidermis and strong YAP staining in HPV-positive cervical cancer tissues.A standardized pan-cancer dataset was downloaded from the UCSC(https://xenabrowser.net/)database: TCGA TARGET GTEx(PANCAN,N=19131,G=60499),from which ENSG00000137693(YAP)gene expression data were extracted in each sample.The difference in expression between normal and tumor samples in each tumor was calculated using R software,and the difference significance was analyzed using unpaired Wilcoxon Rank Sum and Signed Rank Tests.Significant upregulation of YAP was observed in 12 tumors,including cervical cancer CESC(Tumor:-7.65±2.90,Normal: 9.63±1.22,p=0.01).In summary,YAP expression was elevated in both HPV-positive cells and tissues.2)YAP protein expression decreased rapidly after 4 hours of hyperthermia,and then recovered gradually.They return to normal levels after 24 hours.YAP was expressed in the cytoplasmic nucleus of Caski,and YAP gradually moved to the nucleus within 24 hours after 44℃ hyperthermia.However,the expression of TEAD1,a transcription factor that binds YAP in the nucleus,decreased gradually and did not recover after 4 hours of hyperthermia.At 44℃,the binding of YAP and TEAD1 decreased.3)hyperthermia promoted the transcription of YAP and TEAD1 target genes Cyr61,CTGF and BIRC5,but inhibited the transcription of EDN1 and EDN2 in Caski cells.After hyperthermia,EDN1/EDN2 protein expression decreased,which was consistent with PCR results.4)Compared with Ha Cat cells,both EDN1 and EDN2 were highly expressed in Caski cells.Compared with normal tissues,EDN1 was not significantly overexpressed in HPV-positive tissues,while EDN2 was overexpressed(p<0.05).Moreover,the expressions of EDN1 and EDN2 decreased after 4 hours of hyperthermia.5)hyperthermia and interference with EDN1 and EDN2 could reduce Caski cells migration.44 °C hyperthermia and interference with EDN1 and EDN2 could increase the apoptosis of Caski cells.Conclusions: 44 °C hyperthermia interferes with the binding of YAP and TEAD1 to inhibit the expression of its target gene EDN1/2,thus inhibiting the migration of Caski cells and promoting their apoptosis.