The Effect And Mechanism Of HPV On The Malignant Transformation To Immortalized Cervical Epithelial Cells Mediated By Mortalin In Tumor-derived Exosomes

Objectives:To compare the expression of mortalin(Exo-Mortalin)in cervical cancer exosomes of different HPV infection States,and to find out how human papillomavirus(HPV)infection affected the expression of mortalin through transcription factor CTCF regulation;To explore the transformation of malignant phenotype of immortalized cervical epithelial cell H8 by extraction of exosomes;To elucidate the mechanism of oncogenic exosomes acting through mortalin-p53-Gadd45A pathway in immortalized cervical epithelial H8 cells.Methods:The exosomes of cervical cancer cell line were extracted by ultra-centrifugation,and identified by Western blot(WB),transmission electron microscopy(TEM)and nano particle tracing analysis(NTA).The stably transformed cell lines of KoHPVE6/E7-SiHa(Knockout-HPVE6/E7-SiHa)and KdMortalin-SiHa/Caski(Kncokdown-Mortalin-SiHa/Caski)was constructed using CRISPR/Cas9 Technology and lentivirus infection technology.Western blot(WB)was used to verify the expression of Exo-Mortalin,CTCF,HPV-E6/E7,p53 and Gadd45A.After predicted the binding site of transcription factor CTCF in mortalin gene HSPA9 as the promoter,this study confirmed CTCF as the upstream molecule of mortalin by luciferase.After secretion pathway Exos-Mortalin was confirmed by immunofluorescence co-localization,the effect of Exos-Mortalin on the malignant phenotype of H8 cells was further confirmed by transwell co-culture experiments,EdU,flow cytometry experiments.The tumor model of mice was established as in vivo experiment.We used immunehistochemistry and TUNEL detection to confirmed Exos-Mortalin can promote the proliferation of tumor.Then this study used RNA sequencing to analyze the changes of transcriptome expression spectrum and found out the key targets through GO analysis and KEGG pathway.After that we used flow cycle and apoptosis experiments to explore the effect of downstream Gadd45A.Results:This study was divided into 3 parts.The first part:(1)The expression of mortalin in HPV(+)cervical cancer cells and exosomes was higher than that of HPV(-).After knockout of HPVE6/E7,the decreased expression of Exo-mortalin indicated the presence of HPV can up-regulated expression of Exo-mortalin.(2)After transfection of wild-type luciferase plasmid in mortalin promoter region,the proportion of firefly/Hisense signal was significantly increased compared with that of mutant group(P<0.05),which confirmed that CTCF was the upstream transcription component of mortalin and regulates the expression of mortalin;(3)After Knockout of HPVE6/E7,the expression of CTCF decreased.After CTCF was knocked down,the expression of mortalin was decreased.These results indicated that HPV infection could affect the expression of mortalin by regulating CTCF;(4)When immortalized cervical epithelial cell line H8 co-cultured with cervical cancer cell SiHa(HPV positive),the migration ability of H8 cells was significantly improved,and the proportion of apoptosis decreased;the use of exosomes inhibitors can block this phenomenon,indicating that SiHa exosomes can promote the malignant transformation of H8.The proliferation and migration ability of H8 cells decreased after H8 cells co-cultured with KdMortalin exosomes,which indicated that mortalin was the key molecule of malignant transformation of H8 in SiHa exosomes.(5)The use of exosome inhibitors can reduce the expression of Exo-Mortalin in SiHa cells.At the same time,the co-localization of mortalin and CD63 fluorescence was observed in the cells,suggesting that mortalin existed in the exosome precursor and was secreted through exosome pathway.The second part:(1)After we co-cultured exosomes with H8,wild-type exosomes increased the clone formation and edu proliferation of H8 cells,shortened the scratch distance and decreased the apoptosis rate.(2)In vivo experiments showed that the expression of Mortalin in the peripheral epithelium of cervical cancer after treated with HPV(+)wild-type exosomes was higher and the apoptotic fluorescence was lower than that of KdMortalin exosomes,and the expression of mortalin in the blood circulation of the former was also higher with significant difference(P<0.05).The third part:(1)Through comparative analysis of expression profile changes in transcription group before and after knocking down mortalin,We foundp53-Gadd45A pathway was the main downstream pathway;(2)After co-culture of H8 cells with exosomes,the p53 up-regulation could be observed.At the same time,through immunofluorescence comparison,the expression of mortalin in H8 cells after co-culture and the content of Exos-mortalin was in the direct ratio.When mortalin decreased,the fluorescence of p53 returned to the nucleus increased;(3)After H8 cell was knocked down with Gadd45A,the active cell cycle and the decreased apoptosis rate was observed and indicated that the pathway of p53-Gadd45A plays a role in the malignant transformation of cervical epithelial cellsConclusion:HPV in cervical cancer cells can increase the expression of Exo-mortalin by releasing CTCF which was collected during early infection and combining with mortalin promoter,so the expression of mortalin in cervical cancer cells is positively related to HPV(+).Exosomes from cervical cancer can regulate immortalized cell H8,improving the ability of proliferation and migration and reducing the level of apoptosis.In the downstream pathway,we revealed the regulatory pathway of Mortalin-p53-Gadd45A by oncogenic exosomes acting on H8 cells.Mortalin can combine with p53 and inhibit its return to the nucleus to play a role,resulting in malignant transformation of cervical epithelial cells.