The Mechanisms Of HPV16 E6/E7 Inducing Primary Keratinocyte Malignant Transformation By DNA Methylation Pathway

Persistent infection with high risk human papillomaviruses(HR-HPVs),such as HPV16,is the necessary cause for the malignant transformation of HR-HPVs infected cells.The early oncogenes E6 and E7 are the critical molecules in this process.HPV16 can induce tumorigenesis and developing by epigenetic mechanisms.One of the best studied is DNA methylation.We suppose that HPV 16 E6/E7 could induce carcinogenesis by DNA methylation.In this study,HPV 16 E6/E7 was transfected to the primary human foreskin keratinocytes(HFKs)by lentivirus to immortalize HFKs;During the overexpression of HPV 16 E6/E7 and silencing of HP V16 E6 or E7,the expression of 4 DNMTs and promoter methylation of 6 target genes were observed;Finally,the immortalized cell line and HPV 16-positive cervical cancer cell lines(SiHa,CaSki)were subjected to whole genome DNA sequencing,whole genome RNA,and DNA methylation sequencing.Through analyzing the sequencing data,we found out initially that the different gene profiles related to the cellualar immortalization and cellular malignant formation.The latter set up the foundation for further studies of gene function.Part 1 Construction and identification of immortalized cell lineObjective:Construction of an immortalized cell line with the transfection of HPV 16 E6/E7.Methods:(1)Primary HFKs were isolated by sequential two step enzyme digestion(dispase II and 0.25%trypsin-EDTA)and cultured in 0.06 mM Ca2+ EpiLife medium supplemented with HKGS.(2)The recombinant lentiviral vector pLV5-HPV16 E6/E7 and packaging plasmids cotransfected 293 T cells to produce lentiviral supernatant.(3)The lentivirus was used to infect the primary HFKs which passaged only twice and chose with puromycin for 2 weeks.(4)Quantitative PCR(qPCR)and Western Blot were used to detect the mRNA and protein level of HPV16 E6?E7;CCK-8 was added to cells to observe the growth rate of different generations;Cell invasion assay was performed by transwell invasion assay,with the contrast of SiHa;Immortalized HFKs and SiHa were injected subcutaneously to female nude mice respectively to monitor the formation of tumors.Results:(1)The primary HFKs were isolated successfully and could passage continuously in vitro.After 10 subcultures,primary HFKs tended to senesce and died gradually.(2)LV5-HPV16 E6/E7 recombinant plasmid was packaged to 1×109 TU/mL lentiviral supernatant.(3)HPV16 E6/E7 HFKs were able to survive for more than 30 cell passages,which were generally accepted as an immortalized cell line.(4)Contrast to the uninfected HFKs,HPV16 E6?E7 mRNA and protein levels of infected HFKs increased obvisouly.The cells multiplying rate of HPV16 E6/E7 HFKs was significantly faster compared with uninfected ones.In the transwell invasion assay,no violet immortalized HFKs was found on the bottom of the filter.No tumor formed in the female nude mice injected with immortalized HFKs.Conclusions:HPV16 E6 and E7 could be stably expressed in HPV16 E6/E7 HFKs and led to immortalization.The immortalized cell line possessed the capacity to passage for more than 30 times,but it could not pass through the matrigel matrix and form tumor in nude mice.Part 2 The effect of HPV16 E6/E7 to DNA methylationObjective:Detecting the expression of DNMTs and promoter methylation of target genes during the overexpression and slincing of HPV16 E6 or E7.Methods:(1)qPCR and Western Blot were used to detect the mRNA and protein expression of 4 DNMTs in primary HFKs(Blank),different passages of HPV16 E6/E7 HFKs(A1?A10?A20?A30)and SiHa.(2)The promoter methylation of 6 target genes in different cells were detected by bisulfite sequencing PCR,qPCR and Western Blot were used to detect the mRNA and protein expression of 6 target genes.(3)qPCR was used to detect the mRNA expression of 4 DNMTs in different time after silencing HPV16 E6 or E7.(4)The promoter methylation of 6 target genes in different cells were detected by MS-HRM,qPCR was used to detect the mRNA expression of 6 target genes.Results:(1)With the overexpression of HPV16 E6/E7,the mRNA of 4 DNMTs was significantly up-regulated and the expression of proteins was increased.In HPV16 E6 or E7 silenced cells,the mRNA of 4 DNMTs was significantly down-regulated.(2)The promoter methylation of MT1G?RRAD and TFPI2 of HFKs increased obviously,mRNA and protein expression of these three genes were down-regulated.However,SPARC?SFRP1 and NMES1 did not show significant difference between primary HFKs and HPV16 E6/E7 HFKs in methylation and expression.With the silencing of HPV16 E6 or E7,the promoter methylation of 6 target genes were all significantly decreased,and the mRNA expressing were increased obviously.Conclusions:HPV16 E6 and HPV16 E7 could influence the promoter methylation of target genes by regulating of DNMTs expression directly or indirectly,which would change the expression of target genes.Part 3 Whole genome sequencing for immortalized and tumor cellsObjective:To observe the dynamic changes of whole genome DNA,transcriptome sequences,whole genome DNA methylation in the process of immortalization,and the differences between immortalized cells and tumor cells in the above aspects.Screening out genes of interest and functional pathways playing different roles in the process of immortalization and final tumor cell formation.Methods:Cells(Blank,A1?A15?A30,SiHa?CaSki)were subjected to whole genome DNA?transcriptome and DNA methylation sequencing.Results:(1)Few non-synonymous mutations and low degree of DNA variability were detected in the process of cell immortalization.However,more than 300 non-synonymous mutations and high degree of DNA variability were detected between HPV16 E6/E7 HFKs and tumoe.(2)With the increase of the number of immortalizing passages,the difference in methylation between immortalizing cells and tumor cells tended to decrease.However,there were still significant differences of DNA methylation between immortalized cells(in the 30th generation)and tumor cells.(3)Screening out genes and enrichment pathways with significant differences in methylation during cell immortalization.(4)Screening out genes and enrichment pathways with significant differences in methylation between immortalized cells and tumor cells.Conclusion:With the increase of culture passages,the difference in methylation between immortalizing cells and tumor cells shows a decreasing trend.However,there were still significant differences of DNA methylation between immortalized cells(in the 30th generation)and tumor cells.We screen out genes that might play critical roles via DNA methylation in the process of cellular immortalization.DNA mutation and DNA methylation could both play indispensable roles in the process of cellular malignant transformation.