The Role Of WNT/Ca²⁺ Pathway And The DNA Methylation Of ID4 Regulated By UVB In Cutaneous Squamous Cell Carcinoma

Cutaneous squamous cell carcinoma?CSCC?,a keratinocyte or cutaneous appendages-derived skin neoplasm with malignant potential,is the second-most-common cancer in skin malignancy,the incidence of which is second only to basal cell carcinoma and still increasing in recent years.Although early stage of CSCC has a good prognosis with surgical resection,metastatic CSCC cases could be difficult to treat depending on the location of the lesions and the extent of metastasis,with a mortality rate of over 70%in some cases.Besides,for CSCC patients prone to occur on the head and face,they are faced with various challenges such as wound defect repair,cosmetic requirements and organ dysfunction.Therefore,it is still urgent to study its pathogenesis and find new biological markers and treatment methods to diagnose,reduce tumor size and inhibit tumor invasion and recurrence of CSCC.WNT5a mainly participates in the noncanonical WNT pathway through the WNT/Ca²⁺and WNT/PCP pathways.However,the role of WNT5a and its homologous receptors in cancer has been controversial.The aberrant activation or inhibition of WNT5a signaling is emerging as an important event in cancer progression,exerting both oncogenic and tumor suppressive effects depending on the type of cancer.It has been reported that WNT5a expression in skin squamous cell carcinoma is increased compared with that in solar keratosis and normal skin using microarray.But its specific function and mechanism still remains uncovered.In this study,the expression of FZD4 and PLC?4 in CSCC was significantly reduced and NFATc4 was slightly decreased,while WNT5a expression was unchanged by the whole-genome RNA sequencing.Firstly,the sequencing results were verified by expanding tissue samples,and found that the expression of WNT5a in CSCC was generally decreased.Thus,we hypothesized that WNT5a may initiate the noncanonical WNT pathway by binding to the receptor FZD4 and further activate PLC?4,playing anti-carcinomatosis role in CSCC.Lentivirus of WNT5a,FZD4,PLC?4 and NFATc4 was transferred respectivelyto infect A431 and colo16 cells of human skin squamous cell lines cells.Then CCK-8 test,Annexin V-PIdouble staining,scratch test and Transwell Insert were used to detect the changes of cell proliferation,apotosis,migration and invasion before and after infection.Finally,the tumorigenic experiment of nude mice was conducted to observe the effect of cell lines before and after infection on tumorigenesis in vitro.Epigenetics researches on skin cancer confirmed that abnormal DNA methylation is closely related to melanoma,basal cell carcinoma,CSCC and cutaneous lymphoma.Tumor cells are hypomethylated compared with normal cells,and hypemethylation of tumor suppressor promoter is one of the possible causes of tumor,such as p53.DNA methylation is catalyzed by DNA methyltransferase?DNMTs??including DNMT1,DNMT3A and DNMT3B?to generate methylated cytosine.TET,a dioxygenase,was reported to convert 5mC into 5-hydroxymethyl cytosine?5hmC?,5-formyl cytosine?5fC?and 5-carboxyl cytosine?5caC?through three successive oxidation reactions,initiating DNA demethylation.The balance between methylation and demethylation is controlled by a number of factors.In cancer,this balance is often disrupted,leading to altered methylation.The abnormal DNA methylation occurs earlier than the formation of tumor and is reversible.Therefore,DNA methylation can be used as a marker for the early diagnosis,prognosis of tumor and provide a treatment target.Thus,RNA Sequencing?RNA-seq?and Reduced Representation Bisulfite Sequencing?RRBS?were performed on CSCC.We found the expression of inhibitor of DNA binding 4?ID4?in CSCC was decreased,the methylation of which was increased,indicating the negative correlation of hypemethylation and gene expression of ID4.ID4 is a key gene locating in the last position of the BMP-SMAD-ID sub-pathway in the transforming growth factor-??TGF-??pathway which plays an important role in the development,progression and metastasis of liver cancer and cholangiocarcinoma.Ultraviolet irradiation is one of the main pathogenic factors.Studies have shown that continuous UVB irradiation in mice can cause the dysregulation of methylation in the skin.Therefore,we speculated that methylation of ID4 may be regulated by UVB and UVB may play a role by affecting methyltransferase or demethylase.We confirmedthe hypothesis from three aspects,including the tissue,animal and cell level.The results showed that ID4 was hypemethylated with decreased expression after UVB irradiation or exposure.In addition,we found that DNMT1 gene expression increased after UVB irradiation or exposure,while TET1 and TET2 gene expression decreased.Part 1 RNA-seq,RRBS and validationof sequencing resultsObjective:The transcriptome sequence and DNA methylation sequenceof 8 pairs of CSCC tissues and adjacent normal tissue samples were performed,so as to obtain the differentially expressed genes,differential genome methylation and enrichment pathways.Then,the numbers of the CSCC tissue samples and adjacent tissues were expanded to verify the mRNA expression and methylation of differentially expressed genes and methylated differentially expressed genes,laying a foundation for further research on screening pathways and genes.Methods:1.RNA-seqand RRBS were used for genome-wide RNA and methylation sequencing on 8 pairs of CSCC and corresponding adjacent tissues,and the enriched signal pathways of differentially expressed genes and methylated differentially expressed genes were analyzed using KEGG.2.qPCR was applied to detect the mRNA expression of WNT5a,FZD4,FZD8,PLC?4,PPP3CB and NFATc43.qPCR and BSP was applied to detect the methylationlevels of BMP4,BMP8B,BMPR2,SMAD9 and ID4 promoter regions and expression levels in the TGF-? signaling pathway.Results:1.A total of 1053 differentially expressed genes were found,including 123 up-regulated genes 930 down-regulated genes.KEGG showed that the differentially expressed genes were mainly concentrated in the neuroactive ligand-receptor interaction,metabolic pathway and calcium signaling pathway.There were significant differences in DNA methylation.Compared with the corresponding adjacenttissues,gene expression of FZD4,FZD8,PPP3CB and NFATc4 was decreased?p<0.05?,but there was no significant difference in gene expressionof WNT5a?p>0.05?.Compared with the corresponding adjacent tissues,gene expression in all positions of the BMP-SMAD-ID was decreased?p<0.05?,and the methylation level of ID4 in the last position of this pathway was significantly increased?p<0.05?.2.By expanding the sample numbers,we found the mRNA expression of FZD4 was in CSCC were significantly decreased?p<0.001?.The expressionof WNT5a and PLC?4 was decreased?p<0.01?and FZD8,NFATc4 was also decreased?p<0.05?.3.The enlarged samples showed that the methylation of ID4 gene was significantly different in cancer and adjacent tissues which was hypemethylated in CSCC?p<0.001?and the expression of ID4 was was significantly decreased?p<0.001?.The expressionof BMP4?BMPR2?SMAD9 was decreased?p<0.01?,as well as BMP8B?p<0.05?,but the methylation of them showed no obvious differences?p>0.05?.Conclusions:There were significant differences in gene expression and methylation between CSCC and adjacent tissues,which could screen out differentially expressed genes and enriched pathways,and screen out differentially expressed genes and enriched pathways that play key roles through DNA methylation modification.Consistent with RNA-seq and RRBS results,the expression levels ofFZD4,PLC?4,and ID4 were significantly decreased.The methylation of ID4 promoter region in CSCC was negatively correlated with gene expression.Part 2 Construction cell lines of the overexpressiontargeted genes and function studiesObjective:Cell models of the overexpression of WNT5a,FZD4,PLC?4 and NFATc4 were constructed to investigate the effects of overexpressed target genes on cell function and tumorigenesis in nude mice.Methods:1.Lentiviral plasmids of LV-WNT5a,LV-FZD4,LV-PLC?4 and LV-NFATc4 were constructed and co-transfected with packaging plasmids into 293T cells to produce ahigh titer of lentivirus supernatant.2.The lentivirus was infected into A431 and colol6 cell linesand chose with puromycin for 2 weeks.Quantitative PCR?qPCR?and Western Blotwere used to detect the mRNA and protein level of WNT5a,FZD4,PLC?4 and NFATc4.3.CCK8,Annexin V-PI double staining,scratch test and Transwell invasive test were applied to detect the changes of cell proliferation,apotosis,migration and invasion in each group before and after infection.4.The tumorigenic effect of infected cells in nude mice was observed.Results:1.Lentiviruses of LV-WNT5a,LV-FZD4,LV-PLC?4 and LV-NFATc4 weresuccessfully packagedwith titers of 3×108,2×109,5×108,2×109,2×108 TU/mL,respectively.2.Compared with control cells,mRNA and protein levels of each target gene were significantly up-regulated.In the overexpressed FZD4 group,the expression level of NFATc4 was significantly increased in both cells?p<0.05?.In the overexpressed NFATc4 group,the expression of FZD4 was significantly increased in both cells?p<0.05?.3.Compared with the control group,up-regulated expression of FZD4,PLC?4 and NFATc4 can significantly inhibit the cell proliferation level of A431 and colo16,while up-regulated expression of WNT5a has no obvious effect on the cell proliferation level.The ability of cell migration and invasion was decreased in the cancer groups of overexpression of WNT5a,FZD4,PLCP4 and NFATc4,while the ability of cell apotosis was increased.4.Compared with the A431 control group,the upregulation groups of WNT5a,FZD4,PLCP4 and NFATc4 of A431 cells showed significantly reduced tumor volume.Conclusions:Cell lines of A431 and colo16 that overexpressed WNT5a,FZD4,PLC?4 and NFATc4 were successfully constructed.FZD4 and NFATc4 can affect the expression mutually.The increased expression of FZD4,PLC(34 and NFATc4 inhibited the proliferation,migration and invasion ability of CSCC cells,promoted the aoptosis,and could significantly inhibit the formation of tumor in vivo.Part 3 Effect of UVB on methylation of ID4 in CSCCObjective:to investigate the effect of UVB on the methylation of ID4 gene and regulation mechanismin CSCCMethods:The experiment was divided into three parts:tissue level?60 pairs of facial exposure area and unexposed parts?,animal level?6 to 7 weeks of C3H/HeN male mice with a dosage of 150 mJ/cm2of UVB irradiation?and cell level?colo16 cells with10 mJ/cm2 UVB irradiation?.qPCR and BSP were used to detect ID4,DNMT1-3 and TET1-3 mRNA expression and promoter region methylation changes,respectively.In addition,40 pairs of CSCC tissue and paracancer tissue samples were selected for qPCR to verify the mRNA expression levels ofDNMTl-3 and TET1-3.Results:The methylation of ID4 was enhanced and mRNA expression was decreased after exposure or UVB irradiation?p<0.05?.In the face skin samples exposed to UVB or the mouse back skin,the expression level of DNMT1 were increased?p<0.05?,while the expression level of TET1-3 was decreased?p<0.05?.After 8h of UVB exposure in A431 and colo16,the expression of DNMT3A decreased?p<0.01?,while the expression of TET1 and TET2 decreased?p<0.01?.Compared with the skin adjacent samples,DNMT1 expression level was significantly increased in the CSCC tissues?p<0.001?,while TET1 expression level was significantly decreased?p<0.001?and TET2 expression was significantly decreased?p<0.01?.Conclusions:In CSCC,UVB may affect ID4 methylation through DNMT1,TET1 and TET2 to regulate its expression level.Significance and innovation1.We acquired the transcriptional information and DNA methylation of CSCC by RNA-seq and RRBS method.Differentially expressed genes of CSCC were obtained,along with cancer-related enrichment pathways,providing ideas for further research.Meanwhile,differential expression genes and pathways that play key roles in DNA methylation modification were identified.2.The roles of WNT/Ca²⁺signaling related genes in CSCC were systematically studied for the first time.We found that FZD4 and NFATc4 can influence the expression mutually.3.It was found for the first time that ID4 gene methylation in CSCC was regulated by UVB,and its regulatory mechanism was studied for the first time.