Experimental Studies On SFRPs Family Methylation Status And Its Mechanism In Cutaneous Squamous Carcinoma Tissue

Objective: To test methylation status of SFRPs family （SFRP1, SFRP2SFRP4,SFRP5） in Cutaneous squamous cell carcinoma, tissue adjacent to carcinoma, normalskin tissues, and discuss the significance of SFRPs’ methylation in CSCC. To detect theexpression of Axin1and Cyclin D1mRNA in three groups and expression of Wnt1andSFRP1in Cutaneous squamous cell carcinoma and normal skin tissues. To analysemethylation status of SFRPs family as inhibiting factor in Wnt signaling pathways inCutaneous squamous carcinoma and discuss its mechanism. Methods:（1） Using theMassARRAY mass spectrometer EpiTYPER to detect methylation of SFRP1, SFRP2,SFRP4, SFRP5gene in three groups.（2） To detect Axin1and CyclinD1mRNA in inthree groups.using the real-time fluorescence quantitative RT-PCR （Taqman probe）.（3）To detect expression of Wnt1and SFRP1protein in CSCC and normal skin tissues byimmunohistochemical SP method and Werten blot.（4） To analyse SFRPs genemethylation data and data of expression of Wnt1, SFRP1protein and Axin1、CyclinD1mRNA and methylation status of SFRP1SFRP2, SFRP4, SFRP5in CSCC andadjacent tissues and normal skin tissues by SPSS17.0software analysis, and draw ROCcurve of SFRPs gene family. Results:（1） About6801of the CpG units can be analysis（87.19%） in SFRPs family of objiect.（2） Detect the SFRPs family methylation status ofCpG in three groups were statistically significant sites. And in the different level ofpathological CSCC, the methylation of DNA CpG loci are statistically significant.（3）The expression of Axin1mRNA in the CSCC is lower than tissue adjacent to carcinomaand normal skin tissues, the expression of Cyclin D1mRNA is higher than tissueadjacent to carcinoma and normal tissues; the expression differences of Cyclin D1 mRNA in different pathological grading is statistically significant. CpG methylation siteunit of SFRPs family is showed negatively correlated with the expression of Axin1mRNA. in CSCC.（4） Immunohistochemical result show that Wnt1dyeing mainly in thecytoplasm as tan particles, the expression of Wnt1in CSCC were significantly higherthan the normal tissue; SFRP1proteins in the cytoplasm staining positive for tan particles（also expression in the nucleus）, SFRP1expressed in CSCC were significantly lower thannormal skin tissues. Methylation of SFRP1CpG loci showed a negative correlation withSFRP1protein expression. Conclusion:（1） SFRP1, SFRP2SFRP4, SFRP5genepromoter methylation rate in three groups and different pathological classification ofstatistically significant differences in CpG sites suggest SFRPs family methylation statusin CSCC plays an important role in carcinoma’s evolution.（2） Wnt1and SFRP1proteinmay participate in the occurrence of development of CSCC with expression of SFRP1and Wnt1differences in CSCC and normal tissue. High methylation of SFRP1genepromoter caused the deactivation of abnormal chromatin structure, reduces SFRP1protein expressing less or missing.（3） Axin1and Cyclin D1may change the signalingpathways that lead to skin cancer development, Cyclin D1can be used as an indicator ofmalignant transformation. The recognition rate of SFRP1CpG1₁methylation loci ishigher than others in CSCC, provide certain theoretical basis for early diagnosis in CSCC.（4）The study of SFRPs family CpG methylation meaningful site can support cancerepigenetics pathogenesis in future and provide theoretical basis for gene therapy inCSCC.