Roles Of PAG1 Of Palmitoylation Site Mutation In Skin Squamous Cell Carcinoma

Objective: The aim of this thesis is to investigate the effects of phosphoprotein associated with glycosphingolipid-enriched microdomains(PAG,human official gene:PAG1)palmitoylation site mutation(CXXC mutation AXXA)on the biological behavior of cutaneous squamous cell carcinoma(cSCC),and furthermore,the relationship and mechanism of PAG1 and Src signaling in palmitoylation site mutations.Methods: The PAG1 molecular structure was queried by the Uniprot protein database,and the cysteine in the palmitoylation site of PAG1 was mutated to alanine.The A431 cell line was used as the research object,and the wild type PAG1 and the overexpressed wild type PAG1 carrying the enhanced green fluorescent signal were used.Negative control group transfected with negative control group carrying the enhanced green fluorescence.PAG1 mutated by palmitoylation site and Lentiviral vector containing wild-type PAG1(WT-PAG1)or C-A mutant PAG1(mutant-PAG1)were transfected into A431 cells,and A431 cell line with overexpressed WT-PAG1,mutant-PAG1,negative control group(control)and blank control group(parental)were established.The expression of green fluorescence in WT-PAG1 group,mutant-PAG1 group and control group after lentivirus transfection were detected by confocal microscopy.The fluorescence rate of cells after expanded culture was detected by flow cytometry(FCM).The cells carrying green fluorescence from three groups were sorted by sorting FCM,and the monoclonal cell line was screened by limiting dilution method and followed with expansion.The mRNA level of Pag1 in cells was detected by Quantitative Real-time PCR(RT-PCR)and the protein level of PAG1 was detected by Western blot.CCK8 assay was used to detect the proliferation of A431 cells,while Annexin V-APC/PI double staining was accessed for the detection of A431 cells apoptosis,and then studied the mechanism of apoptosis induced by PAG1 palmitoylation site mutation in A431 cell;Transwell migration assay was used to detect the migration ability of A431 cells,while Transwell invasion assay was used to detect the invasion ability of A431 cells.Western Blot was used to detect the expression level of the inhibitory tyrosine kinase Csk that binds to PAG1 and the important signaling molecule Fyn in the Src activation signaling pathway.Result:(1)After 96 hours of transfection with lentivirus,the green fluorescence of WT-APG1,mutant-PAG1 and control group was observed throughout the field by laser confocal microscopy.The results from FCM showed that the fluorescence positive ratesof the control group,WT-PAG1 group and mutant-PAG1 group were 86.2%,88.1% and87.8%,respectively.RT-PCR showed that Pag1 mRNA was carried in WT-PAG1 and mutant-PAG1 transfected cells,and the mRNA levels of PAG1 in WT-PAG1 and mutant-PAG1 A431 cells were 1.65 and 1.68 time compared with control group.The difference was statistically significant(P<0.01).Furthermore,Western Blot showed that the expression of PAG1 protein in A431 cells of WT-PAG1 and mutant-PAG1 group were2.17 and 2.24 time,compared to the control and parental group.The difference in the parental group was statistically significant(P<0.001),which suggested a successful transfection.(2)CCK8 assay showed that the proliferation of A431 cells in WT-PAG1 group was significantly decreased compared with parental and control groups(P<0.001),and the difference between the groups is statistically significant between 2 and 6 days(P<0.01).However,mutated PAG1 loses its inhibitory effect on cell growth,which implied that PAG1 could inhibit the proliferation of A431 cells.(3)Annexin V-APC/PI double staining showed that the proportion of apoptotic cells in the parental and control group was 5.30%±0.04% and 4.20%±0.06%,respectively.The apoptosis of WT-PAG1 transfected A431 cells was 11.20%±0.04%,which has significant difference with parental and control group(P<0.001).The apoptosis rate of the A431 cells in the mutant-PAG1 group was 3.50%±0.04%,which has no significant difference with parental and control group(P>0.05).(4)The results of scratch repair experiments showed that compared with the parental group and the control group,the cell healing ability of the WT-PAG1 group was significantly decreased(P<0.001,P<0.01).There was no significant difference between the mutant-PAG1 group and the parental group and the control group(P>0.05);Moreover,we have further analyzed the cell migration ability Tranwell migration assay,results showed that cell migration ability of the WT-PAG1 overexpressed cells was obviously reducted compared with the parental and control group,while the cell migration ability of the mutant-PAG1 group was significantly increased compared with the WT-PAG1 group(P<0.001).(5)Invasion experiments showed that the invasive ability of the WT-PAG1 group was significantly lower than that of the parental and control group.Compared with WT-PAG1 group,the invasive ability of mutant-PAG1 group was significantly increased(P<0.001).(6)Western blot showed that the expression levels of Csk and Fyn in WT-PAG1 transfected cells were significantly increased compared with parental and control group(P<0.001),and the mutation of PAG1 did not affect the expression of Src related proteins,Csk and Fyn.Conclusion:(1)A431 cell line stably overexpressing the PAG1 palmitoylation sitemutation was successfully constructed.(2)Palmitoylation site mutated PAG1 can weaken or even loses the ability to inhibit the proliferation of skin squamous cell carcinoma A431 cell line,induce apoptosis,and biological behaviors such as movement and migration.(3)Palmitoylation site mutated PAG1 can weaken or even lose the biological functions which regulated by Src family kinase.