The Role And Mechanism Of RNASET2 In The Pathogenesis Of Vitiligo

Part Ⅰ The stress influence the expression of RNASET2 in the human epidermal keratinocytes and melanocytesObjective:To investigate the expression of RNASET2 in the vitiligo tissues; to obtain a large number of high-purity primary keratinocytes (HEKs) and melanocytes (HEMs) by using improved methods, simply and economically isolated from human foreskin tissues; to observe the expression of RNASET2 in HEKs and HEMs under stress factors in vitro.Methods:We first examined whether the expression of RNASET2 was,altered in vitiligo tissues by conducting real-time PCR, western blot analysis and immunohistochemical study using anti-RNaseT2 polyclonal antibodies; we obtained pure primary melanocytes and keratinocytes according to their different tolerance to trypsin and the characteristics of fibroblasts sensitive to G418; we established different stress models within HEKs and HEMs to excessive UV irradiation (NB-UVB), hydrogen peroxide(H2O2) and inflammatory(LPS)factors in vitro-culture system, separately. We then investigated the RNASET2 expression in HEKs and HEMs using real-time PCR, Western blot and immunofluorescence detection technology.Results:Both the transcript (vitiligo vs control:4.056±1.115 vs 1.034±0.166; p<0.001) and protein expressions of RNASET2 were found to be significantly higher in the vitiligo epidermis specimens; Successfully obtained a large number of high-purity primary keratinocytes (HEKs) and melanocytes (HEMs) from human foreskin tissues; Under stress, the expression of RNASET2 in human primary keratinocytes (HEKs) and melanocytes (HEMs) were both increased in a concentration-dependent manner within a certain range.Conclusion:RNASET2 is significntly increased in lesional epidermis of vitiligo patients; HEKs and HEMs can increase the RNASET2 expression in the presence of stress factors.Part Ⅱ The biological function of protein RNASET2 in primary keratinocytes and melanocytesObjective:To investigate the biological functions of RNASET2 overexpression in primary keratinocytes and melanocytes in vitro.Methods:Primary keratinocytes and melanocytes were transfected with the lentiviral empty vector (VECTOR) and RNASET2 overexpression (OE RNASET2); the efficiency of lentivirus-infected cells was identified using fluorescence microscopy; then the overexpression results were confirmed through western blot techniques; the effect of RNASET2 overexpression in primary keratinocytes and melanocytes proliferation and apoptosis were studied using CCK-8 cell proliferation assay, Annexin V-APC/7-AAD apoptosis test and colony-forming method; we observe the effect of oxidative stress to the RNASET2 overexpressed primary keratinocytes and melanocytes through adding appropriate concentration of the exogenous H2O2; we also analyzed the effect of RNASET2 overexpression in melanin synthesis ability using real-time PCR, Western blot, Melanin quantification, and tyrosinase activity.Results:HEKs and HEMs both had>90% transfected efficiency successfully; overexprsing RNASET2 in HEKs and HEMs resulted in a growth defect; The percentage of cell apoptosis for VECTOR versus OE RNASET2 in HEK cells (4.167 ±0.330 vs 23.467±1.482;p=0.003) and HEM cells (5.400±0.455 vs 27.333±1.053; p=0.001) were with statistical significance. HEKs and HEMs overexpressing RNASET2 were hypersensitive to oxidative stress; RNASET2 overexpression in HEKs and HEMs led to reduced melanin synthesis.Conclusion:Overexpressing RNASET2 in HEKs and HEMs results in a growth defect; RNASET2 overexpression in HEKs and HEMs leads to reduced melanin synthesis.Part Ⅲ The mechanism of protein RNASET2 in the pathogenesis of vitiligoObjective:To investigate the mechanism of protein RNASET2 overexpression in the pathogenesis of vitiligo which is caused due to apoptosis and decreased melanin synthesis in primary keratinocytes and melanocytes.Methods:To confirm whether RNASET2 can lead to cell apoptosis when the catalytic activity of RNASET2 was ruled out, we generated a catalytically inactive form of RNASET2 (OE RNASET2-ci) (whose cDNA has been previously mutagenized in the two CAS catalytic sites). Additionally, to verify the predicted TRAF2 site and to evaluate its physiological relevance, we also constructed a TRAF2 binding motif mutant form (OE RNASET2-ti) by successfully changing OE RNASET2 at Positions 222-225 from PKQE to glycines. Then, HEKs and HEMs were transfected with the empty vector (VECTOR), OE RNASET2-ci and OE RNASET2-ti lentiviral particles; the efficiency of lentivirus-infected cells was identified using fluorescence microscopy; then the overexpression results were confirmed through western blot techniques; the effect of RNASET2 overexpression in primary keratinocytes and melanocytes proliferation and apoptosis were studied using CCK-8 cell proliferation assay and Annexin V-APC/7-AAD apoptosis test; to explore whether the pro-apoptotic role of RNASET2 overexpression depends on the interaction between proteins RNASET2 and TRAF2, we used, Western blot to detect apoptosis situation before and after mutations.Results:HEKs and HEMs transfected with the empty vector (VECTOR), OE RNASET2-ci and OE RNASET2-ti lentiviral particles successfully; The results indicated that cells had>90% transfected efficiency; The role of RNASET2 in cell death was independent of its catalytic activity and associated with TRAF2 binding motif; RNASET2 promoted apoptosis via TRAF2-Caspases pathway.Conclusion:The role of RNASET2 in cell death is independent of its catalytic activity and associated with TRAF2 binding motif; RNASET2 promotes apoptosis via TRAF2-Caspases pathway.