Effect And Mechanism Of Human GJB6 Gene And The Mutation On The Proliferation And Apoptosis Of HaCat Cells

Objective Clouston syndrome or hidrotic ectodermal dysplasia (HED) is known as a rare dominant genodermatosis. It is characterized by alopecia, nail dystrophy and palmopl-antar hyperkeratosis. HED is caused by mutations in the human GJB6 gene which encodes connexin30 (Cx30). In order to study the effect and mechanism of human GJB6 gene and the mutation,we have constructed lentiviral vector containing human wild-type GJB6 gene and the mutations(A88V and G11R) stably expressing by Tet-on system on HaCaT cell lines.It can make us to further research the mechanism of apoptosis induced by the expression of mutants by Tet-on system in HaCat cells.Methods The human wildtype and mutant GJB6 gene (A88V and G11R) was amplified by PCR, then constructed to the lentiviral vector expressing by Tet-on system,After enzymatic digestion and sequencing, the recombinant lentivirus was transfected into HaCaT cells, HaCaT cell lines which stably express connexin 30(Cx30) encoded by GJB6 were selected by puromycin, after induced by Doxycycline (DOX),the stable GJB6 expression HaCaT cells were confirmed at mRNA level by RT-PCR and identified by Western blot which used to detect Cx30 expression. Cell proliferation was measured by using CCK8 assay. FITC annexin V and propidium iodide (PI) staining from apoptosis kit were used to determine the levels of apoptosis using flow cytometric analyses. Western blot analyses were done to analyze the relevant clinical indicators of HED and the apoptosis related proteins in transfected HaCat cells.Results The recombinant lentivirus was successfully constructed according to enzymatic digestion and sequencing, RT-PCR detection shows the expression of mRNA encoded by GJB6 Was enhanced significantly inside stably transfected HaCaT cells. The expression of wildtype and mutant GJB6 gene (A88V) in HaCat cells induced by Doxycycline was higher than that in HaCat cells without induced by Doxycycline(P value<0.05),Western blot detect Cx30 protein stably expressing on the Doxycline-induced cells which encoded by GJB6. In different time point,the absorbance value of each subgroup without GJB6 gene were no significant difference(P>0.05),In group with wildtype GJB6 gene,the absorbance value of DOX subgroup were significantly higher than that in control group at 4,8,12,24 and 36 hours(P<0.05), In group with mutant GJB6 gene (A88V and G11R),the absorbance value of DOX subgroup were significantly higher than that in control group at 4.8.12,24,36 and 48 hours(P<0.05). The flow cytometry results showed that The HaCat cells expressing the A88V and G11R mutants showed an increase in apoptosis.(*:P<0.05). After the A88V and G11R mutants were induced to expess by DOX,the expression of involucrin became weak,but the expression of keratin 6b,filaggrin and loricrin was unchange.And the cleaved forms of caspase-3, caspase-8,caspase-9 and PARP (markers of apoptotic activation) were elevated in HaCat cells expressing the A88V and G11R mutants and the cells transfected by negative control virus and expressing wild-type GJB6 gene were unchanged.Meanwhile,the Bax was unchanged.Conclusion The HaCaT cell line stably expressing GJB6 gene and its mutations was successfully constructed. The study conducts the further research to find possible pathway concerning why the mutants can induce HaCaT cells apoptosis.It established a favorable foundation for further functional study.