RNA Interference Of Dsg2Gene And Its Effect On HL-1Cells

Objective To investigate the effects of down-regulation of Dsg2on ultrastructure,apoptosis, location of plakoglobin (PG) protein, AKT/β-catenin signalpaths,adipogenesis and fibrogenesis of HL-1cells. Methods A set of Dsg2-specificsiRNAs and control siRNA (siNC) were synthesized and cloned intopGPU6/GFP/Neo vector. The recombinant plasmids (ShDsg2and ShNC) weretransfected into HL-1cells, and the positive clones were selected with G418. Theefficiency of Dsg2knock down on the levels of mRNA and protein were determinedby RT-PCR and Western blot in order to pick the optimal cell line. The changes ofcell apoptosis and ultrastructure were evaluated by Flow Cytometry and TransmissionElectron Microscope (TEM), respectively. The location of PG was studied byconfocal microscope. RT-PCR and Western blot was used to measure the expressionof Col1a1, Col1a2, Col3a1, Adiponectin, PPAR-γ, and C/EBP-α on mRNA level andAKT/β-catenin signal paths on protein level respectively. Results Fourrecombinant ShDsg2plasmids were constructed successfully and transfected intoHL-1cells, and stable cell lines were established by RNA interference. Comparedwith the control group, the levels of Dsg2knockdown on mRNA and protein inShDsg2-273group were71.67%vs.0.00%(P<0.01) and51.72%vs.0.00%(P<0.01),respectively. In ShDsg2-273group, TEM showed that cellular vacuoles, swollenmitochondria and disappeared cristae and Flow Cytometry further demonstrated thatthe apoptosis was increased in the stable cell lines. confocal microscope showed thelocation of PG was not changed. A marked increase in expression levels of two majorregulators of adipogenesis, namely PPAR-γ, and C/EBP-α, and their target geneadiponectin, were also observed. The expression levels of procollagen genes Col1a1,Col1a2and Col3a1were increases remarkably. Actication of the AKT/β-cateninsignal paths were shown by Western blot. Conclusions We establish Dsg2-deficientHL-1cell line, which exhibited increased apoptosis, adipogenesis and fibrogenesis,thus recapitulating the phenotype of human ARVC. This cellular model could providethe opportunity to explore the pathogenesis and novel therapeutic targets of ARVC in vitro.