Endoplasmic Reticulum Stress And Its Role In The Pathogenesis Of X-linked Spondyloepiphyseal Dysplasia Tarda

Objective: To silence the SEDL gene in HC-a cells using the lentivirus vector-mediated siRNA(LV-siSEDL), investigate the activation of endoplasmic reticulum stress(ERS) in SEDT cell model and its role in the pathogenesis of X-linked spondyloepiphyseal dysplasia tarda(SEDT).Methods: The experiment was divided into 3 groups: Control group(HC-a/PBS), LV- groups(HC-a/LV) and LV groups(HC-a/LV-siSEDL).(1) Human articular chondrocytes(HC-a) were treated with different concentrations of Puromycin(Puro)(0-10ug/ml) for different hours(12h, 24 h, 48 h, 72 h and 96h), the stably transfected cell lines were distinguished by the minimal antibiotic concentration of Puro.(2) The expression of SEDL gene and Sedlin protein were detected by QPCR and Western blot, and to evaluate whether the SEDT cell model was successfully established.(3) The mRNA expression level of stress signaling molecules in ERS signaling pathway were detected by QPCR, to investigate the activation of endoplasmic reticulum stress.(4) According to the results of QPCR, the protein expression level of stress signaling molecules in ERS signaling pathway were verified by Western blot, to investigate the activation of endoplasmic reticulum stress.(5) The apoptosis rates of HC-a were determined by Annexin-FITC/PI double staining method.Results:(1) The minimum antibiotic concentration of Puro kills all HC-a in 72 h was 5ug/ml.(2) Lentivirus were transfected for 4 days, The expression levels of SEDL gene and Sedlin in LV groups were(0.27±0.02) and(0.05± 0.01),which reduced significantly than that in LV- groups and Control groups(P<0.001),and it indicating that SEDT cell model was successfully established.(3) Lentivirus were transfected for 4 days, There was no significant difference between LV group and other group on the relative mRNA expression levels of stress signaling molecules(P>0.05); Lentivirus were transfected for 5 days, The relative mRNA expression levels of XBP-1 and ATF-6 in LV groups were(2.25±0.17) and(1.40±0.08), which were increased significantly than that in LV- groups and Control groups(P<0.05); Lentivirus were transfected for 6 days, The relative mRNA expression levels of GRP78,ATF-4 and CHOP in LV groups were(1.75±0.16),( 1.82± 0.14) and(2.73±0.21), which were up-regulation significantly than that in LV- groups and Control groups(P<0.01); However, There was no significant difference between LV group and other group on the relative mRNA expression levels of NFKBIA,NFKB-1 and RELA(P>0.05).(4) Lentivirus were transfected for 4 days, The relative protein expression levels of GRP78 in LV groups was(3.09±0.09), which was increased significantly than that in LV-group and Control groups(P<0.001), then, it decreased progressively during each succeeding days; Lentivirus were transfected for 6 days, The relative protein expression levels of ATF-4 in LV groups were(0.61±0.03), which was increased significantly than that in LV-group and Control groups(P<0.001); While the relative protein expression levels of CHOP was increased day by day, and the relative protein expression levels of CHOP in LV group was higher than LV- group and Control group(P<0.01).(5) Lentivirus were transfected for 4 days, Compared with LV- group(4.60 + 0.23)% and control group(3.89 + 0.15)%, the apoptosis rates of LV group(9.78 + 0.56)% was up-regulation significantly(P<0.05).Conclusion: Preliminary proves that endoplasmic reticulum stress(ERS) was involved in SEDT cell model, and ERS plays a role in the pathogenesis of SEDT through activating PERK/eIF2α/ATF-4 signaling pathway of the unfolded protein response(UPR), eventually induce apoptosis of human articular cartilage cells.