ABSTRACT
@#AIM: To investigate the effect of silencing SIAH1 gene on H2O2-induced apoptosis of human lens epithelial cells.<p>METHODS: The human lens epithelial cell line HLE-B3 was cultured and divided into normal group, H2O2 group(cultured with medium containing 400μmol/L H2O2)and H2O2+siR-SIAH1 group(transfected with SIAH1 interference sequence, followed by cultured with medium containing 400μmol/L H2O2)and siR-NC group(transfected with negative control sequence, followed by cultured with medium containing 400μmol/L H2O2). The expression of SIAH1 gene in cells was detected by real-time fluorescent quantitative PCR. The apoptosis rate was detected by flow cytometry. The expressions of p38 MAPK, p-p38 MAPK, Bcl-2 and Bax proteins were detected by Western blot.<p>RESULTS: The relative expression levels of SIAH1 mRNA in the H2O2 group, siR-NC group and H2O2+siR-SIAH1 group were higher than that in the normal group(<i>P</i><0.05). The relative expression level of SIAH1 mRNA in H2O2+siR-SIAH1 group was lower than those in the H2O2 group and siR-NC group(<i>P</i><0.05). The apoptosis rates in the H2O2 group, siR-NC group and H2O2+siR-SIAH1 group were higher than that in the normal group(<i>P</i><0.05). The apoptosis rate in the H2O2+siR-SIAH1 group was lower than those in the H2O2 group and siR-NC group(<i>P</i><0.05). The expression levels of p38 MAPK and Bcl-2 proteins in the H2O2 group, siR-NC group and H2O2+siR-SIAH1 group were lower than those in the normal group, while the expression levels of p-p38 MAPK and Bax proteins were higher than those in the normal group(<i>P</i><0.05). The expression levels of p38 MAPK and Bcl-2 proteins in the H2O2+siR-SIAH1 group were higher than those in the H2O2 group and siR-NC group, while the expression levels of p-p38 MAPK and Bax proteins were lower than those in the H2O2 group and siR-NC group(<i>P</i><0.05).<p>CONCLUSION: Down-regulation the expression of SIAH1 gene could inhibit H2O2-induced apoptosis of human lens epithelial cell line HLE-B3, which might be related to inhibition of p38 MAPK signaling pathway activation.
ABSTRACT
Objective To investigate the expression differences and significance of periostin (PN) in eyelid basal cell carcinoma associated fibroblasts (BCAFs) andnormal fibroblasts (NFs) after separation,culture,purification and identification.Methods The third generation of purified BCAFs and NFs was selected,and the concentrations of cell suspensions were modulated to 20 × 106 L-1 by trypsin,and then the cell suspension were seeded and cultured in 6-well plate by 2 mL per well.The cell culture supernatants were collected when BCAFs and NFs were cultured by serum-free medium for 48 h,then the content of PN in cell culture supernatants from BCAFs and NFs was detected by enzyme-linked immunosorbent assay (ELISA).The glass coverslips were placed at the bottom of the 6-well plate to make cell slides,and then the expression of PN in BCAFs and NFs cells were tested by immunofluorescence staining.Results ELISA showed that the content of PN in cell culture supernatants from BCAFs and NFs was (9.26 ± 2.35) μg · L-1 and (2.57 ± 0.41) μg · L-1.And the expression level of PN in BCAFs tested by immunofluorescence staining technology was higher than that in NFs cells,and the differences were statistically significant (all P ＜ 0.05).Conclusion The expression and secretion of PN in the eyelid BCAFs were highly enhanced when compared with NFs,suggesting that periostein may promote or inhibit the occurrence and development of the eyelid basal cell carcinoma in the microenvironment of the eyelid basal cell carcinoma.