Abstract:

Objective To discuss the role of selenoprotein N1 （SEPN1） gene in D-galactose （D-gal）- induced senescence of L929 cells， and to clarify its signaling mechanism on cell proliferation， oxidative stress， endoplasmic reticulum stress， mitochondrial function， and extracellular matrix synthesis（ECM）. Methods D-gal was used to treat the L929 cells to establish a cell senescence model. The experiment was divided into control group and D-gal-induced group （D-gal group）. Small interfering RNA （siRNA） was used to knock down SEPN1 gene， and the cells were divided into negative control group （si-NC group） and si-SEPN1 group. SA-β-gal staining was used to observe the staining condition of L929 cells in various groups and calculate the percentage of SA-β-gal positive cells； Western blotting method was used to detect the expression levels of SEPN1， P21， P16， collagen type Ⅰ（Col Ⅰ）， collagen type Ⅲ（Col Ⅲ）， glucose-regulated protein 78 （GRP78）， and C/EBP homologous protein （CHOP） proteins in the L929 cells in various groups； 2'-7'- dichlorofluorescein diacetate （DCFH-DA） fluorescent probe method was used to detect the levels of reactive oxygen species （ROS） in the cells in various groups； kits were used to detect the mitochondrial membrane potential levels in the cells in various groups； Fluo-4 AM calcium ion （Ca²⁺） fluorescent probe was used to detect the Ca²⁺ levels in the L929 cells in various groups； 5-ethynyl-2'- deoxyuridine （EdU） staining was used to detect the proliferation activity of L929 cells in various groups； immunofluorescence method was used to detect the expression levels of Col Ⅰ and Col Ⅲ proteins in the L929 cells in various groups. Results The SA-β-gal staining results showed that compared with control group， the percentage of SA-β-gal positive cells in L929 cells in D-gal group was significantly increased （P<0.01）； compared with si-NC group， the percentage of SA-β-gal positive cells in si-SEPN1 group was significantly increased （P<0.01）. The Western blotting method results showed that compared with control group， the expression levels of P21 and P16 proteins in L929 cells in D-gal group were significantly increased （P<0.01）， and the expression level of SEPN1 protein was significantly decreased （P<0.01）. The EdU staining results showed that compared with control group， the proliferation activity of L929 cells in D-gal group was significantly decreased （P<0.01）； compared with si-NC group， the proliferation activity of L929 cells in si-SEPN1 group was significantly decreased （P<0.01）. The DCFH-DA fluorescent probe method results showed that compared with control group， the ROS level in L929 cells in D-gal group was significantly increased （P<0.01）； compared with si-NC group， the ROS level in the cells in si-SEPN1 group was significantly increased （P<0.05）. The Western blotting method results showed that compared with si-NC group， the expression levels of SEPN1， Col Ⅰ and Col Ⅲ proteins in the cells in si-SEPN1 group were significantly decreased （P<0.05 or P<0.01）， and the expression levels of P21， P16， GRP78 and CHOP proteins were significantly increased （P<0.05 or P<0.01）. The mitochondrial membrane potential assay results showed that compared with si-NC group， the level of mitochondrial membrane potential in the cells in si-SEPN1 group was significantly decreased （P<0.01）. The Fluo-4 AM calcium ion fluorescent probe results showed that compared with si-NC group， the Ca²⁺ level in the cells in si-SEPN1 group was significantly increased （P<0.05）. The immunofluorescence method results showed that compared with si-NC group， the expression levels of Col Ⅰ and Col Ⅲ proteins in the L929 cells in si-SEPN1 group were significantly decreased （P<0.05 or P<0.01）. Conclusion Knockdown of SEPN1 can promote senescence of L929 cells， and inhibit cell proliferation and synthesis of Col Ⅰ and Col Ⅲ； its mechanism may be related to induction of endoplasmic reticulum stress， impairment of mitochondrial function， Ca²⁺ overload， and increase of intracellular ROS.

Key words: Pelvic floor dysfunction diseases, Selenoprotein N1, Cell senescence, Endoplasmic reticulum stress, Mitochondrial dysfunction, Fibroblasts