Inhibitory effect of asparagine synthetase knockdown on retinal pigment epithelial cell senescence and its underlying mechanism

Objective  To investigate the effect of asparagine synthetase (ASNS) knockdown on the senescence of retinal pigment epithelial (RPE) cells and its potential molecular mechanism.

Methods  Human ARPE-19 RPE cells were divided into four groups: control group, short hairpin RNA targeting ASNS (shASNS) group, control+ (Janus kinase) JAK inhibitor group, and shASNS+ JAK inhibitor group, which were treated with short hairpin RNA control+ dimethyl sulfoxide (DMSO), shASNS+ DMSO, control+ JAK inhibitor and shASNS+ JAK inhibitor for 12 hours, respectively.An RPE cell senescence model was established by cell treatment with 500 μmol/L H ₂O ₂ for 24 hours.The mRNA and protein levels of ASNS and JAK were detected by real-time fluorescent quantitative PCR and Western blot, respectively.Reactive oxygen species (ROS) level within cells was measured using a kit.Cell cycle phase distribution and apoptosis rates were analyzed by flow cytometry.Cell viability from day 1 to day 5 of culturing was assessed via MTT assay.Senescent cell ratio was determined by β-galactosidase staining.Cellular damage was evaluated via immunofluorescence staining.Senescence-associated proteins (p16, pRb), and RPE markers (KRT18, CTNNB1, TJP1, BEST1) were quantified by Western blot.

Results  Compared with the control group, mRNA and protein expression levels of ASNS and JAK were significantly reduced in the shASNS group, control+ JAK inhibitor group, and shASNS+ JAK inhibitor groups (all P<0.05).DCFH-DA staining revealed significantly lower ROS level in the shASNS group, control+ JAK inhibitor group, and shASNS+ JAK inhibitor group than in the control group (all P<0.05).Flow cytometry showed that there were more G2-phase cells and significantly reduced apoptosis rate in the shASNS group, control+ JAK inhibitor group, and shASNS+ JAK inhibitor group compared with the control group (all P<0.01).MTT assay indicated higher cell viability at all time points in the shASNS group, control+ JAK inhibitor group, and shASNS+ JAK inhibitor group compared with the control group, with statistically significant differences (all P<0.01).β-galactosidase-positive cell ratios in the shASNS, control+ JAK inhibitor, and shASNS+ JAK inhibitor groups were (42.36±1.28)%, (43.20±1.89)%, (25.97±1.13)%, respectively, which were significantly lower than (52.25±0.64)% in the control group (all P<0.001).p16 and pRb protein expression were decreased and γ-H2AX fluorescence intensity was attenuated in the shASNS group, control+ JAK inhibitor group, and shASNS+ JAK inhibitor group compared with the control group (all P<0.01).KRT18 and CTNNB1 expressions were upregulated, whereas TJP1 and BEST1 were downregulated in the shASNS group, control+ JAK inhibitor group, and shASNS+ JAK inhibitor group compared with the control group (all P<0.05).The shASNS+ JAK inhibitor group exhibited higher KRT18 and CTNNB1 expressions and lower TJP1 and BEST1 expressions than the shASNS and control+ JAK inhibitor groups (all P<0.05).

Conclusions  ASNS knockdown can promote RPE cell proliferation, mitigate cellular damage, and delay senescence by suppressing the JAK pathway.

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