ObjectiveTo preliminarily investigate the mechanism of MMP-9 blocking CD73 detachment from RPE cells surface and preventing and treating experimental autoimmune pigment membranitis (EAU).MethodsRPE cells isolated from wild-type C57BL/6 and CD73 gene knockout (CD73-/-) mice were cultured in vitro, and treated with lipopolysaccharide and TNF-α to induce CD73 detachment from RPE surface. According to whether MMP-9 inhibitor CTK8G1150 was added at the same time (the final concentration was 5.0 mol/L) or not, RPE cells cultured in the two types of mice were respectively set as MMP-9 inhibitor intervention group and non-intervention control group. The cells in each group were treated with the intervention of a solvent, 1 μmol/L adenosine monophosphate (AMP), 1 μmol/L AMP, and 3 μmol/L 5' -α,β-methylene adenosine diphosphate (APCP) (AMP+APCP). The stimulating effect of RPE cells in different groups on CD4+ T cell proliferation was detected by tritiated thymidine incorporation. Adoptive immune induced EAU in wild-type B6 mice and CD73-/- mice, respectively. The receptor mice were randomly divided into the MMP-9 inhibitor intervention group and the non-intervention control group, and CTK8G1150 or the solvent were injected into the subretinal cavity 4, 7 and 10 days after adoptive immunity. CD73 mRNA and protein expression in RPE cells of recipient mice were detected by real-time quantitative PCR (RT-PCR) and Western blot. One-way ANOVA was used to analyze all experimental data.ResultsWhen the stimulation mode was AMP, the proliferation of CD4+ T cells in the C57BL/6 MMP-9 inhibitor intervention group decreased significantly compared with the non-intervention group (F=13.28, P＜0.01). When the stimulation mode was solvent and AMP+APCP, there was no statistically significant difference in the proliferation capacity of CD4+ T cells between the two groups (F=7.78, 6.58; P＞0.05). There was no statistically significant difference in the proliferation capacity of CD4+ T cells between the CD73-/- MMP-9 inhibitor intervention group and the non-intervention group (F=5.24, 6.12, 7.04; P＞0.05). RT-PCR results showed that there was no statistically significant difference in the relative expression of CD73 mRNA in RPE cells between the MMP-9 inhibitor group and the non-intervention control group (F=6.54, P>0.05). Western blot results showed that the expression of CD73 protein in RPE cells in the MMP-9 inhibitor group of B6 receptor mice was significantly increased compared with the control group (F=15.24, P＜0.01).ConclusionMMP-9 inhibitor blocks CD73 detachment from RPE cells surface and has a protective effect on EAU.
ObjectiveTo systematically investigate the functional roles of the nucleotide metabolism-related gene ecto-5'-nucleotidase (NT5E) in lung adenocarcinoma (LUAD) and to provide potential molecular targets for precision diagnosis and targeted therapy. MethodsMulti-omics analyses were performed using transcriptomic data from The Cancer Genome Atlas (TCGA) and gene expression omnibus (GEO) to identify key prognostic genes and to construct a prognostic model. Tumor tissue specimens from patients with LUAD who underwent surgical resection at the Qingdao Central Hospital of University of Health and Rehabilitation Sciences between May and June 2023 were collected for prognostic gene validation, and tissue microarrays were constructed to evaluate protein expression. In vitro, the biological effects of NT5E on LUAD cells were validated by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), Western blotting, wound-healing, Transwell, EdU, cell-cycle, and apoptosis assays. ResultsA total of 10 paired tumor and adjacent non-tumor tissue samples were used for validation of key prognostic genes, including 4 male and 6 female patients, with a mean age of (58.00±13.06) years. In addition, tissue microarrays comprising 90 patients were analyzed, including 28 male and 62 female patients, with a mean age of (60.07±7.98) years. Bioinformatic screening identified NT5E as a representative prognostic gene that was significantly upregulated in LUAD tissues. Survival analyses demonstrated that high NT5E expression was associated with markedly poorer overall survival and served as an independent risk factor. qRT-PCR and tissue microarray-based immunohistochemistry further confirmed its elevated expression in tumor tissues. Functional experiments showed that NT5E knockdown significantly inhibited cell migration, invasion, and proliferation while promoting apoptosis, whereas NT5E overexpression exerted the opposite effects. ConclusionNT5E markedly promotes LUAD cell proliferation, migration, and invasion and suppresses apoptosis, indicating that NT5E acts as an oncogenic driver in LUAD and may serve as a promising prognostic biomarker and therapeutic target.