Abstract:

Objective To investigate the expression of SPART in gastric cancer tissues and its effects on lipophagy， as well as proliferation and migration in gastric cancer cells. Methods Bioinformatics analysis was performed on gastric cancer transcriptome data from the TCGA database to assess SPART expression level. Five patients diagnosed with gastric cancer and undergoing surgical treatment at the Affiliated Tumor Hospital of Xinjiang Medical University from April 2024 to April 2025 were selected， and Western blotting was used to measure SPART protein levels in gastric cancer and adjacent normal tissues. The experimental groups of human gastric cancer cell lines AGS and NUGC3 were all transfected with SPART overexpression plasmids， while the control groups were all transfected with blank vectors. Western blotting was used to detect the effects of overexpressing SPART on the expression of microtubule-associated protein light chain 3 （LC3）， lysosome-associated membrane protein 2 （LAMP2）， and perilipin 3 （PLIN3）. Cell proliferation and migration capabilities were assessed using CCK-8 and Transwell assays， respectively. Immunofluorescence was used to examine lipid droplet content and LC3 levels after SPART overexpression. Results Bioinformatics analysis revealed that the expression levels of SPART in 375 gastric cancer tissues and 32 normal gastric mucosa tissues were 3.06 （1.62） and 3.54 （2.68）， respectively， with a statistically significant difference （U=7 429.00， P=0.025）. Among the 27 pairs of paired samples， the relative expression level of SPART in gastric cancer tissues was 2.78±0.82， which was lower than that in normal gastric tissues （3.84±1.22）， with a statistically significant difference （t=-3.80， P<0.001）. Immunohistochemical staining data from the Human Protein Atlas database showed that among the 23 gastric cancer tissue samples， SPART was lowly expressed in 16 cases （69.57%）， moderately expressed in 7 cases （30.43%）， and had no high expression （0）. Western blotting analysis showed that the relative expression level of SPART protein in gastric cancer tissues in clinical samples was 0.56 （0.68）， which was lower than that in adjacent tissues 1.38 （1.05）， with a statistically significant difference （U=4.00， P=0.016）. CCK-8 assay results showed that at 48 h， the absorbance （A） values of the control and experimental groups of AGS cells were 0.52±0.01 and 0.41±0.01， respectively； those of the control and experimental groups of NUGC3 cells were 0.67±0.02 and 0.51±0.01， respectively， with statistically significant differences （t=13.47， P<0.001； t=12.39， P<0.001）. At 72 h， the A values of the control and experimental groups of AGS cells were 0.82±0.01 and 0.67±0.01， respectively； those of the control and experimental groups of NUGC3 cells were 1.05±0.01 and 0.78±0.02， respectively， with statistically significant differences （t=18.37， P<0.001； t=20.91， P<0.001）. At 96 h， the A values of the control and experimental groups of AGS cells were 1.04±0.03 and 0.74±0.01， respectively； those of the control and experimental groups of NUGC3 cells were 1.80±0.02 and 1.41±0.08， respectively， with statistically significant differences （t=16.43， P<0.001； t=8.19， P=0.004）. Transwell assay revealed that the numbers of migrating cells in the AGS and NUGC3 cell experimental groups were 500±17 and 276±21， respectively， which were less than those in the control groups （771±33 and 327±12）， with statistically significant differences （t=-12.65， P<0.001； t=-3.65， P=0.030）. Western blotting assay showed that， in the control and experimental groups of AGS cells， the expression levels of LC3 protein were 0.47±0.08 and 1.06±0.32， respectively， and the expression levels of LAMP2 protein were 0.65±0.03 and 0.98±0.11， respectively， the expression levels of PLIN3 protein were 0.82±0.15 and 0.43±0.10， respectively， all with statistically significant differences （t=3.11， P=0.040； t=5.02， P=0.007； t=3.75， P=0.020）. In the control and experimental groups of NUGC3 cells， the expression levels of LC3 protein were 0.89±0.04 and 1.45±0.16， respectively， and the expression levels of LAMP2 protein were 0.68±0.18 and 1.22±0.22， respectively， the expression levels of PLIN3 protein were 1.71±0.45 and 0.35±0.06， respectively， all with statistically significant differences （t=5.89， P=0.004； t=3.29， P=0.030； t=5.19， P=0.007）. Immunofluorescence assay showed that， the lipid droplet levels in the AGS cell control group and the experimental group were 1.08±0.09 and 0.32±0.03， respectively， and the LC3 levels were 0.40±0.01 and 0.88±0.15， respectively， with statistically significant differences （t=13.88， P<0.001； t=-5.53， P=0.033）. Conclusions SPART is lowly expressed in gastric cancer， and its overexpression can inhibit gastric cancer cell proliferation and migration while activating lipophagy.

Key words: Stomach neoplasms, Cell proliferation, Cell movement, Lipophagy, SPART