Fig 2: Analysis of the expression of podocalyxin-like protein (PODXL) and RUN and FYVE domain containing 1 (RUFY1) mRNA in gastric cancer (GC) serum and the association of expression levels with the prognosis of GC patients. A, Serum PODXL levels in GC patients were significantly increased compared with those of healthy volunteers (normal group). B-D, Serum PODXL levels in GC patients with positive lymph node metastases (Lx group), advanced stage (III-IV) or distant metastases (M1) were higher than those without lymph node metastases (L0), of early stage (I-II) or without distant metastases (M0). E, Serum RUFY1 levels in GC patients were significantly increased compared with those of healthy volunteers (normal group). F-H, Serum RUFY1 levels in GC patients with positive lymph node metastases (Lx group), advanced stage (III-IV stage) or distant metastases (M1) were higher than those without lymph node metastases (L0), of early stage (I-II stage) or without distant metastases (M0). I, PODXL mRNA levels in the serum of GC patients were significantly associated with RUFY1 (r = .596 P = .000). J and K, Survival analysis indicated that GC patients with high levels of serum PODXL or RUFY1 had a shorter OS compared with those with low PODXL or RUFY1 levels (*P < .05)

Fig 3: RUN and FYVE domain containing 1 (RUFY1) knockdown attenuated podocalyxin-like protein (PODXL)-induced biological behaviors in vitro. A, The CCK-8 assay revealed that RUFY1 knockdown significantly inhibited the proliferation of gastric cancer (GC) cells at 48, 72 and 96 h and attenuated the proliferative effects of PODXL overexpression at 48, 72 and 96 h. B, Western blotting showed that RUFY1 knockdown altered the levels of caspase-3, Bax, Bcl-2, MMP-2 and MMP9, and attenuated the PODXL-induced changes of these protein expressions. C and D, RUFY1 knockdown inhibited the colony formation and attenuated PODXL-induced colony formation. E, F, Hoechst33342 staining showed that RUFY1 knockdown increased apoptosis and attenuated the PODXL-induced changes in apoptosis (*P < .05)

Fig 4: Restoring podocalyxin-like protein (PODXL) expression promoted the wound healing, migration and invasion of gastric cancer (GC) cells, and activated the PI3K/AKT, NF-?B and MAPK/ERK signaling pathways in vitro. A, Wound healing assay and statistical analysis. C-F, Migration, invasion assay and statistical analysis. G and H, PODXL activated the PI3K/AKT, NF-?B and MAPK/ERK signaling pathways in vitro via upregulation of the levels of phosphorylated PI3K, AKT, MAPK, NF-?B and ERK1, whereas the total levels of PI3K, AKT, MAPK, NF-?B and ERK1 were unchanged (*P < .05)

Fig 5: Podocalyxin-like protein (PODXL) interacted with RUN and FYVE domain containing 1 (RUFY1) in gastric cancer (GC) cells in vitro. A, B, Mass spectrometry identified MFF, GRP78, TGM3, RUFY1, FSH6Q2, CALL5, TBA1B and ACTB as possible interacted partners of PODXL. C, Co-immunoprecipitation assays detected PODXL/RUFY1 complexes in SGC-7901 cells. D, Western blotting revealed that re-expressing PODXL significantly increased the levels of RUFY1, while knocking down RUFY1 expression did not change the PODXL expression in siRNA-RUFY1 or PODXL-siRNA-RUFY1 cells (*P < .05)