Fig. 4: DIAPH2-AS1 promotes malignant behavior and NI potential of GC cells through NTN1 in vitro.

a Heatmap exhibiting the DEGs in AGS cells transfected with DIAPH2-AS1 or not. b Kaplan–Meier survival analysis of OS of 172 GC patients with low expression of NTN1 versus 170 GC patients with high expression of NTN1 by log-rank (Mantel-Cox) test. c, d qRT-PCR analysis of NTN1 mRNA level in HGC-27 cells after transfection of si-DIAPH2-AS1 or DIAPH2-AS1. e Western blot analysis of NTN1 protein level in HGC-27 cells transfected with si-DIAPH2-AS1, DIAPH2-AS1, or corresponding control. f NTN1 mRNA level was quantified in 84 pairs GC and adjacent normal tissue via qRT-PCR. g NTN1 mRNA level was quantified in 46 NI-positive GC tissues compared with 38 NI-negative GC tissues via qRT-PCR. h Protein level of NTN1 was detected by western blot using 8 NI-positive GC tissues and 8 NI-negative GC tissues. i Correlation analysis was carried out to assess the correlation between the expression level of DIAPH2-AS1 and the mRNA level of NTN1 in 84 GC tissues. N = 84, R = 0.366, P < 0.001. j Transwell assay was performed utilizing indicated engineered HGC-27 cells. Scale bar: 100 μm. Data and error bars are shown as mean ± SD of triplicate independent replicate experiments and all data were analyzed by Student’s t test. k–n Representative D1 (day1) and D7 (day7) images of the DRG-GC cells co-culture model utilizing designated HGC-27 and AGS cells and corresponding statistic charts were shown. Scale bar: 250 μm. NI potential was measured according to the relative area covered by neurites calculated by ImageJ. Error bars represented the mean ± SD of six independent duplicate experiments and all data was analyzed by Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001).