ASAP3 disrupts ASAP1-ARHGAP12 to inhibit RhoA and yes-associated protein/transcriptional coactivator with PDZ-binding motif, suppressing gastric cancer progression

Figure 1 ASAP3 expression in gastric cancer, and cell transfection efficiency. A: Relative expression of ASAP3 in gastric cancer (GC) cell lines (AGS and HGC-27 cells); B: Cell transfection efficiency assessed by determining the messenger RNA (mRNA) expression of ASAP3 in AGS transfected with over-expression-ASAP3 plasmids; C: Cell transfection efficiency assessed by determining the mRNA expression of ASAP3 in HGC-27 transfected with short hairpin-ASAP3 plasmids; D: Proliferation of GC cells (HGC-27 and AGS) after transfection using cell counting kit-8 after cultured for 24 hours, 48 hours, and 72 hours. ^aP < 0.05 vs AGS cell. ^bP < 0.05 vs HGC-27 cell. ^cP < 0.05 vs short hairpin-negative control-HGC-27 cell. ^dP < 0.05 vs over-expression-negative control-AGS cell. mRNA: Messenger RNA; oe: Over-expression; sh: Short hairpin; NC: Negative control.

Figure 2 Effects of ASAP3 on the growth of gastric cancer cells. A: Cell cycle of HGC-27 and AGS cells after transfection with short hairpin (sh)-ASAP3 or over-expression (oe)-ASAP3 plasmids using a flow cytometry; B: Apoptosis of HGC-27 and AGS cells after transfection with sh-ASAP3 or oe-ASAP3 plasmids using a flow cytometry; C: Migration of HGC-27 and AGS cells after transfection with sh-ASAP3 or oe-ASAP3 plasmids by cell scratch assay; D: Migration of HGC-27 and AGS cells after transfection with sh-ASAP3 or oe-ASAP3 plasmids by Transwell assay; E: Invasion of HGC-27 and AGS cells after transfection with sh-ASAP3 or oe-ASAP3 plasmids by Transwell assay; F: The colony-forming ability of HGC-27 and AGS cells after transfection with sh-ASAP3 or oe-ASAP3 plasmids using clone formation assay. ^cP < 0.05 vs short hairpin-negative control-HGC-27 cell. ^dP < 0.05 vs over-expression-negative control-AGS cell. oe: Over-expression; sh: Short hairpin; NC: Negative control.

Figure 3 Effects of ASAP3 on gastric cancer tumor growth in vivo. A: Body weight changes of mice after injected with HGC-27 or AGS cells after transfection with short hairpin (sh)-ASAP3 or over-expression (oe)-ASAP3 plasmids for different days; B: Tumor size of mice after injected with HGC-27 or AGS cells after transfection with sh-ASAP3 or oe-ASAP3 plasmids; C: Tumor volume changes of mice after injected with HGC-27 or AGS cells after transfection with sh-ASAP3 or oe-ASAP3 plasmids for different days; D: Hematoxylin and eosin staining of tumor tissues with different treatments. Purple arrow: Irregular cavity; Red arrow: Necrosis; Black arrow: Nuclear fission phase; E: ASAP3 positive cells in tumor tissues of mice after injected with HGC-27 cells after transfection with sh-ASAP3 plasmids using immunofluorescence; F: ASAP3 positive cells in tumor tissues of mice after injected with oe-ASAP3 plasmids using immunofluorescence. ^cP < 0.05 vs short hairpin-negative control-HGC-27 cell. ^dP < 0.05 vs over-expression-negative control-AGS cell. oe: Over-expression; sh: Short hairpin; NC: Negative control.

Figure 4 Underlying mechanisms of ASAP3 in HGC-27 cells using transcriptome sequencing. A: The principal component analysis of the short hairpin (sh)-negative control (NC)-HGC-27 and sh-ASAP3-HGC-27 cells; B: The volcano plot of the differential expressed messenger RNAs (DEMs) between the sh-NC-HGC-27 and sh-ASAP3-HGC-27 cells; C: The heatmap of the identified DEMs; D: The Gene Ontology terms enriched by the identified DEMs; E: Kyoto Encyclopedia of Genes and Genomes pathway analysis of the identified DEMs. PCA: Principal component analysis; PC: Principal component; GO: Gene Ontology; NADPH: Nicotinamide adenine dinucleotide phosphate hydrogen; KEGG: Kyoto Encyclopedia of Genes and Genomes; BP: Biological process; MF: Molecular function; CC: Cellular component; cAMP: Cyclic adenosine monophosphate; AGE-RAGE: Advanced glycation end products-receptor for advanced glycation end products; TNF: Tumor necrosis factor; FoxO: Forkhead box O; sh: Short hairpin; NC: Negative control.

Figure 5 Underlying mechanisms of ASAP3 in AGS cells using transcriptome sequencing. A: The principal component analysis of the over-expression (oe)-negative control (NC)-AGS and oe-ASAP3-AGS cells; B: The volcano plot of the differential expressed messenger RNAs (DEMs) between the oe-NC-AGS and oe-ASAP3-AGS cells; C: The heatmap of the identified DEMs; D: The Gene Ontology terms enriched by the identified DEMs; E: Kyoto Encyclopedia of Genes and Genomes pathway analysis of the identified DEMs. PCA: Principal component analysis; PC: Principal component; oe: Over-expression; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; BP: Biological process; MF: Molecular function; CC: Cellular component; TNF: Tumor necrosis factor; IL: Interleukin; MAPK: Mitogen-activated protein kinase; NC: Negative control.

Figure 6 Underlying mechanisms of ASAP3 in HGC-27 cells using proteomics. A: The principal component analysis of the short hairpin (sh)-negative control (NC)-HGC-27 and sh-ASAP3-HGC-27 cells; B: The volcano plot of the differential expressed proteins (DEPs) between the sh-NC-HGC-27 and sh-ASAP3-HGC-27 cells; C: The heatmap of the identified DEPs; D: Subcellular localization analysis of the DEPs; E: The Gene Ontology terms enriched by the identified DEPs, including biological process, cellular component, and molecular function; F: Kyoto Encyclopedia of Genes and Genomes pathway analysis of the identified DEPs. PC: Principal component; sh: Short hairpin; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; BP: Biological process; MF: Molecular function; CC: Cellular component; NC: Negative control.

Figure 7 Underlying mechanisms of ASAP3 in AGS cells using proteomics. A: The principal component analysis of the over-expression (oe)-negative control (NC)-AGS and oe-ASAP3-AGS cells; B: The volcano plot of the differential expressed proteins (DEPs) between the oe-NC-AGS and oe-ASAP3-AGS cells; C: The heatmap of the identified DEPs; D: Subcellular localization analysis of the DEPs; E: The Gene Ontology terms enriched by the identified DEPs, including biological process, cellular component, and molecular function; F: Kyoto Encyclopedia of Genes and Genomes pathway analysis of the identified DEPs. PC: Principal component; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; BP: Biological process; MF: Molecular function; CC: Cellular component; oe: Over-expression; NC: Negative control.

Figure 8 Validation of the related mechanisms using real-time quantitative polymerase chain reaction, western blot, co-immunoprecipitation and pull down. A: The messenger RNA expression of CCN1, CCN2 and AMOTL2 in gastric cancer (GC) cells with different treatments measured using real-time quantitative polymerase chain reaction; B: The protein expression of phosphorylated (p)-MST1/MST2, p-LATS1 + LAST2, p-yes-associated protein, p-transcriptional coactivator with PDZ-binding motif, ASAP1 and ARHGAP12 in GC cells with different treatments detected by western blot; C: The relationship between ASAP3 and ASAP1-ARHGAP12 interaction determined by co-immunoprecipitation; D: The correlation between ASAP3 and RhoA activity examined by RhoA activation pull down. ^cP < 0.05 vs short hairpin-negative control-HGC-27 cell. ^dP < 0.05 vs over-expression-negative control-AGS cell. mRNA: Messenger RNA; oe: Over-expression; sh: Short hairpin; NC: Negative control; YAP: Yes-associated protein; TAZ: Transcriptional coactivator with PDZ-binding motif; p-YAP: Phosphorylated yes-associated protein; p-TAZ: Phosphorylated transcriptional coactivator with PDZ-binding motif; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IP: Immunoprecipitation; WB: Western blot.

Figure 9 Correlation between ASAP3/ASAP1/ARHGAP12 and clinicopathologic features using bioinformatics. A: Correlation between ASAP3/ASAP1/ARHGAP12 and age; B: Correlation between ASAP3/ASAP1/ARHGAP12 and gender; C: Correlation between ASAP3/ASAP1/ARHGAP12 and tumor node metastasis stage; D: Correlation between ASAP3/ASAP1/ARHGAP12 and tumor size (mm); E: Correlation between ASAP3/ASAP1/ARHGAP12 and patient overall survival. TNM: Tumor node metastasis; FPKM: Fragments per kilobase of transcript per million mapped reads.