br ANGPTL expression was associated with the
3.2. ANGPTL6 expression was associated with the microvessel density of AFPGC patients
To investigate the importance of ANGPTL6 in gastric cancer, com-pared to patients with a low expression of ANGPTL6 (Fig. 2A), patients with high levels of ANGPTL6 had a poorer prognosis. In addition, we found that the expression of ANGPTL6 was positively correlated with the expression of CD34 (a specific biomarker of vascular endothelial cells) (Fig. 2B). These findings revealed that the upregulation of ANGPTL6 might have a significant role in AFPGC progression and is associated with angiogenesis.
3.3. ANGPTL6 regulates HUVEC migration and tube formation in vitro
Previous studies have reported that ANGPTL family members (ANGPTL2 , and ANGPTL4 ) can exert angiogenic or growth-promoting eﬀects on endothelial cells. Therefore, we performed pro-liferation, chemotactic migration, and tubule formation of endothelial SR 11302 to investigate whether ANGPTL6 has a similar biological eﬀect on
endothelial cells in vitro.
We used the CCK8 assay to evaluate the pro-proliferation eﬀect of endothelial cells administered ANGPTL6. The proliferation of en-dothelial cells treated with ANGPTL6 was increased with no significant diﬀerence compared with that in the control group, even with a high dose of recombinant ANGPTL6 (Fig. 3A), whereas ANGPTL6 induced a concentration-dependent chemotactic migration of endothelial cells in vitro (Fig. 3B). Finally, we performed tube formation assays to detect the eﬀect of ANGPLT6. Consistent with the results above, ANGPLT6 facilitated the formation of a loop (Fig. 3C). To gain insight into the molecular mechanisms underlying this ANGPTL6-mediated chemotaxis, we conducted western blotting analysis to examine several important signaling pathways involved in angiogenesis. We found that transient stimulation of HUVECs with recombinant ANGPTL6 caused a sig-nificant increase in the phosphorylation of pro-survival kinase (AKT) and extracellular signal-related kinase 1 and (ERK1/2) (Fig. 3D). Fur-thermore, pretreating HUVECs with a specific AKT inhibitor or ERK1/2 inhibitor for two hours significantly abolished ANGPTL6-induced mi-gration and tube formation, suggesting that the activation of AKT and ERK1/2 signaling is involved in ANGPTL6-mediated endothelial cell chemotaxis (Fig. 3E, F). Above all, ANGPTL6 can promote the chemo-taxis, migration, and tube-forming ability of endothelial cells.
3.4. ANGPTL6 decreased the apoptosis but not the mitotic activity, migration and invasion of AFPGC
To interrogate the loss-of-function regulation of ANGPTL6, we performed shRNA-mediated knockdown of ANGPTL6 through control siRNA (shCtrl) or two independent shRNAs against ANGPTL6(shANGPTL6-1, shANGPTL6-2) in GCIY cells (an AFPGC cell line). qRT-PCR and immunoblotting were performed to assess the ef-fective knockdown (Fig. 4A, B). Compared to the condition medium of GCIY-shControl, the conditioned medium of GCIY-shANGPTL6-1 or shANGPTL6-2 significantly impaired the tube formation ability of HUVECs (Fig. 4C). Previous studies have reported that some ANGPTL family members can exert anti-apoptotic eﬀects on cells. Therefore, investigated whether the biological behavior changed in AFPGC cells. As expected, our results showed that silencing of ANGPTL6 significantly increased the levels of cell apoptosis compared to that in the control (Fig. 4D). In addition, we investigated how ANGPTL6 influences AFPGC cell viability in vitro. As shown in Fig. 4 E and F. ANGPTL6 loss led to reduced cell viability measured by cell migration and invasion assay. Above results suggest that ANGPTL6 may prevent cancer cells from undergoing apoptosis and promote cell viability.
Fig. 2. ANGPTL6 has clinical significance. (A) A high expression of ANGPTL6 confers a poor prognosis. (B) The level of ANGPTL6 is positively correlated with CD34 (an endothelial cell marker).
Fig. 3. ANGPTL6 promotes endothelial cell migration and tube formation via ERK1/2 and AKT. (A) The proliferation of endothelial cells was increased without statistical significance after exposure to ANGPTL6. (B) ANGPTL6 si-mulated the migration of endothelial cells. (C) Tube formation ability was increased by pre-treatment with ANGPTL6. (D) Western blotting revealed that phospho-AKT and ERK1/2 were upregulated on exposure to ANGPTL6. (E) AKT or ERK1/2 inhibitor abrogated the pro-migra-tion eﬀect of ANGPTL6. (f) AKT or ERK1/2 inhibitor attenuated the pro-formation ability of ANGPTL6. *P < 0.05; **P < 0.01 com-pared with the control. The data are expressed as the mean ± SD and are representative of three independent experiments.
3.5. Targeting ANGPTL6 impairs AFPGC tumor growth in vivo
To further assess the contribution of ANGPTL6 to AFPGC tumor-igenesis, we performed in vivo studies using a lentivirus shRNA ap-proach. As expected, the mean tumoral volume or weight of the shANGPTL6 group was significantly smaller than that of the control group (Fig. 5A, B and C). Moreover, the expression of Ki-67 (an cell proliferation markers) and the expression of CD34 was remarkably decreased in the shANGPTL6 group compared to that in the controls (Fig. 5 D). These results indicated that ANGPTL6 may be a possible therapeutic target in AFPGC.