3.1. LINC01224 is upregulated in GC cells and promotes GC development in vitro
Firstly, we used qRT-PCR to measure LINC01224 expression in four lines of GC cells (MKN-45, MKN-28, HGC-27, and NCI-N87) and one line of normal gastric mucosal cells (GES-1). All four lines of GC cells had greater expression of LINC01224 than the GES-1 cells, especially the HGC-27 cells (all P < 0.05, Fig. 1A). Thus, we further examined the effect of LINC01224 by focusing on HGC-27 cells. We then performed transient transfection experiments using different transfectants to alter the expression of LINC01224, and then used qPCR to measure changes in expression (Fig. 1B).
To establish the effects of LINC01224 in GC, we performed assays that measured the viability, proliferation, apoptosis, migration, and invasion of HGC-27 cells that had overexpression or inhibition of LINC01224 (Fig 1. C-H). Relative to the si-NC group, down-regulation of LINC01224 impeded the viability, colony formation, migration, and invasion of GC cells, but increased their apoptosis. Conversely, overexpression of LINC01224 had the opposite effects, in that it promoted cell viability, colony formation, migration, invasion, and suppressed apoptosis. These in vitro results demonstrated that LINC01224 promotes the carcinogenesis and metastasis of GC. However, these results did not identify the mechanism by which LINC01224 promotes GC.
3.2 LINC01224 activates the PI3K/AKT/mTOR/HIF-1α axis in GC
Activation of the PI3K/AKT/mTOR/HIF-1α axis has an important role in the pathogenesis of numerous cancers[15-17]. Thus, we examined the role of LINC01224 on the PI3K/AKT/mTOR/HIF-1α axis in HCG-27 cells by transfecting these cells with si-NC, si-LINC0 1224, oe-NC, or oe-LINC01224. We then used qRT-PCR and western blotting to measure the expression of mRNAs and proteins in the PI3K/AKT/mTOR/HIF-1α axis. After transfection for 48 h, there were significant changes in the mRNA levels of PI3K, AKT, mTOR and HIF-1α (Fig. 2A). However, the total protein levels of PI3K (PI3K3β), AKT, and mTOR did not change (Fig. 2B). Treatment with oe-LINC01224 led to an increase in the levels of p-PI3K, p-AKT, p-mTOR, and HIF-1α (Fig. 2B). We then examined the role of LINC01224 on the PI3K/AKT/mTOR/HIF-1α pathway using a PI3K activator (740-YP) and a PI3K inhibitor (LY294002) in HGC-27 cells (Fig. 2C and Fig. 2D). The results indicated that the 740-YP significantly reversed the effect of LINC01224 down-regulation (Fig. 2C), and LY-294002 significantly reversed the effect of LINC01224 overexpression (Fig. 2D).
3.3 LINC01224 regulates GC by altering the PI3K/AKT/mTOR/HIF-1α axis
We then further assessed the function of the PI3K/AKT/mTOR/HIF-1α pathway and LINC01224 on the malignant behavior of GC cells using the CCK8 assay, migration assay, and invasion assay. 740-YP treatment greatly increased cell viability, cell migration, and cell invasion in the si-LINC01224 group (Fig. 3A, 3E and 3G), but inhibited the apoptosis ((Fig. 3C). Conversely, LINC01224 dramatically altered cell viability, migration, and invasion, but these effects were alleviated by addition of LY294002 (Fig. 3B, 3F and 3H). Treatment with LY294002 significantly increased the apoptosis rate of HGC-27 cells after transfection with oe-LINC01224 (Fig. 3D). In general, LINC01224 exerted an oncogenic function by facilitating GC progression and up-regulating the PI3K/AKT/mTOR/HIF-1α axis.
3.4 LIN01224 enhances the Warburg effect through the PI3K/AKT/mTOR/HIF-1α axis in vitro
Several studies showed that aerobic glycolysis (Warburg effect) provides a basic fuel source and supports the rapid proliferation of malignant cells as an adaptation to the tumor environment[18-20]. We therefore examined the effect of LINC01224 on inducing the Warburg effect by measurement of glucose uptake and lactate production. The results showed that LINC01224 overexpression promoted glucose uptake (Fig. 4A) and lactate production (Fig. 4B). In line with this, the levels of key glycolysis proteins (GLUT1, HK2, ENO1, PKM2, and LDHA) were significantly increased in the oe-LINC01224 group compared with the negative controls (Fig. 4G). In the si-LINC01224 group, glucose uptake (Fig. 4C), lactate production (Fig. 4D), and the levels of key glycolysis proteins (Fig. 4H) were all greatly reduced, but this effect was mostly restored by the addition of 740-YP. In addition, LINC01224 enhanced glucose uptake (Fig. 4E), lactate production (Fig. 4F), and the levels of key glycolysis proteins (Fig. 4I), and this was suppressed by LY-294002.
These results demonstrated that LINC01224 modulated the glycolytic phenotype of GC cells. In other words, LINC01224 upregulated glycolysis in GC cells by activating the PI3K/AKT/mTOR/HIF1α axis, and inactivation of the PI3K/AKT/mTOR/HIF-1α axis reversed the pro-tumor effects of LINC01224. Taken together, these results demonstrate that targeting PI3K/AKT/mTOR/HIF-1α altered the Warburg effect in GC cells.
3.5 Role of LINC01224 on the Warburg effect via the PI3K/AKT/mTOR/HIF-1α axis in vivo
Our in vitro results showed that LINC01224 promoted GC progression and induced the Warburg effect by activating the PI3K/AKT/mTOR/HIF-1α axis. To determine if this effect also occurred in vivo, we administered HGC-27 cells by subcutaneous injection into different groups of BALB/c nude mice: control (HGC-27 cells); si-NC (negative control HGC-27 cells); and si-LINC01224 (HGC-27 cells with down-regulation LINC01224). We then measured tumor volume two times per week (Fig. 5A). After euthanasia, we removed the tumor tissues and weighed them (Fig. 5B) and took photographs (Fig. 5C). We also used qRT-PCR to measure the expression of LINC01224 in the different xenografts. The results showed that si-LINC01224 group had reduced the expression of LINC01224 (Fig. 5D), and that down-regulation of LINC01224 led to smaller tumors. In addition, the in vivo down-regulation of LINC01224 led to suppression of the PI3K/AKT/mTOR/HIF-1α axis (Fig. 5E) and attenuation of the Warburg effect (Fig. 5F). Thus, the results of these in vivo experiments of a tumor xenograft model are consistent with the results of our in vitro experiments with HGC-27 cells.