3.2 Correlation between F11-AS1 expression and cliniopathological markers of glioma
The analysis results showed that the low expression level in glioma tissues is not correlated to patients’ age (P = 0.514) and gender (P = 0.517), but is partly correlated to tumor number (P = 0.048), tumor size (P = 0.015), KPS score (P = 0.006) and patients’ postoperative recurrence (P = 0.011), see Table 1 for details.
3.3 Gene expression level of F11-AS1 in various cell strains and groups
Figure 2A showed that, compared with normal glial cell HEB, the expression level of F11-AS1 mRNA in glioma cell lines U87, U251, T98G, HS683 and SW1783 was significantly lower (P < 0.001, respectively), and the gene expression of F11-AS1 in U87 and U251 cells were the lowest; compared with the NC group, after F11-AS1 was transfected into U87 and U251 cells, the expression level of F11-AS1 mRNA in F11-AS1 group was significantly higher (P < 0.0001, respectively, Fig. 2B & 2C).
3.4 Impacts of F11-AS1 on proliferation, apoptosis, and cycle of glioma cells
Compared with the NC group, the proliferation rate of U87 and U251 cells in the F11-AS1 group was significantly lower (P < 0.001, respectively, Fig. 3A); the apoptosis rate and G1 cell retention rate increased significantly (P < 0.001, respectively, Fig. 3B-3E).
3.5 Impacts of F11-AS1 on invasion and migration of glioma cells
Compared with the NC group, the number of invading U87 and U251 cells in the F11-AS1 group after transfection into F11-AS1 was significantly lower (P < 0.001, respectively, Fig. 4A & 4B); the observation of the impacts of F11-AS1 on the migration ability of glioma cells by wound healing showed that, compared with the NC group, the 24 h and 48 h wound healing rate of U87 and U251 cells in F11-AS1 group were significantly lower (P < 0.001, respectively, Fig. 4C & 4D).
3.6 Impacts of F11-AS1 on relevant mRNA
The results of RT-qPCR detection showed that, compared with the NC group, in U87 and U251 cell lines, the expression level of miRNA-3146, PI3K, AKT and MMP-9 mRNA in the F11-AS1 group was significantly lower, but the expression level of PTEN and P53 mRNA was significantly higher (P < 0.001, respectively, Fig. 5A & 5B).
3.7 Impacts of F11-AS1 on relevant proteins
The results of WB detection showed that, compared with the NC group, in U87 and U251 cell lines, the expression level of PI3K, AKT and MMP-9 proteins in the F11-AS1 group was significantly lower, but the expression level of PTEN and P53 proteins was significantly higher (P < 0.001, respectively, Fig. 6A & 6B).
3.8 Difference in expression of F11-AS1 and miRNA-3146 among groups
The results of RT-qPCR detection showed that, compared with the NC group, the expression level of F11-AS1 mRNA in U87 and U251 cell lines in F11-AS1, F11-AS1 + miR-NC and F11-AS1 + miR-3146 groups was significantly higher (P < 0.001, respectively, Fig. 7A); the expression level of mRNA-3146mRNA in U87 and U251 cell lines in F11-AS1 and F11-AS1 + miR-NC groups was significantly lower (P < 0.001, respectively, Fig. 7B); compared with the F11 + AS1 + miR-NC group, the expression level of miRNA-3146mRNA was significantly higher (P < 0.001, Fig. 7B).
3.9 Role of miRNA-3146 in F11-AS1’s inhibiting proliferation of glioma cells and promoting apoptosis and cell cycle
The results of MTT detection showed that, compared with the NC group, the cell proliferation rate in U87 and U251 cell lines in F11-AS1 and F11-AS1 + miR-NC groups was significantly lower (P < 0.001, respectively, Fig. 8A); compared with the F11-AS1 + miR-NC group, the cell proliferation rate in the F11-AS1 + miRNA-3146 group was significantly higher (P < 0.001, respectively, Fig. 8A). Flow cytometry was used to detect the apoptosis and cycle of cells in each group. The results showed that, compared with the NC group, the apoptosis rate of U87 and U251 cells in F11-AS1 and F11-AS1 + miR-NC groups without intervention of miRNA-3146 significantly increased (P < 0.001, respectively, Fig. 8B & 8C), accompanied by a significant increase in G1 phase cells and a significant decrease in G2 phase cells (P < 0.001, respectively, Fig. 8D & 8E). After miRNA-3146 was transfected into U87 and U251 cells, compared with the F11-AS1 + miR-NC group, the apoptosis and cycle of cells in the F11-AS1 + miR-3146 group significantly reversed (P < 0.001, respectively, Fig. 8B-8E).
3.10 Role of miRNA-3146 in F11-AS1’s inhibiting invasion and migration of glioma cells
Transwell experiment proved that, compared with the NC group, the number of invading U87 and U251 cells in F11-AS1 and F11-AS1 + miR-NC group was significantly inhibited (P < 0.001, respectively, Fig. 9A & 9B). After the cotransfection of miRNA-3146, compared with the F11-AS1 + miR-NC group, the number of invading U87 and U251 cells in the F11-AS1 + miR-3146 increased significantly (P < 0.001, respectively, Fig. 9A & 9B). Wound healing experiment proved that, compared with the NC group, the 24 h and 48 h wound healing rate in U87 and U251 cell lines in F11-AS1 and F11-AS1 + miR-NC groups was significantly inhibited (P < 0.001, respectively, Fig. 9C & 9D). However, after the transfection of miRNA-3146 into cells, compared with the F11-AS1 + miR-NC group, the 24 h and 48 h wound healing rate in U87 and U251 cell lines in the F11-AS1 + miR-3146 increased significantly (P < 0.001, respectively, Fig. 9C & 9D).
3.11 Expression of relevant genes in groups in RT-qPCR detection
Compared with the NC group, the expression of PTEN and P53mRNA in F11-AS1 and F11-AS1 + miR-NC groups increased significantly, but the expression of PI3K, AKT and MMP-9 genes was significantly inhibited (P < 0.001, respectively, Fig. 10A & 10B); however, after the transfection of miRNA-3146 into cells, compared with the F11-AS1 + miR-NC group, the expression of PTEN and P53mRNA in the F11-AS1 + miR-3146 was significantly inhibited, but the expression of PI3K, AKT and MMP-9 genes increased significantly (P < 0.001, respectively, Fig. 10A & 10B).
3.12 Expression of relevant proteins in groups in WB detection
Compared with the NC group, the expression of PTEN and P53 proteins in F11-AS1 and F11-AS1 + miR-NC groups increased significantly, but the expression of PI3K, AKT and MMP-9 proteins was significantly inhibited (P < 0.001, respectively, Fig. 11A & 11B); however, after the transfection of miRNA-3146 into cells, compared with the F11-AS1 + miR-NC group, the expression of PTEN and P53 proteins in the F11-AS1 + miR-3146 was significantly inhibited, but the expression of PI3K, AKT and MMP-9 proteins increased significantly (P < 0.001, respectively, Fig. 11A & 11B).
3.13 Analysis of correlation among F11-AS1, miRNA-3146 and PTEN
Luciferase report analysis was used to analyze the correlation among F11-AS1, miRNA-3146 and PTEN. The results showed that, F11-AS1 can realize the targeted regulation of miRNA-3146 (P < 0.001, Fig. 12A); the further analysis of the correlation between miRNA-3146 and PTEN showed that miRNA-3146 can realized the targeted regulation of PTEN (P < 0.001, Fig. 12B).