Fig 2: Administration of siRNA-AEG-1/MTDH reduces tumor progression in the rat glioma model(A) The magnification of the striatum with a representative part of the tumor by H & E staining. The siRNA-AEG-1/MTDH-treated rats show significantly smaller tumor volumes compared to untreated control rats (Scale bar: 1 mm, upper); The siRNA-AEG-1/MTDH-treated rats show less histopathological changes of the malignant C6 glioma compared to untreated animals (Scale bar: 100 µm, below). (B) Frontal brains bearing with C6 glioma and T2-weighted MR images after administration for 14 days. MRI is undertaken on a 7.0 T animal MRI scanner using a 38 mm birdcage rat brain quadrature resonator for radiofrequency transmission and reception as previously described. Images from representative glioma-bearing rats show well established tumor after implantation. Image from siRNA-AEG-1/MTDH administration show the reduced tumor zone (white arrow). (C) The percent survival of the glioma-bearing rats. (D) C6 glioma volumes in mm3 of AEG-1/MTDH siRNA-treated rats compared with untreated control rats. (E) Reduced proliferation and increased apoptosis caused by siRNA-AEG-1/MTDH administration. Ten-micrometer thick cryostat sections are obtained and stained for proliferating cell nuclear antigen (PCNA) or terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL), and counterstained with hematoxylin. (F) PCNA and TUNEL density mean and Log10IOD determined by Image Pro-Plus (IPP). IOD indicates integrated optical density.

Fig 3: AEG-1 overexpression enhances TGF-ß1-induced autophagy and EMT in malignant glioma cells(A) Transmission electron microscopy images of U87 malignant glioma cells after transfection with pcDNA3.1-AEG-1/pcDNA3.1. N, nuclear; ER, endoplasmic reticulum; M, mitochondrion; AP, initial autophagic phagophore (isolation membrane); AV1, autophagosomes as autophagic vacuoles 1; AV2, autolysosomes as autophagic vacuoles 2. Scale bars: 500 nm (upper) and 200 nm (lower). (B) U251 and U87 cells are transfected with pcDNA3.1-GFP-MAP-LC3 and pcDNA3.1-AEG-1/pcDNA3.1 using Lipofectamine™ 2000 for 24 hours and then treated with TGF-ß1 (5 ng/ml) or vehicle for another 24 hours. GFP-MAP-LC3-labeled cells are monitored by fluorescence microscopy at 600 × magnification. Scale bar: 200 µm. (C) Quantification of the percentage of cells with focal GFP-MAP-LC3 at the indicated times after treatment. Error bars correspond to SD of three independent experiments (200 cells were counted per experiment). **, ##P < 0.01 compared to vehicle-treated cells. $$, &&P < 0.01 compared to pcDNA3.1-transfected cells. (D and E) U251 and U87 cells are transfected with pcDNA3.1-AEG-1/pcDNA3.1 using Lipofectamine™ 2000 for 24 hours, the expression of AEG-1 and cyclin D1 and MAP-LC3 lipidation and BECN1, E-cadherin, and vimentin are analyzed by western blot. ß-actin is used as internal controls to ascertain equal loading. (F and H) U251 and U87 cells are transfected with pcDNA3.1-AEG-1/pcDNA3.1 using Lipofectamine™ 2000 for 24 hours and then treated with TGF-ß1 (5 ng/ml) or vehicle for another 24 hours. The expression of AEG-1, MAP-LC3 lipidation and BECN1, E-cadherin, vimentin, Snail, Slug and Twist are analyzed by western blot. ß-actin is used as internal controls to ascertain equal loading. (G and I) Representative quantitative data of densitometric analyses. Data represent the mean ± SD of three different experiments. *P < 0.05 and **P < 0.01 compared to vehicle-treated cells or si Control-transfected cells.

Fig 4: miR-3662 inhibitor rescues the inhibitory effect of shMTDH on osteosarcoma cells. (A) Flow cytometry was used to detect the apoptotic rate of the four groups in the recovery experiment and to verify that miR-3662 could reverse the apoptotic effect of MTDH on osteosarcoma cells. (B) Transwell assays were used to detect the migration of four groups in the recovery experiment and to verify that miR-3662 could reverse the migration effect of MTDH on osteosarcoma cells. (C) Transwell assay was used to detect the invasion of the four groups in the recovery experiment and to verify that miR-3662 could reverse the invasion effect of MTDH on osteosarcoma cells. (D) Western blotting was used to detect the expression of EMT-related proteins (E-cadherin, N-cadherin, and Vimentin) in four groups and to verify that miR-3662 could reverse the effect of MTDH on the EMT of osteosarcoma cells. Data are presented as the mean ± standard deviation of three independent experiments, ***p < 0.001.

Fig 5: Circ-03955 acts as a ceRNA by sponging miR-3662 and indirectly regulates MTDH expression. (A) The differential expression of circ-03955 in MG-63 cytoplasm and nucleus of osteosarcoma cells verified by qRT-PCR. (B) Analysis of the correlation between circ-03955 and miR-3662 expression in osteosarcoma. (C) qRT-PCR was used to detect the effect of OE-circ-03955 and sh-circ-03955 on the expression of miR-3662 in osteosarcoma MG-63 cells 48 hours after transfection. (D) Western blotting was used to detect the effects of OE-circ-03955 and sh-circ-03955 on the expression of MTDH protein in osteosarcoma cells MG-63 and U2OS at 48 hours after transfection. (E) The expression of MTDH in osteosarcoma MG-63 and U2OS was detected by qRT-PCR after 48 hours of transfection. Western blotting was used to detect the expression of MTDH protein in osteosarcoma MG-63 and U2OS at 48 hours after transfection. (F) The binding relationship between circ-03955 and miR-3662 was detected by the dual luciferase reporting system. (G) The binding relationship between miR-3662 and MTDH was detected by the dual luciferase reporting system. (H) Ago2-RIP assays were used to detect the relationship between circ-03955 and miR-3662. (I) Western blot assays were used to verify that circ-03955 reverses the inhibitory effect of miR-3662 on MTDH. Data are presented as the mean ± standard deviation of three independent experiments, NS, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.