Fig 1: MicroRNA (MiR)-664 suppresses PLP2 expression via direct targeting of the 3'UTR. (A) Predicted miR-664 target sequence in the 3'UTR of PLP2 (PLP2-3'UTR) and PLP2-3'UTR mutant containing 4 altered nucleotides in the seed sequence (PLP2-3'UTR-mut). (B) Western blot analysis of PLP2, p-AKT, AKT, p21, cyclin D1, and PCNA expression by miR-664 overexpression or miR-664 inhibition. ß-actin served as the loading control. (C) Western blot analysis of GFP expression in indicated cells. (D) Luciferase assay of indicated cells transfected with the pGL3-PLP2-3'UTR reporter with increasing amounts (10, 50 nM) of miR-664 mimic or miR-664 inhibitor. (E) Luciferase assay of indicated cells transfected with pGL3-PLP2-3'UTR or pGL3-PLP2-3'UTR-mut reporter with increasing amounts (10, 50 nM) of miR-664 oligonucleotides. (F) Analysis of expression (up) and correlation (down) of miR-664 and PLP2 expression in 8 freshly collected human CMM tissues. Each bar represents the mean ± standard deviation of 3 independent experiments. *P < 0.05. 3'UTR = untranslated region, AKT = protein kinase B, CMM = cutaneous malignant melanoma, p-AKT = AKT phosphorylation, PLP2 = proteolipid protein 2.
Fig 2: Exosomes originated from CD45RO-CD8+ T cells partly suppress UCEC development in a miR-765/PLP2-dependent manner. After Ishikawa cells were treated with estrogen and/or CD45RO-CD8+T cell-derived exosomes for 24 h, A Levels of miR-765 were tested by RT-qPCR. B-C Levels of PLP2 were tested by RT-qPCR and western blotting. D Cell viability was measured by the CCK-8 assay at 12 h, 24 h, 36 h or 48 h. E Ki67 expression was determined with by flow cytometry. F Levels of EMT related markers (Vimentin and E-cadherin) were measured by western blotting and were quantified in (G-H), respectively. I Cell invasion was detected by the transwell assays. Scale bars: 100 µm. J Ultimate tumor volume of Ishikawa cells xenografts in mice were measured. K Survival curves of Ishikawa cells xenografts-bearing mice was plotted. Data were presented as mean ± SEM and analyzed by t test or ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, NS: no significance.
Fig 3: PLP2 promotes the EMT process and invasion of UCEC. A Cell viability of NC or siPLP2 Ishikawa (up) and KLE (down) cells was measured by the CCK-8 assay at 12 h, 24 h, 36 h, 48 h. B mRNA levels of EMT related markers (COL1A, COL3A1, FN1, CDH1, CDH2, S100A, MMP2, MMP9, SNAIL, VIMENTIN, ZEB1, TJP1) in NC or siPLP2 Ishikawa and KLE cells by RT-qPCR. C Western blotting images of EMT-related markers (E-cadherin and Vimentin) in NC or siPLP2 Ishikawa and KLE cells. D Cell invasion was detected in NC or siPLP Ishikawa and KLE cells at 24 h. Scale bars: 100 µm. E In vivo imaging of tumor metastasis after injection of NC or siPLP2 transfected Ishikawa cells in mice. F Survival curves of NC and siPLP2 Ishikawa cells xenografts-bearing mice. G Kaplan-Meier plots were drawn to visualize the survival outcomes for UCEC patient with high or low level of PLP2 (p = 0.0028).
Fig 4: Hematoxylin and eosin staining of (A) normal brain tissue, (B) pilocytic astrocytoma, (C) diffuse astrocytoma, (D) anaplastic astrocytoma, and (E) glioblastome multiforme. IHC analysis of PLP2 in (F) normal brain tissue, (G) pilocytic astrocytoma, (H) diffuse astrocytoma, (I) anaplastic astrocytoma, and (J) glioblastoma. (Original magnification × 400).
Fig 5: (A) Influence of the possible signaling pathway in the LN229 glioma cell line after siPLP2 and siRNA-A (NC)transfection. p-p38 and p-ERK showed a significant decrease after knockdown PLP2 expression. (*** p < 0.001); (B) Assessment of tumor migration ability in GBM8401 and LN229 glioma cell lines after siPLP2 and NC transfection. Glioma cells with a PLP2 knockdown revealed slow tumor migration after 16 h. (*** p < 0.001).
Supplier Page from Abcam for Anti-PLP2 antibody [EPR14238(B)]