Fig 1: PLP2 is a direct target of miR-765. (A) and (B) PLP2 was negatively correlated with miR-765 in TCGA-KIRC and clinical ccRCC samples. (C-F) qRT-PCR and western blotting analysis of PLP2 expression in miR-765 mimic-, inhibitor- and NC-transfected A498 and Caki-1 cells. (G) and (H) Luciferase reporter assays showing approximately 50% decreased reporter activity after transfection of the wild-type PLP2 3'UTR reporter construct into A498 and Caki-1 cells in combination with miR-765 mimics. Data indicate the means ± SEM. *P<0.05; **P<0.01 (t-test).
Fig 2: PLP2 is involved in the biological pathogenesis of ccRCC. (A-D) PLP2 is upregulated in ccRCC tissues and renal cancer cells, as assessed by qRT-PCR and western blotting analysis. (E) Gene set enrichment analysis (GSEA) was used to compare high and low PLP2 expression groups in the TCGA database. Enrichment curves are shown for activated gene sets related to EMT and the G2M checkpoint. (F) Silencing of PLP2 with small RNA interference technology (si-PLP2-1 and si-PLP2-2). (G) si-PLP2 significantly repressed the migration of A498 cells. (H) GSEA enrichment curves showed that low PLP2 expression was associated with fatty acid triacylglycerol metabolism, lipid catabolic processes and neutral lipid metabolic processes. (H) and (I) Silencing of PLP2 significantly promoted neutral lipid catabolic processes and eliminated abnormal lipid accumulation in A498 cancer cells. Original magnification 200x. Data indicate the means ± SEM. **P<0.01, *** P < 0.001 (t-test).
Fig 3: Target genes of miR-765 predicted with bioinformatics analysis in ccRCC. (A) Bioinformatic prediction of the top 15 candidate mRNAs targeting miR-765 via the TargetScan and miRDB platforms. (B) Column chart depicting the expression of the 6 target genes in TCGA-KIRC. (C) and (D) Overall survival (OS) and disease-free survival (DFS) analysis showed 3 target genes in TCGA-KIRC. (E) and (F) Univariate and multivariate survival analysis of OS and DFS indicated that high PLP2 expression was a potential independent prognostic factor in ccRCC patients. Data indicate the means ± SEM.****P < 0.0001 (t-test).
Fig 4: MiR-765 inhibits the malignant potential of renal cancer cells and eliminates lipids in renal cancer cells via PLP2 in vivo. (A), (B) and (C) Tumour size, weight and volume curves from the xenograft formation assay in which Caki-1 cells were subcutaneously injected into the flanks of mice and grown for 30 days. Tumour size was measured every 3 days. (D) PLP2 reversed the effect of miR-765 on tumour liver metastasis. (E) Oil Red O (ORO) staining showed PLP2 reversed the effect of miR-765 on tumour lipid accumulation. (F) PLP2 reversed the angiogenesis-inhibiting effect with vascular endothelial growth factor A (VEGFA) and tumour metastasis ability by Ki67 of miR-765. Original magnification x200. Data indicate the means ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001 (t-test).
Fig 5: PLP2 reverses the function of miR-765 in renal cancer. (A) and (B) The expression of miR-765 and PLP2 was measured by qRT-PCR in A498 and Caki-1 cells. (C) The expression of PLP2 was measured by western blotting in A498 cells. (D) PLP2 overexpression impaired the effects of the miR-765 mimic on cell proliferation in A498 and Caki-1 cells. (E) and (F) PLP2 overexpression impaired the effects of the miR-765 mimic on migration and invasion in A498 cells. (G) and (H) PLP2 overexpression impaired the effects of the miR-765 mimic on lipid elimination in A498 cells. Original magnification 200x. Data indicate the means ± SEM. *P<0.05; **P<0.01, *** P < 0.001 (t-test).
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