Fig 2: RASAL2 regulates PCa cell proliferation via PI3K/AKT/cyclin D1 signalling.A Western blotting analysis of AKT, p-AKT and cyclin D1 expression levels in established cell lines. B Western blotting analysis of AKT, p-AKT and cyclin D1 expression in the LNCaP cell line treated with an AKT inhibitor. C Flow cytometry analysis of AKT inhibitor-treated LNCaP cell lines. D Western blotting analysis of cyclin D1 expression levels in the siCCND1-treated LNCaP cell line. Cell viability (E), colony formation (F), cell cycle (G) and cell proliferation (H) assays in siCCND1-treated LNCaP cell lines.

Fig 3: miR135b-5p Regulates RASAL2(A) BxPC3 cells were treated with 10 μM sulforaphane (SF) or were left untreated (CO). Twenty-four hours later, mRNA was harvested, followed by gene expression profiling in triplicate using the GeneChip miRNA 4.0 Array. A heatmap of candidate mRNAs was created, which combined the array and database results. The most significant upregulated candidate gene RASAL2 is marked. The scale from −0.7 to 0.7 marks the intensity of differential regulation of genes: high expression (red), middle expression (black), and low expression (green). (B) BxPC-3, PANC-1, and AsPC-1 cells were treated as described above, and RNA was harvested. The fold change in RASAL2 expression was determined by qRT-PCR and normalized to GAPDH. The controls were set to 1. (C) The cells were treated as described above, followed by harvest of the proteins 24 h later and examination of RASAL2 protein expression by western blot analysis. The expression of β-actin served as a control for equal conditions. The band intensities (pixel intensities) were measured using ImageJ and are shown in the diagram on the left, after normalization to β-actin. Means ± SD are shown; *p < 0.05, **p < 0.01.

Fig 4: Correlation of RASAL2 and cyclin D1 in clinical specimens.A Immunohistochemical analysis of the expression levels and correlation of RASAL2 and cyclin D1 in clinical samples of PCa (n = 19). B Molecular correlation analysis of RASAL2 and cyclin D1 (CCND1) from the GEO database (GSE29079, GSE8511 and GSE62293). C Correlation analysis of RASAL2 and cyclin D1 (CCND1) from TCGA database.

Fig 5: Expression of RASAL2 in PCa cell lines and the effects of RASAL2 on the proliferation of PCa cell lines.A Western blotting analysis of RASAL2 expression levels in human nontumor prostate and PCa cell lines. B, C Quantitative real-time RT–PCR and Western blotting analysis of RASAL2 expression in 22RV1 or PC3 cell lines transfected with RASAL2 shRNAs and shControl and LNCaP cell lines infected with RASAL2 lentivirus and negative control. D Cell activity assay for established cell lines. E Clone formation experiments with quantitative results on established cell lines. F Cell proliferation experiments on established cell lines. G, H Cell cycle analysis of PC3 and LNCaP cell lines using flow cytometry.