Journal: Scientific Reports

Article Title: Loss of RBMS1 as a regulatory target of miR-106b influences cell growth, gap closing and colony forming in prostate carcinoma

doi: 10.1038/s41598-020-75083-9

Figure Lengend Snippet: RBMS1 as putative target for miR-106b is downregulated in PCa cell lines and primary prostate tumour tissue. ( A ) The predicted binding site for miR-106b inside the RBMS1 3′UTR including the mutation site of its seed sequence and ( B ) the secondary structure of the miR-106b-RBMS1 hybridization created using RNAhybrid online tool ( https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid ). ( C ) Expression levels of RBMS1 in DU145 and LNCaP cells relative to primary PNF-08 cells. Total mRNA was extracted and the relative levels of RBMS1 was determined by qRT-PCR. Levels were quantified relative to the amounts observed in PNF-08 cells. ( D ) In total RNA, extracted from 5 pairs of primary CaP (tumor) and corresponding non-tumor (normal) prostate tissues, the relative expression of RBMS1 was determined by qRT-PCR. The data is shown as mean ± SEM performed in triplicates.

Article Snippet: The gene specific primer/probe combinations used were HPRT1 (Hs99999909_m1, Thermo Scientific) and RBMS1 (Hs00249930_s1, Thermo Scientific, Darmstadt, Germany).

Techniques: Binding Assay, Mutagenesis, Sequencing, Hybridization, Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Loss of RBMS1 as a regulatory target of miR-106b influences cell growth, gap closing and colony forming in prostate carcinoma

doi: 10.1038/s41598-020-75083-9

Figure Lengend Snippet: Response of RBMS1 3′UTR and protein expression towards miR-106b. ( A ) The RBMS1 3′UTR fragment was cloned behind the luciferase reporter gene of the pMIR vector. ( B ) The reporter gene construct was expressed with the miRNA expression constructs of the miR-17 family or with the empty pSG5 vector as control in the indicated combinations. Results represent the mean of at least four independent experiments performed in duplicates. The dashed line represents the luciferase activity of the empty luciferase reporter plasmid with the empty pSG5 vector which was set to 100% (***p < 0.001). ( C ) Reporter gene vector containing mutated miR-106b binding site in the RBMS1 3′UTR was co-expressed with miR-106b expression plasmid. Luciferase activity of the reporter vector without miRNA expression was set to 100% (***p < 0.001). ( D ) DU145 or LNCaP cells were transfected either with control vector or miR-106b expression vector. 48 h post-transfection, the protein expression of RBMS1 was determined by Western blot using ß-actin as loading control. Representative cropped Western Blots of RBMS1 detection after miRNA overexpression in DU145 and LNCaP cells from four independent experiments. Full-length blot is presented in Supplementary Figure S5. ( E ) For determination of relative RBMS1 downregulation, each four Western Blots of DU145 and LNCaP cells transfected either with control vector or miR-106b expression vector were densitometrically quantified in relation to the corresponding ß-actin band as loading control. The data is shown as mean ± SEM whereas the control lane intensity was set to 1.

Article Snippet: The gene specific primer/probe combinations used were HPRT1 (Hs99999909_m1, Thermo Scientific) and RBMS1 (Hs00249930_s1, Thermo Scientific, Darmstadt, Germany).

Techniques: Expressing, Clone Assay, Luciferase, Plasmid Preparation, Construct, Control, Activity Assay, Binding Assay, Transfection, Western Blot, Over Expression

Journal: Scientific Reports

Article Title: Loss of RBMS1 as a regulatory target of miR-106b influences cell growth, gap closing and colony forming in prostate carcinoma

doi: 10.1038/s41598-020-75083-9

Figure Lengend Snippet: RBMS1 has an inhibiting effect on cell growth of PCa cells. DU145 or LNCaP cells were transfected either with RBMS1 expression vectors or RBMS1 targeting siRNAs and their corresponding controls, respectively, and cells were seeded in a limited cell number. Cell growth was determined by automated cell counting of parallel experiments at 24, 48 and 72 h post-transfection for three independent experiments. The data is shown as mean ± SEM (*p < 0.05; ***p < 0.001).

Article Snippet: The gene specific primer/probe combinations used were HPRT1 (Hs99999909_m1, Thermo Scientific) and RBMS1 (Hs00249930_s1, Thermo Scientific, Darmstadt, Germany).

Techniques: Transfection, Expressing, Cell Counting

Journal: Scientific Reports

Article Title: Loss of RBMS1 as a regulatory target of miR-106b influences cell growth, gap closing and colony forming in prostate carcinoma

doi: 10.1038/s41598-020-75083-9

Figure Lengend Snippet: Overexpression of RBMS1 leads to a reduced colony forming ability. DU145 or LNCaP cells were transfected either with RBMS1 expression vectors or RBMS1 targeting siRNAs and their corresponding controls, respectively, and cells were seeded in a limited cell number. 10 days (DU145, ( A )) or 15 days (LNCaP, ( B )) after seeding, colonies were stained with crystal violet. Colony formation was quantified by densitometry analyses. Data show the mean and ± SEM of the densitometry analysis of three independent experiments (***, p < 0.001).

Article Snippet: The gene specific primer/probe combinations used were HPRT1 (Hs99999909_m1, Thermo Scientific) and RBMS1 (Hs00249930_s1, Thermo Scientific, Darmstadt, Germany).

Techniques: Over Expression, Transfection, Expressing, Staining

Journal: Scientific Reports

Article Title: Loss of RBMS1 as a regulatory target of miR-106b influences cell growth, gap closing and colony forming in prostate carcinoma

doi: 10.1038/s41598-020-75083-9

Figure Lengend Snippet: Reduction of gap closing by RBMS1 in PCa cells. DU145 ( A , C ) or LNCaP cells ( E , G ) were transfected either with RBMS1 expression vectors or RBMS1 targeting siRNAs and their corresponding controls, respectively, and cells were seeded into a ibidi Culture-Insert system to create a defined cell-free gap. Gap closing was monitored for 36 h (DU145) or 96 h (LNCaP) in three independent experiments. For each experiment, the gap width after removing the ibidi culture insert at 0 h was normalized to 0% and three independent visual fields per time point and replicate were examined (*p < 0.05; ***p < 0.001). ( B , D , F , H ) Representive pictures at different time points are depicted.

Article Snippet: The gene specific primer/probe combinations used were HPRT1 (Hs99999909_m1, Thermo Scientific) and RBMS1 (Hs00249930_s1, Thermo Scientific, Darmstadt, Germany).

Techniques: Transfection, Expressing

Journal: Epigenetics

Article Title: Suppressing circIDE/miR-19b-3p/RBMS1 axis exhibits promoting-tumour activity through upregulating GPX4 to diminish ferroptosis in hepatocellular carcinoma

doi: 10.1080/15592294.2023.2192438

Figure Lengend Snippet: RBMS1 is downregulated in HCC and low expression of RBMS1 correlates with poor HCC patient survival. (a) RT-qPCR analysis of RBMS1 expression in 25 cases paired HCC tissues and adjacent liver tissues. (b) Comparison of RBMS1 expression between patients with T stage 1–2 (T1+T2, n =25) and T stage 3–4 (T3+T4, n =12), detected by RT-qPCR. (c) Comparison of RBMS1 expression between patients with TNM stage I-II ( n =23) and TNM stage III-IV ( n =14), detected by RT-qPCR. (d) Western blotting analysis of RBMS1 expression in HCC tissues and normal liver tissues. ( e, f) Representative images and statistics of IHC staining of RBMS1 in HCC tissue of T stage T1 and T3 and clinical TNM stage I and III. Scale bar, 50 µm. (g) Kaplan–Meier analysis of correlation between RBMS1 expression and OS, RFS. (h) Forrest plot of univariate or multivariate Cox proportional hazard regression indicated the impact of different characteristics on OS and RFS. Data are denoted as mean ± SD from three independent experiments.

Article Snippet: Blocking the sections in PBS with 5% BSA for 25 min at room temperature and then incubating the sections with antibodies specific for RBMS1 (1:100, Cat#11061–2-AP, Proteintech), GPX4 (1:200, Cat#A1933, ABclonal), 4HNE (1:100, Cat#MBS808701), and Ki67 (1:100, Cat#16667, Abcam) overnight.

Techniques: Expressing, Quantitative RT-PCR, Comparison, Western Blot, Immunohistochemistry

Journal: Epigenetics

Article Title: Suppressing circIDE/miR-19b-3p/RBMS1 axis exhibits promoting-tumour activity through upregulating GPX4 to diminish ferroptosis in hepatocellular carcinoma

doi: 10.1080/15592294.2023.2192438

Figure Lengend Snippet: RBMS1 overexpression promotes ferroptosis in HCC cells. (a) RT-qPCR analysis for the expression of RBMS1 in normal liver cell line and HCC cell lines. (b) RT-qPCR analysis of efficiency of RBMS1 overexpression in HepG2 and Hep3B cell lines, compared to empty vector. (c) Western blotting of analysis of RBMS1, GPX4, and SLC7A11 expression in HepG2 and Hep3B as indicated treatments. (d) Half-life of GPX4 mRNA after treatment with ActD in HepG2 and Hep3B cells with or without RBMS1 overexpression. (e) Schematic of GPX4 luciferase reporter plasmids. GPX4-RLuc-FL (the full length of 3’-UTR); GPX4-RLuc-D1 (1–69 nt region of 3’UTR); and GPX4-RLuc-D2 (70–139 nt region of 3’UTR). (f-g) The relative luciferase activity of HepG2 and Hep3B cells with or without RBMS1 overexpression after transfecting GPX4-RLuc-FL (f) , GPX4-RLuc-D1, and GPX4-RLuc-D2 (g) luciferase reporter vectors. (h) Representative images of H2DCFDA staining (green) and quantification of ROS level in HepG2 and Hep3B cells. Scale bar, 5 µm. (i) The assessment of MDA level in HepG2 and Hep3B cells. (j) Relative cell viability of HepG2 and Hep3B cells with or without RBMS1 overexpression after treated with erastin, ferrostatin-1, or both combined compared to corresponding control group. (k) Immunohistochemistry staining and correlation analysis of protein levels of RBMS1, GPX4, and 4HNE in clinical HCC samples. Scale bar, 50 µm. Data are denoted as mean ± SD from three independent experiments.

Article Snippet: Blocking the sections in PBS with 5% BSA for 25 min at room temperature and then incubating the sections with antibodies specific for RBMS1 (1:100, Cat#11061–2-AP, Proteintech), GPX4 (1:200, Cat#A1933, ABclonal), 4HNE (1:100, Cat#MBS808701), and Ki67 (1:100, Cat#16667, Abcam) overnight.

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Plasmid Preparation, Western Blot, Luciferase, Activity Assay, Staining, Control, Immunohistochemistry

Journal: Epigenetics

Article Title: Suppressing circIDE/miR-19b-3p/RBMS1 axis exhibits promoting-tumour activity through upregulating GPX4 to diminish ferroptosis in hepatocellular carcinoma

doi: 10.1080/15592294.2023.2192438

Figure Lengend Snippet: GPX4 rescues the inhibiting proliferation effect of HCC cells induced by RBMS1 overexpression in vitro and in vivo . (a) Western blotting analysis of RBMS1 and GPX4 expression in RBMS1-overexpressed HepG2 and Hep3B cells with or without GPX4 overexpression, compared to vector control. (b-d) Effects of RBMS1 overexpression with or without GPX4 overexpression on the proliferation of HepG2 and Hep3B cells were, respectively, assessed by CCK-8 assay (b) , colony formation assay (c), and EdU staining (d) , scale bar, 20 µm. (e-g) Hepa 1–6 cells (transduced with empty vector lentivirus, or vector overexpressing RBMS1 withor without overexpressing GPX4) were subcutaneously injected into the right flank of C57BL/6 mice. RT-qPCR analysis for the expression of RBMS1 and GXP4 in Hepa 1–6 cells (e) . Tumor growth was monitored by Xenogen IVIS 200 imaging system (f) . Western blotting analysis for the expression of RBMS1, GPX4, and 4HNE in Hepa 1–6 cells (g) . Data are denoted as mean ± SD from three independent experiments.

Article Snippet: Blocking the sections in PBS with 5% BSA for 25 min at room temperature and then incubating the sections with antibodies specific for RBMS1 (1:100, Cat#11061–2-AP, Proteintech), GPX4 (1:200, Cat#A1933, ABclonal), 4HNE (1:100, Cat#MBS808701), and Ki67 (1:100, Cat#16667, Abcam) overnight.

Techniques: Over Expression, In Vitro, In Vivo, Western Blot, Expressing, Plasmid Preparation, Control, CCK-8 Assay, Colony Assay, Staining, Transduction, Injection, Quantitative RT-PCR, Imaging

Journal: Epigenetics

Article Title: Suppressing circIDE/miR-19b-3p/RBMS1 axis exhibits promoting-tumour activity through upregulating GPX4 to diminish ferroptosis in hepatocellular carcinoma

doi: 10.1080/15592294.2023.2192438

Figure Lengend Snippet: MiR-19b-3p inhibits ferroptosis and potentiates proliferation of HCC cells by repressing RBMS1 expression. (a) Prediction of miRNAs targeting RBMS1 by miRmap, PicTar, TargetScan, and PITA databases. (b) The expression of miR-19a-3p, miR-19b-3p, miR-124-3p, miR-129-5p, miR-376-3p, miR-383-5p, miR-487a-3p, and miR-501-3p in HCC and normal liver tissues through TCGA database. (c, d) RT-qPCR and western blotting analysis for the expression of RBMS1 in HepG2 and Hep3B cells after transfecting miR-19b-3p inhibitor. (e) Schematic diagram of luciferase reporter vector containing wild-type (WT) or mutant (MUT) putative miR-19b-3p binding sites of the 3’ UTR of RBMS1. (f) The dual luciferase reporter assays demonstrated that miR-19b-3p inhibitor influenced the luciferase activity of luciferase reporter vectors containing WT or MUT 3’UTR of RBMS1. (g, h) RBMS1 knockdown plasmids were added to the basis of miR-19b-3p inhibitor transfection in HepG2 and Hep3B cells. The ROS and MDA level were measured as indicated treatments. (i) Proliferation of HepG2 and Hep3B cells as indicated treatments was evaluated by colony formation assay. (j) RBMS1, GPX4, and 4HNE expression in HepG2 and Hep3B as indicated treatments was evaluated by western blotting. Data are denoted as mean ± SD from three independent experiments.

Article Snippet: Blocking the sections in PBS with 5% BSA for 25 min at room temperature and then incubating the sections with antibodies specific for RBMS1 (1:100, Cat#11061–2-AP, Proteintech), GPX4 (1:200, Cat#A1933, ABclonal), 4HNE (1:100, Cat#MBS808701), and Ki67 (1:100, Cat#16667, Abcam) overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Plasmid Preparation, Mutagenesis, Binding Assay, Activity Assay, Knockdown, Transfection, Colony Assay

Journal: Epigenetics

Article Title: Suppressing circIDE/miR-19b-3p/RBMS1 axis exhibits promoting-tumour activity through upregulating GPX4 to diminish ferroptosis in hepatocellular carcinoma

doi: 10.1080/15592294.2023.2192438

Figure Lengend Snippet: CircIDE enhances ferroptosis and attenuates proliferation of HCC cells via miR-19b-3p/RBMS11 axis. (a-e) After circIDE overexpression, HepG2 and Hep3B cells were transfected with miR-19b-3p mimic or RBMS1 knockdown plasmid, respectively. (a, b) Western blotting analysis of RBMS1, GPX4, and 4HNE expression in HepG2 and Hep3B as indicated treatments. (c, d) the assessment of ROS and MDA level in HepG2 and Hep3B cells as indicated treatments. (e) Proliferation of HepG2 and Hep3B cells as indicated treatments was evaluated by EdU staining. Scale bar, 20 µm. Data are denoted as mean ± SD from three independent experiments.

Article Snippet: Blocking the sections in PBS with 5% BSA for 25 min at room temperature and then incubating the sections with antibodies specific for RBMS1 (1:100, Cat#11061–2-AP, Proteintech), GPX4 (1:200, Cat#A1933, ABclonal), 4HNE (1:100, Cat#MBS808701), and Ki67 (1:100, Cat#16667, Abcam) overnight.

Techniques: Over Expression, Transfection, Knockdown, Plasmid Preparation, Western Blot, Expressing, Staining

Journal: Epigenetics

Article Title: Suppressing circIDE/miR-19b-3p/RBMS1 axis exhibits promoting-tumour activity through upregulating GPX4 to diminish ferroptosis in hepatocellular carcinoma

doi: 10.1080/15592294.2023.2192438

Figure Lengend Snippet: GPX4 overexpression restores circIDE induced inhibition of tumor growth. (a, b) RT-qPCR analysis of circIDE and GPX4 expression of circIDE overexpression of Hepa 1–6 cells with or without GPX4 overexpression, compared to vector control. (c) Tumor growth was monitored by Xenogen IVIS 200 imaging system. (d) Representative IHC staining and corresponding statistics for RBMS1, GPX4, 4HNE and Ki67 expression in the xenografts tumors. Scale bars, 20 µm. Data are denoted as mean ± SD from three independent experiments.

Article Snippet: Blocking the sections in PBS with 5% BSA for 25 min at room temperature and then incubating the sections with antibodies specific for RBMS1 (1:100, Cat#11061–2-AP, Proteintech), GPX4 (1:200, Cat#A1933, ABclonal), 4HNE (1:100, Cat#MBS808701), and Ki67 (1:100, Cat#16667, Abcam) overnight.

Techniques: Over Expression, Inhibition, Quantitative RT-PCR, Expressing, Plasmid Preparation, Control, Imaging, Immunohistochemistry

Journal: iScience

Article Title: DNA-guided photoactivatable probe-based chemical proteomics reveals the reader protein of mRNA methylation

doi: 10.1016/j.isci.2021.103046

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti- RBMS1 , ABclonal , Cat#A3079; RRID: AB_2764882.

Techniques: Recombinant, Purification, Bicinchoninic Acid Protein Assay, Binding Assay, Plasmid Preparation