Journal: Molecular Therapy. Nucleic Acids

Article Title: FOXM1-Mediated LINC-ROR Regulates the Proliferation and Sensitivity to Sorafenib in Hepatocellular Carcinoma

doi: 10.1016/j.omtn.2019.04.008

Figure Lengend Snippet: LINC-ROR Acts as a Competing Endogenous RNA for miR-876-5p to Regulate FOXM1 Expression (A) RIP experiments revealed that LINC-ROR and FOXM1 mRNA coexisted in the anti-Ago2 complex in the cell cytoplasm. IgG was used as the negative control. (B) A Venn diagram shows the overlapping miRNAs that are predicted to bind with both LINC-ROR and the 3′ UTR of FOXM1 mRNA. (C) The expression levels of the miRNAs were detected with quantitative real-time PCR in a normal liver cell line and six HCC cell lines. (D) The expression levels of the six miRNAs were measured individually by quantitative real-time PCR methods in LINC-ROR-transfected HepG2 cells. (E) Quantitative real-time PCR and (F) western blot analysis of FOXM1 expression after all six miRNA mimics were introduced. (G) Binding sequences of LINC-ROR and miR-876-5p based on bioinformatics analysis and schematic constructions of WT-Luc (LINC-ROR 3′ partial region), Mut1-Luc, Mut2-Luc, and Mut1/2-Luc. (H) Dual-luciferase reporter assays were performed to examine the potential combination of LINC-ROR and miR-876-5p. (I) WT and mutant binding sites of miR-876-5p and 3′ UTR of FOXM1 mRNA from StarBase ( http://starbase.sysu.edu.cn/ ). (J) Dual-luciferase reporter assays were conducted to confirm the association of FOXM1 and LINC-ROR or miR-876-5p and FOXM1. All data are presented as the mean ± SD of three independent experiments. The p values represent comparisons between groups (*p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant).

Article Snippet: Has-miR-876-5p mimics and miR-NC/mimics and has-miR-876-5p inhibitor and miR-NC/inhibitor were purchased from Ribobio (Guangzhou, China).

Techniques: Expressing, Negative Control, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Binding Assay, Luciferase, Mutagenesis

Journal: Molecular Therapy. Nucleic Acids

Article Title: FOXM1-Mediated LINC-ROR Regulates the Proliferation and Sensitivity to Sorafenib in Hepatocellular Carcinoma

doi: 10.1016/j.omtn.2019.04.008

Figure Lengend Snippet: miR-876-5p Attenuated Proliferation and Promoted Apoptosis and Chemosensitivity of HCC Cells (A) A correlation plot of IC 50 of sorafenib and relative miR-876-5p levels in six HCC cell lines. (B) CCK-8 assays were performed to detect the proliferation of HepG2 cells transfected with miR-NC/inhibitor or miR-876-5p/inhibitor. (C) Quantification of colony-formation ability of HepG2 cells treated with miR-NC/inhibitor or miR-876-5p/inhibitor, with sorafenib or DMSO added. (D) Quantification of the total apoptosis rate of HepG2 cells after transfection of miR-NC/inhibitor or miR-876-5p/inhibitor, supplemented with sorafenib or DMSO. The percentage represents the sum of both the Annexin- and 7-AAD-positive populations. (E) CCK-8 assays were performed to detect the proliferation of HCCLM3 cells after transfection with miR-NC/mimics or miR-876-5p/mimics. (F) Quantification of colony-formation ability of HCCLM3 cells treated with miR-NC/mimics or miR-876-5p/mimics, with sorafenib or DMSO added. (G) Quantification of total apoptosis rate of HCCLM3 cells after transfection with miR-NC/mimics or miR-876-5p/mimics, supplemented with sorafenib or DMSO. The percentage represents a sum of the Annexin- and 7-AAD-positive populations. All data are presented as the mean ± SD of three independent experiments. The p values represent comparisons between groups (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Has-miR-876-5p mimics and miR-NC/mimics and has-miR-876-5p inhibitor and miR-NC/inhibitor were purchased from Ribobio (Guangzhou, China).

Techniques: CCK-8 Assay, Transfection

Journal: Molecular Therapy. Nucleic Acids

Article Title: FOXM1-Mediated LINC-ROR Regulates the Proliferation and Sensitivity to Sorafenib in Hepatocellular Carcinoma

doi: 10.1016/j.omtn.2019.04.008

Figure Lengend Snippet: FOXM1 Suppression, Mediated by miR-876-5p, Was Involved with LINC-ROR-Mediated Sorafenib Tolerance (A) Quantitative real-time PCR and (B) western blotting were utilized to examine FOXM1 mRNA and protein levels in HCCLM3 cells, transfected with shROR or co-transfected with shROR and miR-876-5p inhibitor, or in HepG2 cells, transfected with LINC-ROR or co-transfected with LINC-ROR and miR-876-5p mimics. (C) CCK-8 assays were carried out to detect the proliferation of HCCLM3 cells transfected with shROR or co-transfected with shROR and miR-876-5p inhibitor or co-transfected with shROR, miR-876-5p inhibitor, and shFOXM1. (D) Quantification of colony-formation ability of HCCLM3 cells transfected with shROR or co-transfected with shROR and miR-876-5p inhibitor or co-transfected with shROR, miR-876-5p inhibitor, and shFOXM1 under sorafenib treatment. (E) Quantification of total apoptosis rate of HCCLM3 cells treated with shROR or co-transfected with shROR and miR-876-5p inhibitor or co-transfected with shROR, miR-876-5p inhibitor, and shFOXM1, with the addition of DMSO or sorafenib. The percentage represents a sum of the Annexin- and 7-AAD-positive populations. (F) CCK-8 assays were carried out to detect the proliferation of HepG2 cells transfected with LINC-ROR or co-transfected with LINC-ROR and miR-876-5p mimics or co-transfected with LINC-ROR, miR-876-5p mimics, and FOXM1. (G) Quantification of colony-formation ability of HepG2 cells transfected with LINC-ROR or co-transfected with LINC-ROR and miR-876-5p mimics or co-transfected with LINC-ROR, miR-876-5p mimics, and FOXM1 after sorafenib treatment. (H) Quantification of total apoptosis rate of HepG2 cells transfected with LINC-ROR or co-transfected with LINC-ROR and miR-876-5p mimics or co-transfected with LINC-ROR, miR-876-5p mimics, and FOXM1, with the addition of DMSO or sorafenib. The percentage represents the sum of the Annexin- and 7-AAD-positive populations. All data are presented as the mean ± SD of three independent experiments. The p values represent comparisons between groups (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Has-miR-876-5p mimics and miR-NC/mimics and has-miR-876-5p inhibitor and miR-NC/inhibitor were purchased from Ribobio (Guangzhou, China).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Transfection, CCK-8 Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: FOXM1-Mediated LINC-ROR Regulates the Proliferation and Sensitivity to Sorafenib in Hepatocellular Carcinoma

doi: 10.1016/j.omtn.2019.04.008

Figure Lengend Snippet: Schematic Overview of FOXM1 and LINC-ROR Regulatory Signaling LINC-ROR, which was activated by FOXM1 through promoter binding and transferred to the cytoplasm, then upregulated FOXM1 expression by competitively sponging miR-876-5p, forming a positive regulatory signaling pathway to promote proliferation and induce chemoresistance in HCC cells.

Article Snippet: Has-miR-876-5p mimics and miR-NC/mimics and has-miR-876-5p inhibitor and miR-NC/inhibitor were purchased from Ribobio (Guangzhou, China).

Techniques: Binding Assay, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Prolonged Absence of Mechanoluminal Stimulation in Human Intestine Alters the Transcriptome and Intestinal Stem Cell Niche

doi: 10.1016/j.jcmgh.2016.12.008

Figure Lengend Snippet: Primary and Secondary Antibodies

Article Snippet: LGR5 , Rabbit , Miltenyi (San Diego, CA) , 130-104-945 , 1:200.

Techniques: Transduction, Plasmid Preparation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Prolonged Absence of Mechanoluminal Stimulation in Human Intestine Alters the Transcriptome and Intestinal Stem Cell Niche

doi: 10.1016/j.jcmgh.2016.12.008

Figure Lengend Snippet: qPCR Primer List

Article Snippet: LGR5 , Rabbit , Miltenyi (San Diego, CA) , 130-104-945 , 1:200.

Techniques: Sequencing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Prolonged Absence of Mechanoluminal Stimulation in Human Intestine Alters the Transcriptome and Intestinal Stem Cell Niche

doi: 10.1016/j.jcmgh.2016.12.008

Figure Lengend Snippet: Absence of mechanoluminal stimulation increases LGR5 mRNA expression and downstream Wnt signaling gene expression. ( A and B ) Quantification and in situ hybridization of LGR5 ISCs within the crypts of fed and unfed intestine. Inset : High-magnification photographs label LGR5-positive in situ hybridization with purple arrows . qPCR comparison of ( C ) LGR5 , ( D ) CCND1 , and ( E ) MYC mRNA expression between 6 and 7 pairs of matched fed vs unfed intestine with outliers excluded. ( F ) Representative images of Western blot analysis of STAT3 phosphorylation from 3 pairs of matched tissue. Images were obtained on an upright Leica DM5500B immunofluorescence microscope using Leica Suite Advanced Fluorescence (LAS AF) 6000 software, processed with ImageJ software. Scale bars : 50 μm. * P < .05. Grey bars on plots indicate median with 95% confidence interval.

Article Snippet: LGR5 , Rabbit , Miltenyi (San Diego, CA) , 130-104-945 , 1:200.

Techniques: Expressing, Gene Expression, In Situ Hybridization, Comparison, Western Blot, Phospho-proteomics, Immunofluorescence, Microscopy, Fluorescence, Software

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Prolonged Absence of Mechanoluminal Stimulation in Human Intestine Alters the Transcriptome and Intestinal Stem Cell Niche

doi: 10.1016/j.jcmgh.2016.12.008

Figure Lengend Snippet: Global decrease of LGR5+, SOX9+, and OLFM4+ intestinal stem cell populations occurs in the absence of mechanoluminal flow. ( A–H ) Quantification and immunofluorescence staining of LGR5+ rapidly cycling intestinal stem cells per crypt with proliferating cell nuclear antigen counterstain in fed vs unfed intestine. ( C and F ) LYZ+ Paneth cells with E-cadherin (Ecad) counterstain of same crypt shown in panels A and D , respectively, for comparison. ( I–N ) Quantification and immunofluorescence staining of SOX9 and OLFM4 intestinal stem cells per crypt in fed vs unfed intestine. Images were obtained on an upright Leica DM5500B immunofluorescence microscope using Leica Suite Advanced Fluorescence (LAS AF) 6000 software, processed with ImageJ software. Scale bars : ( B , C , E , and F ) 50 μm, and ( A , D , I , J , L , and M ) 100 μm. * P < .05. Grey bars on plots indicate median with 95% confidence interval. PCNA, proliferating cell nuclear antigen.

Article Snippet: LGR5 , Rabbit , Miltenyi (San Diego, CA) , 130-104-945 , 1:200.

Techniques: Immunofluorescence, Staining, Comparison, Microscopy, Fluorescence, Software