MALAT1 regulates cell proliferation and apoptosis in HepG2 cells through binding with DBC1. (A) The growth curve of MALAT1-downregulated cells (left panel) and MALAT1-overexpressed cells (right panel) over 9 days. Cells (2 × 105) were seeded in each well, and the cell numbers were counted every 48 h. Data are from three independent assays. Bar, mean; error bar, SD (*P < 0.05, **P < 0.01, ***P < 0.001, by repeated measures ANOVA). (B) Colony-formation assay of HepG2-MALAT1 and HepG2 cells co-transfected with both MALAT1 and DBC1 or DBC1-N4. Representative images show the results of the colony-formation assay (left panel). Histogram shows the relative colony numbers (right panel). Data are from three independent assays. Bar, mean; error bar, SD (***P < 0.001, by one-way ANOVA). BrdU incorporation analysis of MALAT1-knockdown HepG2 cells (C) and MALAT1-overexpressing cells with or without the overexpression of full-length or N4 fragment of DBC1 respectively (D). Representative images showing alterations in S phase distribution (left panel). Histogram shows the percentage of BrdU staining (right panel). Data are from three independent assays. Bar, mean; error bar, SD (**P < 0.01, ***P < 0.001, by Student's t-test or one-way ANOVA). (E) Annexin V assay for analyzing cell apoptosis in HepG2 cells transfected with shMALAT1 plasmid (left panel) or MALAT1-overexpressing plasmid with or without co-transfection of full-length or N4 fragment of DBC1 respectively (right panel). Data are from three independent assays. Bar, mean; error bar, SD (*P < 0.05, **P < 0.01, ***P < 0.001, by Student's t-test or one-way ANOVA).

Gene ontology analysis of the identified MALAT1-interacting proteins and the validation using RNA immunoprecipitation (RIP). (A) Diagram showing the molecular functions of the identified MALAT1-interacting proteins. (B) Diagram showing the related biological processes of the identified MALAT1-interacting proteins. The annotation information was acquired through the Panther database (http://www.pantherdb.org). Bar graphs present the P values of the enriched protein domains (C) and signaling pathways (D). The protein domain and pathway analyses were performed using DAVID (https://david.ncifcrf.gov/). (E) Agarose gel electrophoresis analysis of the qRT-PCR products of MALAT1, 18S rRNA and NEAT1 in the immunoprecipitates of the selected proteins. IgG was used as negative control.

DBC1 and DBC1-N4 inhibit cell proliferation and promote apoptosis. (A) The overexpression of DBC1 and DBC1-N4 was examined by qRT-PCR. Data represent the means ± SD of triplicate independent analyses (***P < 0.001, by student's t-test). (B) Western blot analysis indicates that DBC1 overexpression increases p53 acetylation. GAPDH expression was used as a control. (C) Growth curve assay using DBC1-overexpressing HepG2 cells. Data represent the means ± SD of triplicate independent analyses (**P < 0.01, by repeated measures ANVOA). (D) BrdU incorporation analysis using DBC1-overexpressing HepG2 cells. Histogram shows the means ± SD from two independent experiments (**P < 0.01, by student's t-test). (E) Colony-formation assay was performed to study the proliferation of HepG2 cells overexpressing full-length DBC1 or DBC1-N4, and their corresponding control cells. Data represent the means ± SD of two independent analyses (**P < 0.01, by one-way ANOVA). (F) Apoptosis analysis of HepG2 cells overexpressing full length DBC1 or DBC1-N4. Apoptotic cells were detected by Annexin V/PI staining assay and examined by flow cytometry. Data represent the means ± SD of triplicate independent analyses (**P < 0.01, ***P < 0.001, by one-way ANOVA).