Additional file 1 of HDAC6 involves in regulating the lncRNA-microRNA-mRNA network to promote the proliferation of glioblastoma cells
Creators: name: Wu, An-Chih
name: Yang, Wen-Bin
name: Chang, Kwang-Yu
name: Lee, Jung-Shun
name: Liou, Jing-Ping
name: Su, Ruei-Yuan
name: Cheng, Siao Muk
name: Hwang, Daw-Yang
name: Kikkawa, Ushio
name: Hsu, Tsung-I
name: Wang, Chih-Yang
name: Chang, Wen-Chang
name: Chen, Pin-Yuan
name: Chuang, Jian-Ying
Publisher: name: figshare
Year: 2022
Subjects: subject: Biophysics
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Hematology

description: Additional file 1: Figure S1. MPT0B291 commonly alters two lncRNAs (LINC00461 and LINC01559) in stem-like and TMZ-resistant U87MG cells. Volcano plots show overlapping lncRNAs between two microarray data (MS1040791 and MS1060987). Relative expression level (log2) to control (DMSO) of each lncRNA is shown in the x-axis. y-axis indicates SNR (signal-to-noise ratio) value (log2) of each lncRNA. Genes with significant upregulation (red) and downregulation (green) in both microarray datasets are highlighted. Figure S2. MPT0B291 decreases Ki-67 expression in vivo. (A) IHC detected the protein expressions of proliferation (Ki-67) and apoptosis (cleaved caspase-3) marker. Serial sections of control and MPT0B291-treated xenografts were hybridized with either an antibody against Ki-67 (upper two panels) or cleaved caspases-3 (lower two panels), and cell nuclei were stained with hematoxylin. Scale bars indicate 20 μm and 200 μm. Mean intensity of Ki-67 and cleaved caspases-3 staining in the cells of control and MPT0B291-treated xenografts were semi-quantitative determined. Unpaired Student’s t-test. Figure S3. Interaction between HDAC6 and RNA-binding proteins (CNOT6 and FUS). (A) Flag-tagged CNOT6-expressed U87MG cells were treated with 10 μM MPT0B291, 10 μM Trichostatin A (TSA), or DMSO for 2 h. Flag-CNOT6 protein was immunoprecipitated and the precipitated samples were then analyzed by immunoblotting analysis with antibodies of HDAC6 or Flag-M2. (B) Enrichment of HuR from the lysate by desthiobiotin-labeled androgen receptor (AR) 3′-UTR validates the efficiency of RNA-protein pull-down assay (left panel). Protein lystaes from TMZ-resistant Pt#3 cells were also used for the RNA-protein pull-down assay with desthiobiotin-labeled LINC00461, and both poly(A)-binding protein (PABP) and HuR were slightly detected in the pull-down sample, but desthiobiotin-labeled LINC00461 did not bind HDAC6 directly. (L = lysate; FT1 = flow-through in first wash; FT2 = flow-through in second wash; E = eluate). (C) Quantification of CNOT6 and FUS protein expression from the parental and TMZ-resistant Pt#3 cells either treated with MPT0B291 or si-HDAC6. Unpaired Student’s t-test. The results are shown as mean ± SEM for triplicate samples in each group. Figure S4. Cell division-related genes show poor prognostic implications in high-grade glioma patients. (A) Kaplan–Meier curves compare the survival outcomes in high-grade glioma patients with high (red) and low (green) expressions of cell division-related genes. All survival curves were obtained from PROGgeneV2. Log-rank test. Figure S5. Functional analysis of the conserved markers in cluster 5 from scRNA-seq data reveals gene classes associated with cell division and survival. Core analysis using IPA revealed the top ten molecular and cellular functions of highly conserved marker genes in cluster 5 of patient-derived GBM cells. Log(p-value) indicates the significance of enrichment for highly expressed marker genes from our dataset. The threshold for significance was set at a p-value &lt; 0.05. Figure S6. Three datasets identify potential interaction networks between 13 miRNAs and cell division-related genes. (A) Pipeline for the identification of potential regulatory LINC00461-miRNA-mRNA networks. (B) The miRNA-mRNA networks were identified using three public databases (miRDB, miRWALK, and starBase). Figure S7. The regulation of LINC00461 stability. Protein expression levels of HDAC1 and HDAC2 in parental and TMZ-resistant U87MG cells treated with either si-NC or si-HDAC1/2. Effect of either HDAC1 or HDAC2 depletion on LINC00461 expression in parental and TMZ-resistant U87MG cells. n.s., not significant, unpaired Student’s t-test. (B) A schematic diagram illustrates the proposed regulatory mechanism underlying HDAC6 controls the LINC00461 stability via regulating both the RNA-binding activity of FUS (fused in sarcoma)/PABP and the activity of deadenylases of human Ccr4-Not complex. Figure S8.LINC00461 knockdown downregulates the expressions of cell division-related molecules. (A) Expressions of the cell division-related mRNAs (cyclin D1, CDK4, cyclin A2, cyclin E1, MELK, and MCM10) in parental and TMZ-resistant R-U87MG cells that were treated with either si-NC or si-LINC00461. Unpaired Student’s t-test. (B) Effect of LINC00461 depletion on the protein expression levels of cell division-related molecules (cyclin D1 and CDK1) in parental and TMZ-resistant U87MG cells. Table S1. siRNA targeted-sequences for each gene. Table S2. The primer or probe sequences for each gene used in SYBR green and TaqMan qPCR assays. Table S3. Custom LNA™ detection probes sequences. Table S4. List of biomarkers with fold-change more significant than 1.5 in cluster 5.