Additional file 1 of Long non-coding RNA LPP-AS2 promotes glioma tumorigenesis via miR-7-5p/EGFR/PI3K/AKT/c-MYC feedback loop
Creators: name: Zhang, Xiaoming
name: Niu, Wanxiang
name: Mu, Maolin
name: Hu, Shanshan
name: Niu, Chaoshi
Publisher: name: figshare
Year: 2020
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Figure S1. Identification of differentially expressed lncRNAs and mRNAs in high-throughput RNA sequencing. a Heatmap of reported lncRNAs and mRNAs in high-throughput RNA sequencing. b Hierarchical cluster analysis of differentially expressed mRNAs in three glioblastoma and normal samples. c Volcano plot of differentially expressed mRNAs in three glioblastoma and normal samples. Figure S2. LPP-AS2 is upregulate in glioblastoma and regulated by c-MYC. a Four dysregulated lncRNAs obtained from RNA-seq were validated in 6 normal brain tissues and 15 GBM tissues by RT-qPCR. b Six dysregulated mRNAs obtained from RNA-seq were validated in 6 normal brain tissues and 15 GBM tissues by RT-qPCR. c Co-expression network of 35 mRNAs in the neuron apoptotic process and the top 50 lncRNAs. d Relative expression level of LPP-AS2 in TCGA (207 normal brain tissues and 163 glioma tissues). e Relative LPP-AS2 expression in glioma tissues (n = 75) and normal brain tissues (n = 13) of the GEO databases (GSE50161 and GSE33331). f Relative expression of LPP-AS2 in U251 and SHG44 cells treated with siRNAs of c-MYC. Error bars, s.e.m. from three independent experiments. *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; ****P &lt; 0.0001 (Student’s t test). Figure S3. LPP-AS2 promotes glioblastoma progression in vitro. a Transwell assays in U251 and SHG44 cells transfected with siRNAs of LPP-AS2 and treated with Ara-C. Representative staining images are presented. b Transwell assays in U251 and SHG44 cells transfected with overexpression of LPP-AS2 and treated with Ara-C. Representative staining images are presented. c Wound healing assays in U251 and SHG44 cells transfected with siRNAs/plasmids of LPP-AS2 and treated with Ara-C. Data represent the mean ± s.e.m. of three independent experiments. **P &lt; 0.01; ***P &lt; 0.001; ****P &lt; 0.0001 (Student’s t test). Figure S4. Regulation relationship between LPP-AS2 and EGFR. a Relative expression of LPP in U251 and SHG44 cells treated with siRNAs of LPP-AS2. b Relative expression of LPP in U251 and SHG44 cells with overexpression of LPP-AS2. c Relative expression of EGFR in seven glioma cell lines and HEB by RT-qPCR. d Overexpression of LPP-AS2 significantly increased EGFR expression in U251 and SHG44 cells. e Protein level of EGFR in U251 and SHG44 cells with overexpression of LPP-AS2. Error bars, s.e.m. from three independent experiments. ns (no significance); *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; ****P &lt; 0.0001; (Student’s t test). Figure S5. LPP-AS2 requires EGFR to facilitate tumor progression. a Relative expression of EGFR in U251 and SHG44 cells transfected with siRNAs of EGFR alone or co-transfection of pEGFP-C1/LPP-AS2. b CCK8 assays in U251 and SHG44 cell treated with siRNAs of EGFR alone or co-transfection of pEGFP-C1/LPP-AS2. c Transwell assays in U251 and SHG44 cells treated with siRNAs of EGFR alone or co-transfection of pEGFP-C1/LPP-AS2. d Migration assays in U251 and SHG44 cells treated with siRNAs of EGFR alone or co-transfection of pEGFP-C1/LPP-AS2. e Colony formation assays in U251 and SHG44 cells treated with siRNAs of EGFR alone or co-transfection of pEGFP-C1/LPP-AS2. f Flow cytometry assays in U251 and SHG44 cells treated with siRNAs of EGFR alone or co-transfection of pEGFP-C1/LPP-AS2. Error bars, s.e.m. from three independent experiments. *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; ****P &lt; 0.0001; #P &lt; 0.05; ##P &lt; 0.01; ###P &lt; 0.001 (Student’s t test). Figure S6. Characterization of miR-7-5p in glioma. a Relative expression of miR-7-5p in 17 normal brain tissues and 17 glioblastoma tissues. b Relative expression of miR-7-5p in seven glioma cell lines and HEB by RT-qPCR. c Relative expression of miR-7-5p in U251 and SHG44 cells treated with Overexpression of LPP-AS2. d Knockdown or overexpression of miR-7-5p in U251 and SHG44 cells have no significantly effect on LPP-AS2 expression. e Relative expression of miR-7-5p in U251 and SHG44 cells treated with miR-7-5p mimics alone or co-transfection of pEGFP-C1/LPP-AS2. Data represent the mean ± s.e.m. of three independent experiments. ns (no significance); **P &lt; 0.01; ***P &lt; 0.001; ****P &lt; 0.0001; ##P &lt; 0.01; ###P &lt; 0.001; ####P &lt; 0.0001 (Student’s t test). Figure S7. Functions of miR-7-5p in glioma. a CCK8 assays in U251 and SHG44 cell transfected with miR-7-5p mimics or miR-7-5p inhibitor. b Colony formation assays in U251 and SHG44 cells transfected with miR-7-5p mimics or miR-7-5p inhibitor. c Transwell assays in U251 and SHG44 cells transfected with miR-7-5p mimics or miR-7-5p inhibitor. d Migration assays in U251 and SHG44 cells transfected with miR-7-5p mimics or miR-7-5p inhibitor. e Flow cytometry assays in U251 and SHG44 cells transfected with miR-7-5p mimics or miR-7-5p inhibitor. Error bars, s.e.m. from three independent experiments. *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; ****P &lt; 0.0001 (Student’s t test). Figure S8. LPP-AS2 decoys miR-7-5p to regulate glioma progression. a CCK8 assays in U251 and SHG44 cell transfected with miR-7-5p mimics alone or co-transfection of pEGFP-C1/LPP-AS2. b Migration assays in U251 and SHG44 cells transfected with miR-7-5p mimics alone or co-transfection of pEGFP-C1/LPP-AS2. Migration assays in U251 and SHG44 cells transfected with miR-7-5p inhibitor alone or co-transfection of siRNAs of LPP-AS2. c Transwell assays in U251 and SHG44 cells transfected with miR-7-5p mimics alone or co-transfection of pEGFP-C1/LPP-AS2. Representative staining images are presented. d Colony formation assays in U251 and SHG44 cells transfected with miR-7-5p mimics alone or co-transfection of pEGFP-C1/LPP-AS2. e Flow cytometry assays in U251 and SHG44 cells transfected with miR-7-5p mimics alone or co-transfection of pEGFP-C1/LPP-AS2. Error bars, s.e.m. from three independent experiments. *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; ****P &lt; 0.0001; #P &lt; 0.05; ##P &lt; 0.01; ###P &lt; 0.001 (Student’s t test). Figure S9. LPP-AS2 functions as an oncogene via miR-7-5p/EGFR/PI3K/AKT/c-MYC axis. a Relative protein levels of EGFR in U251 and SHG44 cells treated with miR-7-5p mimics or miR-7-5p inhibitor. b Relative protein levels of EGFR in U251 and SHG44 cells treated with pEGFP-C1/EGFR alone or co-transfection of si-LPP-AS2–1. c Western blotting analysis of EGFR, PI3K, AKT, p-PI3K, p-AKT, c-MYC in U251 and SHG44 cells treated with miR-7-5p mimics alone or co-transfection with pEGFP-C1/LPP-AS2. d Histogram of EGFR protein levels in U251 and SHG44 cells treated with miR-7-5p mimics alone or co-transfection with pEGFP-C1/LPP-AS2. e Relative expression of EGFR in U251 and SHG44 cells treated with miR-7-5p mimics alone or co-transfection with pEGFP-C1/LPP-AS2 were validated by RT-qPCR. f, g Ratio of p-PI3K/PI3K and p-AKT/AKT in U251 and SHG44 cells. h Western blotting analysis of EGFR, PI3K, p-PI3K, AKT, p-AKT and c-MYC in U251 and SHG44 cells treated with siRNAs of EGFR alone or co-transfection with pEGFP-C1/LPP-AS2. i Histogram of EGFR protein levels in U251 and SHG44 cells treated with siRNAs of EGFR alone or co-transfection with pEGFP-C1/LPP-AS2. j Ratio of p-PI3K/PI3K and p-AKT/AKT in U251 and SHG44 cells treated with siRNAs of EGFR alone or co-transfection with pEGFP-C1/LPP-AS2. Data represent the mean ± s.e.m. of three independent experiments. *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; ****P &lt; 0.0001; #P &lt; 0.05; ##P &lt; 0.01; ###P &lt; 0.001; ####P &lt; 0.0001 (Student’s t test).