Additional file 1 of A novel LncRNA transcript, RBAT1, accelerates tumorigenesis through interacting with HNRNPL and cis-activating E2F3
Creators: name: He, Xiaoyu
name: Chai, Peiwei
name: Li, Fang
name: Zhang, Leilei
name: Zhou, Chuandi
name: Yuan, Xiaoling
name: Li, Yongyun
name: Yang, Jie
name: Luo, Yingxiu
name: Ge, Shengfang
name: Zhang, He
name: Jia, Renbing
name: Fan, Xianqun
Publisher: name: figshare
Year: 2020
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Supplementary Table 1. Clinicopathological features and demographics of retinoblastoma (Rb) patient cohorts. Supplementary Table 2. Clinicopathological features of Bladder Urothelial Carcinoma (BCa) patient cohorts. Supplementary Table 3. Primers, oligos, shRNAs, GapmeRs and probes used in the experiment. Supplementary Table 4. The list of lncRNAs that shared an overlap region with tumor related genes in retinoblastoma. Supplementary Table 5. Interactions between E2F3 and its targeting genes. Supplementary Table 6. Information of GO (Biological Process) Enrichment Analysis. Supplementary Table 7. ChIRP-MS identified lncRNA RBAT1 specifically binding proteins in Y79. Supplementary Table 8. ChIRP-MS identified lncRNA RBAT1 specifically binding proteins in 5637. Fig. S1. RBAT1 is a novel transcript in retinoblastoma (Rb) and bladder cancer (BCa). (A) Volcano plots of differentially expressed protein-coding genes. X axis represents log fold changes. Y axis represents log p values. Blue points denote significantly downregulated genes and red points denote significantly upregulated genes. (B) Agarose gel electrophoresis of PCR products generated by 3′ (left) and 5′ (right) RACE. (C) Schematic diagram of RACE assay. Fig. S2. RACE and coding potential analyses of RBAT1. (A) The full sequence of RBAT1. (B) Coding Potential Calculator (CPC) analysis suggested that RBAT1 is a noncoding RNA. GAPDH, CRTC1 and ACTB genes were used as protein-coding controls. XIST and HOTAIR genes were used as noncoding RNA controls. (C) RBAT1 was predicted by PhyloCSF to have no protein coding potential. The peaks showed the PhyloCSF score for each codon in each of 6 frames. Regions with a score less than 0 are predicted to be noncoding while Regions with a score greater than 0 are predicted to be coding. The protein coding gene ACTB and the noncoding RNA gene HOTAIR were used as controls. (D) Fractionation of tumor cell lines (Y79, WERI-Rb-1 and 5637) followed by RT-PCR. RBAT1 was mainly expressed in the nucleus. GAPDH and U6 RNA served as positive controls for the cytoplasmic and nuclear fractions, respectively. Fig. S3. Effect of RBAT1 on colony formation ability and migration ability of tumor cell lines. (A) A colony formation assay was performed to determine the colony formation ability of RBAT1-silenced tumor cell lines (Y79, WERI-Rb-1 and 5637). For colony formation assays, 500 cells were seeded in 6-well plates (Poly-L-lysine-coated 6-well plates for retinoblastoma cell lines). 7–14 days later, the colonies were washed with PBS, fixed and stained for 20 min with a 1% crystal violet solution. Images were captured by a scanner, and the percentage of cell occupancy was counted and analyzed by ImageJ software. (B) The migration and invasion abilities displayed no significant changes in RBAT1-silenced tumor cell lines (5637) compared with ctrl group. (C) A colony formation assay was performed to test sustained effect after removing GapmeRs at the 3rd day. (D) A real-time PCR was performed to determine the expression level of RBAT1 in tumors from GapmeR1/2 treated groups and Ctrl. The results are shown as the mean ± SD in three independent experiments. *p &lt; 0.05 and **p &lt; 0.01. Fig. S4. Gene expression patterns associated with cell proliferation in RBAT1-silenced Rb and BCa cell lines. (A) Gene expression profiles in 5637 cells and WERI-Rb-1 cells transfected with GapmeR1/2 or a ctrl GapmeR. (B) The interaction of genes in cell cycle pathway. The down-regulated genes in red semitransparent area were verified in our study. Fig. S5. The overexpression of RBAT1 and E2F3 in normal cell lines. (A) Realtime-PCR showed the expression levels of RBAT1 after transfecting pcDNA3.1- RBAT1 in ARPE-19 and SV-HUC-1. (B-C) Western blot was performed to test E2F3 expression after transfecting pcDNA3.1-RBAT1 or pcDNA3.1-E2F3 in ARPE-19 and SV-HUC-1, respectively. (D-E) Colony formation assays were performed to measure the colony formation ability of ARPE-19 and SV-HUC-1 with RBAT1 or E2F3 overexpression. (F) CCK8 assay was performed to assess cell proliferation of RBAT1 or E2F3 over-expressed normal cell lines (ARPE-19 and SV-HUC-1). The results were shown as Mean ± SD, *p &lt; 0.05; **p &lt; 0.01. Fig. S6. ChIRP-MS analysis of RBAT1-interacting proteins. The protein peptides isolated by ChIRP. U6 was selected as control, and scrambled oligos were selected as negative controls. Fig. S7. HNRNPL expression analysis. (A) Western blot analysis showed that RBAT1 depletion did not influence the expression level of HNRNPL. (B) HNRNPL was silenced in tumor cell lines (Y79, WERI-Rb-1 and 5637) by two independent shRNAs. Fig. S8. Alignment of RBAT1 sequences and human genome sequences. lncRNA RBAT1 could not only align to E2F3 promoter (chr6:20401729–20,481,937, 100% similarity, 1st panel), but also 5 kb upstream from TSS of SLFN12L (chr17:35490212–35,490,325, 94.8% similarity, 2nd panel) and an intronic region of DNAH7 (chr2:195995428–195,995,503, 87.1% similarity, 3rd panel). Fig. S9. Identification of the E2F3 promoter region. (A) Schematic of the luciferase assay of E2F3 promoter. 2000 bp upstream to E2F3 TSS was selected for detection. The full-length 5′ to 3′ sequence was used as positive control (lane a). The untreated group (NC) and empty vector group (Mock) served as negative control groups. Different segments of E2F3 promoter (lane b-e) were constructed into pGL3 vector and subjected to luciferase reporter assays in HEK-293 T cells. Through luciferase reporter assays, we identified a − 500 to − 1000 bp segment of TSS upstream region as the core promoter of E2F3 with transcriptional activity. Fig. S10. Copy number variation (CNV) analysis of Rb cell lines. (A and B) CNV microarray showed increased E2F3 gene copy number in Y79 and WERI-Rb-1 compared with ARPE-19. RBAT1 interference did not influence the CNV of retinoblastoma cell lines. The Affymetrix OncoScan CNV microarray was used to detect the CNV of cells. The arrays were scanned with a GeneChip Scanner 3000 7G System and the data were analyzed using Chromosome Analysis Suite (ChAS). Fig. S11. Copy number variation (CNV) analysis of BCa cell lines. (A and B) CNV microarray of bladder cancer cell line showed increased E2F3 gene copy number in 5637 compared with SV-HUC-1. RBAT1 silencing did not influence the CNV of bladder cancer cell lines. Fig. S12. HT1376, a bladder cancer cell line without E2F3 copy number amplification, represent highly-expressed RBAT1. (A) HT-1376, a bladder cancer cell line without E2F3 locus amplification. (B) Real-time PCR showed that RBAT1 was also highly expressed in HT-1376. (C) Real-time PCR showed that RBAT1 was knockdown by GapmeR1. (D and E) After RBAT1 knockdown the expression of E2F3 was largely reduced (D) and the tumor proliferation was also suppressed (E). The results were shown as Mean ± SD, **p &lt; 0.01. Fig. S13. E2F3 associates with Rb and BCa clinical characteristics and predict disease prognosis. (A) Box plot represented E2F3 expression in Rb (left) and BCa (right) specimens at stage I (Rb: n = 5; BCa: n = 6) and at stage II/III/IV (Rb: n = 15; BCa: n = 19). *p &lt; 0.05; **p &lt; 0.01. (B) Kaplan-Meier survival analysis was performed in 224 cases of bladder cancer using R2 platform ( https://hgserver1.amc.nl ). (C)The normalized expression value of E2F3 in 224 BCa patients were automatically calculated in R2 platform.