Additional file 1 of Long noncoding RNA SGO1-AS1 inactivates TGFβ signaling by facilitating TGFB1/2 mRNA decay and inhibits gastric carcinoma metastasis
Creators: name: Huang, Donglan
name: Zhang, Ke
name: Zheng, Wenying
name: Zhang, Ruixin
name: Chen, Jiale
name: Du, Nan
name: Xia, Yuanyuan
name: Long, Yan
name: Gu, Yixue
name: Xu, Jianhua
name: Deng, Min
Publisher: name: figshare
Year: 2021
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Mathematical Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Fig. S1 SGO1-AS1 was downregulated in GC tissues. Expression levels of SGO1-AS1 and 12 other lncRNAs validated by qRT-PCR in 18 paired gastric cancer tissues and adjacent normal tissues (Cohort 1). The results are expressed as -∆Ct. **P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001. Fig. S2 Characterization of human SGO1-AS1 as a long noncoding RNA. a. Schematic diagram of the genomic locus and isoforms of SGO1-AS1 in the UCSC Genome Browser ( http://genome.ucsc.edu/ ). b. Identification of full-length SGO1-AS1 by 5′ and 3′ RACE. Left: Representative images of the PCR products from 5′ RACE and 3′ RACE. Right: Nucleotide sequence of full-length human SGO1-AS1. c. Coding potential of SGO1-AS1 as predicted by the CPAT and CPC tools. The lncRNA MALAT1 and the protein-coding genes GAPDH and ACTB are also shown. d. Relative distribution of SGO1-AS1 in gastric cell lines as determined by RT-PCR. SGO1-AS1 was mainly expressed in the cytoplasm of epithelial cells. Fig. S3 SGO1-AS1 inhibits GC cell migration and growth. a. qRT-PCR showed the relative levels of SGO1-AS1 expression in human gastric cancer cell lines compared to those in the immortalized human gastric epithelial cell line GES1. b. qRT-PCR analysis was used to confirm the level of SGO1-AS1 in cells with stable overexpression of SGO1-AS1. c. qRT-PCR analysis was performed to assess the inhibition efficiency in MKN28 cells infected with SGO1-AS1 shRNA lentiviruses targeting different regions of SGO1-AS1. d. Cell migration in BGC823 cells stably expressing SGO1-AS1 and control cells was examined by a wound healing assay. Scale bar, 150 μm. e. Cell proliferation assay in SGC7901 and BGC823 cells stably expressing SGO1-AS1, MKN28 cells stably silencing SGO1-AS1 and the respective control cells. f-g. A soft agar colony formation assay was carried out in the indicated cells. Scale bars, 100 μm. h. Overexpression of SGO1-AS1 inhibits GC tumor growth in a nude mouse model. SGC-7901 cells with stable overexpression of SGO1-AS1 or the control were inoculated subcutaneously into nude mice (n = 6 mice/group). The mice were sacrificed at 28 days postinoculation, and the tumors were weighed. Error bars indicate SD. *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001. Fig. S4 Effects of SGO1-AS1 on the expression of the sense gene SGO1. a-b. qRT-PCR and Western blotting analyses of the SGO1 levels in SGC7901 and BGC823 cells stably expressing SGO1-AS1, MKN-28 cells stably silencing SGO1-AS1 and the related control cells. Error bars indicate SDs from three independent experiments. *P &lt; 0.05, **P &lt; 0.01. Fig. S5 SGO1-AS1 reduces TGFB1/2 mRNA stability. a. Western blot analysis was used to evaluate PTBP1 expression in human gastric cancer cell lines and normalgastric epithelial cell line. b. qRT-PCR analysis was performed to examine the expression levels of genes related to the TGFβ pathway in SGC7901 cells transfected with PTBP1 shRNA or the control. c. PTBP1 RIP assay was performed to analyze the interactions between PTBP1 and TGFB1/2 or ID3 in SGC7901 cells. The relative fold enrichment of these mRNAs compared to IgG was determined by qRT-PCR. SMAD5 and GAPDH were used as negative controls. d. RIP-qPCR assay was used to evaluate the interaction between PTBP1 and ID2 mRNA in MKN28 cells with SGO1-AS1 knockdown or SGC7901 cells with SGO1-AS1 overexpression. e. Deletion mapping was performed to determine the binding domain of PTBP1 to TGFB1/2 mRNA using full-length or truncated PTBP1. f-g. mRNA (f) and protein (g) expression levels of TGFβ1/2 in xenograft tumor tissues recovered from nude mice. #1–3 denote individual tumors grown in different mice. h. Western blot analysis of AGO2, TGFβ1 and TGFβ2 in control and PTBP1-KO MKN28 cells transfected with siAGO2 or control siRNA. i. RIP-qPCR assay of the interaction between the AGO2 protein and TGFB1/2 mRNA in MKN28 cells with PTBP1 knockout or SGO1-AS1 knockdown. Error bars are SDs from three independent experiments. *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001 and ns, not significant. Fig. S6 SGO1-AS1 changes cell morphology and suppresses cell invasion and stemness. a-c. Representative phase-contract (a), cell invasion (b) and tumor spheroid images (c) of the indicated cells are shown. a, Scale bars,100 μm; b-c, Scale bars, 150 μm. Fig. S7 Expression level of PTBP1 and its correlation with SGO1-AS1, TGFB1/2 and ZEB1 expression in GC tissues. a. Relative expression levels of PTBP1 in 92 paired GC and normal tissues from Cohort 2 were quantified by qRT-PCR. The pie chart shows the proportions of samples in the downregulation (blue), upregulation (red) and no change (yellow) categories. b. The expression level of PTBP1 in GCs according to their clinical stage and status of lymph node or distant metastasis. c. Correlation between PTBP1 expression and SGO-AS1, TGFB1, TGFB2 and ZEB1 expression in GC specimens. a-c, Error bars indicate SDs. *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001 and ns, not significant. Table S1. Clinicopathological characteristics of Cohort 2 samples. Table S2. Clinicopathological characteristics of Cohort 3 samples. Table S3. Primers used for quantitative PCR. Table S4. Primers for rapid amplification of cDNA ends analysis. Table S5. Correlation between clinicopathological parameters and SGO1-AS1 levels in 95 cases of GC tissues (Cohort 3). Table S6. Univariate and multivariate analyses of factors associated with overall survival. Table S7. Mass spectrometry protein identification results for biotinylated SGO1-AS1 RNA pull down.