The online version of this article (https://doi.org/10.1186/s12943-017-0756-y) contains supplementary material, which is available to authorized users.
Long noncoding RNAs (lncRNAs) are an important class of functional regulators involved in human cancers development, including gastric cancer (GC). Studying aberrantly expressed lncRNAs may provide us with new insights into the occurrence and development of gastric cancer by acting as oncogenes or tumor suppressors. In this study, we aim to examine the expression pattern of lncRNA HAGLROS in GC and its clinical significance as well as its biological role in tumor progression.
Bioinformatics analysis and qRT-PCR were performed to detect the relative expression of HAGLROS in GC tissues and cell lines. Gain or loss of function approaches were used to investigate the biological functions of HAGLROS. The effect of HAGLROS on proliferation was evaluated by MTT, colony formation assay and nude mouse xenograft model. Wound healing and Transwell assays were used to study the invasion and migration of GC cells. FISH, RIP, RNA-seq, Luciferase report assays, RNA pulldown and Western blot were fulfilled to measure molecular mechanisms. Results are shown as means ± S.D. and differences were tested for significance using Student’s t-test (two-tailed).
We screened out HAGLROS, whose expression was significantly increased and correlated with outcomes of GC patients by publicly available lncRNAs expression profiling and integrating analyses. Exogenous down-regulation of HAGLROS expression significantly suppressed the cell proliferation, invasion and migration. Mechanistic investigations showed that HAGLROS was a direct target of transcriptional factor STAT3. Moreover, HAGLROS knockdown decreased mTOR expression and increased autophagy-related genes ATG9A and ATG9B expression. Further investigation showed that HAGLROS regulated mTOR signals in two manners. In the one hand, HAGLROS competitively sponged miR-100-5p to increase mTOR expression by antagonizing miR-100-5p-mediated mTOR mRNA inhibition. On the other hand, HAGLROS interacted with mTORC1 components to activate mTORC1 signaling pathway which was known to be an important negative signal of autophagy. Here activation of mTORC1 signaling pathway by HAGLROS inhibited autophagy, thereby promoted excessive proliferation and maintained the malignant phenotype of GC cells.
The present study demonstrates that HAGLROS overexpression contributes to GC development and poor prognosis and will be a target for GC therapy and further develop as a potential prognostic biomarker.
Additional file 1: Table S1. The relationship between HAGLROS expression and clinicopathological factors of GC patients. (DOCX 14 kb)12943_2017_756_MOESM1_ESM.docx
Additional file 2: Table S2. Primers used for qRT-PCR,RT-PCR, CHIP and siRNAs/shRNA oligonucleotides. (XLSX 12 kb)12943_2017_756_MOESM2_ESM.xlsx
Additional file 3: Figure S1. The relative lncRNAs expression from 12 GC patients were validated by qRT-PCR. Error bars indicate the means ± S.E.M. *P < 0.05, **P < 0.01 for carcinoma vs paracarcinoma. (TIFF 165 kb)12943_2017_756_MOESM3_ESM.tiff
Additional file 4: Table S3. Univariate and multivariate analyses of the clinicopathological factors for overall survival in 84 patients with GC. (DOCX 16 kb)12943_2017_756_MOESM4_ESM.docx
Additional file 5: Figure S2. (a) Relative areas of the wound scratch assay by Image J software. *P < 0.05 for siRNAs vs Ctrl siRNAs and pcDNA3.1-HAGLROS vs vector. (b) Transcription efficiencies of siRNAs, shRNAs and pcDNA3.1-HAGLROS. **P < 0.01. Error bars indicate the means ± S.E.M. (TIFF 1170 kb)12943_2017_756_MOESM5_ESM.tif
Additional file 6: Figure S3. RNA-sequencing analysis of HAGLROS siRNA vs Ctrl. (a) 194 differentially expressed genes upon HAGLROS siRNA vs Ctrl. (b) GO analysis of differentially expressed genes. (c) Pathway enrichment analysis of differentially expressed genes. (TIFF 3460 kb)12943_2017_756_MOESM6_ESM.tif
Additional file 7: Figure S4. The relative expression levels of downstream signals were validated by qRT-PCR upon HAGLROS knockdown in accordance with RNA high-throughput sequencing guidelines. (a) Down-regulated genes were validated by qRT-PCR upon HAGLROS knockdown. (b) Up-regulated genes were validated by qRT-PCR upon HAGLROS knockdown. Error bars indicate the means ± S.E.M. *P < 0.05, **P < 0.01. (TIFF 1840 kb)12943_2017_756_MOESM7_ESM.tiff
Torre LA, Siegel RL, Ward EM, Jemal A. Global cancer incidence and mortality rates and trends--an update. Cancer Epidemiol Biomark Prev. 2016;25(1):16–27. CrossRef
Liu S, Song L, Zeng S, Zhang L. MALAT1-miR-124-RBG2 axis is involved in growth and invasion of HR-HPV-positive cervical cancer cells. Tumor Biol. 2016;37(1):633–40. CrossRef
Quagliata L, Matter MS, Piscuoglio S, Arabi L, Ruiz C, Procino A, Kovac M, Moretti F, Makowska Z, Boldanova T, et al. Long noncoding RNA HOTTIP/HOXA13 expression is associated with disease progression and predicts outcome in hepatocellular carcinoma patients. Hepatology. 2014;59(3):911–23. CrossRefPubMedPubMedCentral
Stephen A. Bustin. Why the need for qPCR publication guidelines?—the case for MIQE. Methods. 2010;50:217–6. CrossRef
Willems L, Tamburini J, Chapuis N, Lacombe C, Mayeux P, Bouscary D. PI3K and mTOR signaling pathways in cancer: new data on targeted therapies. Curr Oncol Rep 2012; 14(2):129–138.
Bussemakers MJ, van Bokhoven A, Verhaegh GW, Smit FP, Karthaus HF, Schalken JA, Debruyne FM, Ru N, Isaacs WB. DD3: a new prostate-specific gene, highly overexpressed in prostate cancer. Cancer Res. 1999;59(23):5975–9. PubMed
Yecies JL, Manning BD. mTOR links oncogenic signaling to tumor cell metabolism. J Mol Med (Berl). 2011;89(3):221–8. CrossRef
- STAT3-induced lncRNA HAGLROS overexpression contributes to the malignant progression of gastric cancer cells via mTOR signal-mediated inhibition of autophagy
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