The online version of this article (doi:10.1186/s12943-017-0674-z) contains supplementary material, which is available to authorized users.
Oct4, a key stemness transcription factor, is overexpressed in lung cancer. Here, we reveal a novel transcription regulation of long non-coding RNAs (lncRNAs) by Oct4. LncRNAs have emerged as important players in cancer progression.
Oct4 chromatin-immunoprecipitation (ChIP)-sequencing and several lncRNA databases with literature annotation were integrated to identify Oct4-regulated lncRNAs. Luciferase activity, qRT-PCR and ChIP-PCR assays were conducted to examine transcription regulation of lncRNAs by Oct4. Reconstitution experiments of Oct4 and downstream lncRNAs in cell proliferation, migration and invasion assays were performed to confirm the Oct4-lncRNAs signaling axes in promoting lung cancer cell growth and motility. The expression correlations between Oct4 and lncRNAs were investigated in 124 lung cancer patients using qRT-PCR analysis. The clinical significance of Oct4/lncRNAs signaling axes were further evaluated using multivariate Cox regression and Kaplan-Meier analyses.
We confirmed that seven lncRNAs were upregulated by direct binding of Oct4. Among them, nuclear paraspeckle assembly transcript 1 (NEAT1), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and urothelial carcinoma-associated 1 (UCA1) were validated as Oct4 transcriptional targets through promoter or enhancer activation. We showed that lung cancer cells overexpressing NEAT1 or MALAT1 and the Oct4-silenced cells reconstituted with NEAT1 or MALAT1 promoted cell proliferation, migration and invasion. In addition, knockdown of NEAT1 or MALAT1 abolished Oct4-mediated lung cancer cell growth and motility. These cell-based results suggested that Oct4/NEAT1 or Oct4/MALAT1 axis promoted oncogenesis. Clinically, Oct4/NEAT1/MALAT1 co-overexpression was an independent factor for prediction of poor outcome in 124 lung cancer patients.
Our study reveals a novel mechanism by which Oct4 transcriptionally activates NEAT1 via promoter and MALAT1 via enhancer binding to promote cell proliferation and motility, and led to lung tumorigenesis and poor prognosis.
Additional file 1: Table S1. Supplementary methods. Anchorage-independent growth assay, Tumor-sphere formation assay, Tumor formation assay, Western blot analysis. Table S1. The plasmids and their characteristics used in the current study. Table S2. The ChIP-PCR primers used in the current study. Table S3. The cDNA primers used in the current study. Table S4. The construction primers of promoters and enhancers used in the current study. Figure S1. Oct4 promoted lung cancer tumorigenesis in vitro and in vivo. A Anchorage-independent assays in empty vector stably-transfected cell line (vector) and two biological replicates of Oct4 stably-overexpressed A549 and CL1-0 cell lines (Oct4#1, Oct4#2). Results were photographed (left) and quantified (right). B Transwell migration and invasion assay analysis of stably-transfected cell lines in A549 and CL1-0 cells. Results were photographed (left) and quantified (right). P-values were determined by two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001. C In vitro tumor sphere formation assay of A549 lung cancer cells stably expressing Oct4#1 or vector photographed (top) and quantified (middle). In vivo tumor formation assay using limited cell number (100, 1000, and 5000 cells) of vector and Oct4#1 cells. Tumor incidence of mice was analyzed at 8 weeks after implantation. D The immunoblots (upper) and qRT-PCR (lower) confirmed Oct4 expression in A549 and CL1–0 stable clones. Figure S2. Expression of lncRNAs in CL1–0 lung cancer cells manipulated for Oct4. A, B qRT-PCR analysis of eight lncRNAs expression in CL1-0 cells stably overexpressing Oct4 (Oct4#1, Oct4#2) (A) or Oct4-silenced CL1-0 cells (si-Oct4#1, si-Oct4#2) (B). Target lncRNA expression levels were normalized to GAPDH expression levels. Data represent mean ± SEM. P-values were determined by two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001. Figure S3. Oct4 positively regulated NEAT1 and UCA1 lncRNAs transcription in normal bronchial epithelial BEAS-2B cells. qRT-PCR analysis of selected lncRNAs expressions in BEAS-2B cells overexpressing Oct4. Target lncRNA expression levels were normalized to GAPDH expression levels. Data represent mean ± SEM. P-values were determined by two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001. Figure S4. RNA expression level of the manipulated NEAT1 and MALAT1 in A549 lung cancer cells. A549 cells transfected with NEAT1 expression vector (A) or MALAT1 expression vector (B), si-NEAT1 oligo (si-NEAT1) (C) or si-MALAT1 oligo (si-MALAT1) (D) were harvested and subjected to qRT-PCR assays for NEAT1 and MALAT1 RNA expression. Data are mean ± SEM. P-values were determined by two-tailed Student’s t-test. *P < 0.05; ***P < 0.001. Figure S5. RNA and protein expression level of the manipulated Oct4, NEAT1 and MALAT1 in A549 lung cancer cells. A549 cells were transfected with expression vectors of NEAT1 (A) or MALAT1 (B) alone or together with si-Oct4 oligo (si-Oct4). A549 cells were transfected with si-NEAT1 oligo (si-NEAT1) (C) or si-MALAT1 oligo (si-MALAT1) (D) alone or together with Oct4 expression vector. Cell lysates were subjected to qRT-PCR assays for Oct4, NEAT1 and MALAT1 RNA expression or Western blot analysis for Oct4 protein expression (inset). GAPDH serves as an internal control. Data are mean ± SEM. P-values were determined by two-way ANOVA. **P < 0.01; ***P < 0.001. (PDF 911 kb)12943_2017_674_MOESM1_ESM.pdf
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- Oct4 transcriptionally regulates the expression of long non-coding RNAs NEAT1 and MALAT1 to promote lung cancer progression
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