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. 2022 Feb 15;12(1):2548.
doi: 10.1038/s41598-022-06154-2.

E2F1-induced lncRNA, EMSLR regulates lncRNA LncPRESS1

Priyanka Priyanka  1 , Madhur Sharma  2 , Sanjeev Das  1 , Sandeep Saxena  3   4
Affiliations

E2F1-induced lncRNA, EMSLR regulates lncRNA LncPRESS1

Priyanka Priyanka et al. Sci Rep. .

Abstract

E2F1 induces hundreds of protein-coding genes influencing diverse signaling pathways but much less is known about its non-coding RNA targets. For identifying E2F1-dependent oncogenic long non-coding RNAs (lncRNAs), we carried out genome-wide transcriptome analysis and discovered an lncRNA, EMSLR, which is induced both in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). EMSLR depletion blocks the cells in G1 phase and inhibits the clonogenic ability indicating that it is essential for the tumor-related phenotypes. We discovered that EMSLR represses the promoter activity of another lncRNA, LncPRESS1, which is located 6.9 kb upstream of EMSLR and they display an inverse expression pattern in lung cancer cell lines. Depletion of C-MYC results in downregulation of EMSLR and simultaneous upregulation of EMSLR target LncPRESS1, exemplifying how C-MYC and E2F1 signal transduction pathways control the network of lncRNA genes to modulate cell proliferation and differentiation.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Transriptome analysis identifies EMSLR, an E2F1-dependent lncRNA that is upregulated in TCGA-LUAD and TCGA-LUSC datasets. (A) Strategy used for the identification of E2F1-dependent dysregulated lncRNAs in LUAD and LUSC, determination of their gene targets and their effect on cell proliferation and oncogenesis. (B) Comparison of transcriptional profiles identifies 111 lncRNAs upregulated in both LUAD and LUSC. The expression of around 12,000 lncRNAs was compared between 488 tumor and 58 normal samples of LUAD as well as 220 tumor and 17 normal samples of LUSC to identify upregulated (fold change > 2; upright arrow) or downregulated (fold change < 0.5; inverted arrow) lncRNAs. 111 lncRNAs that were upregulated in both cancers have been depicted by an intersecting Venn diagram. (C) From the 111 upregulated lncRNAs, 6 lncRNAs were selected which were previously reported to have effects on cell proliferation and oncogenesis. The plot displays the relative expression of the selected lncRNAs, namely ZFAS1, SNHG17, VPS9D1-AS1, PCAT6, LINC00467 and EMSLR in 488 tumor and 58 normal samples of LUAD dataset. Each point refers to the levels of selected lncRNAs in one sample, whereas long and short horizontal bars represent the mean and S.D., respectively. Due to exceptionally high expression of ZFAS1, its log2 FPKM values has been plotted with respect to the left vertical axis (range from 0–300), while the other lncRNAs have been plotted with respect to the right vertical axis (range from 0–30). p values calculated using Student’s t test display that expression of the selected lncRNAs is significantly different in LUAD samples compared to their respective normal samples (*p < 0.05; **p < 0.01, ***p < 0.001). (D) Relative expression of the selected lncRNAs in 220 tumor and 17 normal samples of LUSC dataset. Details are same as part (C).
Figure 2
Figure 2
EMSLR depletion blocks the cell cycle progression. (A) Identification of E2F1-dependent lncRNA, EMSLR. A549 cells were transfected on three consecutive days with control GL2 siRNA or E2F1 siRNA and the levels of indicated lncRNAs were quantified by individual quantitative real-time PCR. The bar-graph indicates the levels of individual lncRNA in E2F1 depleted samples relative to control GL2 samples. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the endogenous control for normalization of lncRNA expression. (B) RNAi mediated depletion of EMSLR. A549 cells were transfected on three consecutive days with control GL2 siRNA or siRNA targeting different regions of EMSLR: siRNA (I) and siRNA (II) followed by quantification of EMSLR levels by individual quantitative real-time PCR. GAPDH was used as the endogenous control for normalization of EMSLR expression. (C) Depletion of EMSLR reduces the rate of cell proliferation. MTT assay displaying the growth rates of EMSLR-depleted A549 cells as described in part (B) were seeded at equal numbers after transfection. The graph represents the growth rate with respect to control siRNA transfected cells at the indicated time after seeding the cells. (D) Overexpression of EMSLR enhances the rate of cell proliferation. A549 cells were infected with lentivirus expressing EMSLR or GFP followed by seeding of equal number of cells at 0 h and measurement of growth rates by MTT assay till 96 h. (E) Depletion of EMSLR leads to an accumulation of cells in the G1 phase. EMSLR-depleted A549 cells, as described in part (B) were obtained followed by treatment with nocodazole for 16 h and stained with propidium iodide (PI) for analysis of cell cycle distribution by flow cytometry. The percentage of cells in the G1 phase is significantly different in EMSLR siRNA samples compared to control GL2 siRNA samples. (F–G) EMSLR-depleted H1299 cells were obtained by transfecting EMSLR siRNA (I) or siRNA (II) followed by treatment with nocodazole for 16 h and stained with propidium iodide (PI) for analysis of cell cycle distribution by flow cytometry. The efficiency of siRNA depletion of EMSLR in H1299 cells is shown in part (G). (H) Depletion of EMSLR impedes S phase progression. Flow cytometry of control GL2 or EMSLR siRNA (I)-depleted A549 cells, as described in part (B), pulsed with BrdU followed by staining with FITC-conjugated anti-BrdU antibody along with propidium iodide. Dot plot displays BrdU incorporation (y-axis) and DNA content (x-axis) and inset shows the cells incorporating BrdU. The top-left panel (asynchronous) shows background FITC signal in the absence of anti-BrdU antibody. (I) EMSLR depletion results in downregulation of major cell-cycle related genes. A549 cells were transduced with lentiviral particles expressing either control shRNA or EMSLR shRNA followed by puromycin selection to obtain stable knockdown cells. The level of EMSLR and other cell-cycle related genes was quantified by individual quantitative real-time PCR. GAPDH was used as the endogenous control for normalization. The data is represented as mean of two independent experiments ± S.D. p values were calculated using two-tailed t-test (*p < 0.05; **p < 0.01, ***p < 0.001).
Figure 3
Figure 3
EMSLR depletion inhibits the tumor-related phenotypes of LUAD cells. (A) Depletion of EMSLR induces cell death in A549 cells. Flow cytometry of control GL2 or EMSLR-depleted A549 cells, obtained by transfecting EMSLR siRNA (I) or siRNA (II), stained with FITC-conjugated anti-Annexin V antibody along with propidium iodide. Dot plot displays PI (y-axis) and Annexin V (x-axis) incorporation. Criteria were set to distinguish between viable (bottom left), early apoptotic (bottom right) and late apoptotic (top right) cells. (B–D) Clonogenic assay to evaluate the effect of EMSLR manipulation on A549 cells. (B) EMSLR siRNA (I)-depleted A549 cells as described in Fig. 2B were allowed to grow for 12 days, stained with crystal violet and the colonies were counted. (C) A549 cells were infected with lentivirus expressing EMSLR and grown for 12 days, followed by staining with crystal violet and the colonies were counted. Quantification of parts (B) and (C) is shown in part (D). The data is represented as mean of two independent experiments ± S.D. p values were calculated using two-tailed t-test (*p < 0.05; **p < 0.01, ***p < 0.001).
Figure 4
Figure 4
EMSLR represses a closely located lncRNA, LncPRESS1. (A) G-banded ideogram representing human chromosome 7, showing the cytogentic location of LncPRESS1, EMSLR, IFT22, SERPINE1 and VGF genes at the 7q22.1 cytogenetic band. The genomic coordinates of LncPRESS1 gene are 101,299,613..101,301,346 while the EMSLR gene lies within 101,308,270..101,314,800 nt as per Genome Reference Consortium Human Build 38 patch release 13 (GRCh38.p13). Three alternatively spliced variants of EMSLR have been identified, namely NR 110115.2, ENST00000419422.2 and ENST00000663483.1. NR 110115.2 variant has been characterized as the functional EMSLR in a previous study. The LncPRESS1 and EMSLR gene are expressed in the same direction from the "forward" strand of chromosome 7. The IFT22 gene which lies within the complement strand from 101,310,914..101,321,812 has a partial overlap with EMSLR gene. Also shown in the discontinuous ideogram are the locations of SERPINE1 (101,127,104..101,139,247) and VGF (complement: 101,162,509..101,169,956) genes. (B) Effect of EMSLR depletion on the expression of the genes located in its vicinity. A549 cells were transduced with lentiviral particles expressing either control shRNA or EMSLR shRNA followed by puromycin selection to obtain stable knockdown cells and the levels of indicated genes were quantified by individual quantitative real-time PCR. (C–D) Depletion of EMSLR leads to upregulation of lncRNA LncPRESS1. A549 cells were transfected with control GL2 or EMSLR siRNA (I) followed by quantification of EMSLR and LncPRESS1 levels by individual quantitative real-time PCR. (E–F) Depletion of EMSLR by shRNA that targets a different region in EMSLR compared to EMSLR siRNA (I) or EMSLR siRNA (II) leads to upregulation of lncRNA LncPRESS1. A549 cells were transduced with lentiviral particles expressing either control shRNA or EMSLR shRNA followed by puromycin selection to obtain stable knockdown cells and quantification of EMSLR and LncPRESS1 levels by individual quantitative real-time PCR. (G–H) Ectopic expression of EMSLR represses endogenous LncPRESS1 expression. A549 cells were infected with lentiviral expressing EMSLR followed by quantification of EMSLR and LncPRESS1 levels by individual quantitative real-time PCR. The data of part (B–H) is from mean of two independent experiments ± S.D. GAPDH was used as the endogenous control for normalization. (I–J) Relative expression of LncPRESS1 and EMSLR in different lung cell lines with respect to MRC-5, a human lung fibroblast cell line derived from normal lung tissue which is used as a control for non small cell lung cancer. The quantification of EMSLR and LncPRESS1 levels in different cell lines was carried out by individual quantitative real-time PCR and normalized with 18 s RNA expression from two technical replicates. p values were calculated using two-tailed t-test (*p < 0.05; **p < 0.01, ***p < 0.001, ns, non-significant).
Figure 5
Figure 5
EMSLR mediates the transcriptional repression of LncPRESS1. (A) Schematic representation of the reporter plasmid containing the human LncPRESS1 upstream region: The LncPRESS1 promoter and 5′UTR region spanning − 1500 bp to + 50 bp with respect to transcriptional start site (TSS) was used to drive expression of the firefly luciferase gene (Fluc) in promoterless pGL4.20 vector (promega). (B) Depletion of EMSLR, by transfecting EMSLR siRNA (I) or siRNA (II), relieves the repression of LncPRESS1 promoter in A549 cells. The pGL4.20 vector containing LncPRESS1 promoter was transfected into control or EMSLR-depleted A549 cells together with a renilla luciferase (pRL-TK) reporter vector and both luciferase activities were measured after 24 h. The relative luciferase activity in each sample is expressed as a ratio of firefly to renilla luminescence. (C) A549 cells were infected with lentivirus expressing EMSLR followed selection with puromycin to obtain stable cells. The pGL4.20 vector containing LncPRESS1 promoter was transfected into control or EMSLR-expressing stable A549 cells together with a renilla luciferase (pRL-TK) reporter vector and both luciferase activities were measured after 24 h. The relative luciferase activity in each sample is expressed as a ratio of firefly to renilla luminescence. (D) Depletion of EMSLR, by transfecting EMSLR siRNA (I) or siRNA (II), relieves the repression of LncPRESS1 promoter in H1299 cells. The pGL4.20 vector containing LncPRESS1 promoter was transfected into control or EMSLR-depleted H1299 cells together with a renilla luciferase (pRL-TK) reporter vector and both luciferase activities were measured after 24 h. The relative luciferase activity in each sample is expressed as a ratio of firefly to renilla luminescence. (E) H1299 cells were infected with lentivirus expressing GFP or EMSLR followed by selection with puromycin to obtain stable cells. The pGL4.20 vector containing LncPRESS1 promoter was transfected into GFP or EMSLR-expressing stable H1299 cells together with a renilla luciferase (pRL-TK) reporter vector and both luciferase activities were measured after 24 h. The relative luciferase activity in each sample is expressed as a ratio of firefly to renilla luminescence. (F) Kaplan–Meier estimates of the survival of patients with low or high levels of expression of EMSLR in LUAD. The expression and survival information was downloaded from GEPIA (Gene Expression Profiling Interactive Analysis) platform. (G) Kaplan–Meier estimates of the survival of patients with low or high levels of expression of LncPRESS1. The sample size of LUAD was insufficient for correlating survival probability with LncPRESS1 expression. Thus the survival probability was calculated in a combination of samples from all 33 cancers available at GEPIA with a high group cutoff of 10% and low group cutoff of 90%. The data is represented as mean of two independent experiments ± S.D. p values were calculated using two-tailed t-test (*p < 0.05; **p < 0.01, ***p < 0.001, ns, non-significant).
Figure 6
Figure 6
C-MYC signal transduction pathways control LncPRESS1 expression. (A) The transcriptional repression of LncPRESS1 mediated by EMSLR is dependent on DNA Methyltransferase 1. A549 cells were transduced with lentiviral expressing either control vector or EMSLR followed by puromycin selection to obtain stable cells. Control or EMSLR stably overexpressing A549 cells were then transfected with control GL2 siRNA or siRNA targeting DNMT1, DNMT3B or EZH2 followed by quantification of LncPRESS1 levels by individual quantitative real-time PCR with GAPDH as an endogenous control. The levels of LncPRESS1 observed after different siRNA transfections in EMSLR expressing stable cells were normalized to non-EMSLR expressing cells. (B) Effect of DNMT1 depletion on the expression of genes located in the vicinity of LncPRESS1. A549 cells were transfected on three consecutive days with control GL2 or DNMT1 siRNA and the levels of indicated genes were quantified by individual quantitative real-time PCR. GAPDH was used as the endogenous control for normalization of lncRNA expression. (C–D), Myc signal transduction pathways control LncPRESS1 expression. A549 cells were transfected with control GL2 or siRNA targeting C-MYC followed by quantification of C-MYC, E2F1, EMSLR, LncPRESS1 and VPS9D1-AS1 levels by individual quantitative real-time PCR. GAPDH was used as the endogenous control for normalization. The data is represented as mean of two independent experiments ± S.D. p values were calculated using two-tailed t-test (*p < 0.05; **p < 0.01, ***p < 0.001, ns, non-significant). (E) An illustration depicting that positive feedback loops connect C-MYC and E2F1 signals to cell cycle control. The model is based on the following observations: (i) C-MYC and E2F1 activate each other’s transcription, depicted by a ‘ + ’ sign; (ii) C-MYC induces EMSLR which stabilizes E2F1 mRNA; (iii) Our observations that EMSLR expression is dependent on E2F1 and EMSLR represses LncPRESS1. The LncPRESS1 gene is located 6.9 kb upstream of the E2F1-dependent lncRNA, EMSLR and both are expressed in the same direction from the cytogenic band 7q22.1. CFU stands for colony-forming units.
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