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. 2018 May;109(5):1346-1356.
doi: 10.1111/cas.13587. Epub 2018 Apr 22.

Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 promotes lung adenocarcinoma by directly interacting with specificity protein 1

Shufeng Li  1 , Fang Ma  1 , Kunpeng Jiang  1 , Haitao Shan  1 , Minke Shi  2 , Baojun Chen  2
Affiliations

Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 promotes lung adenocarcinoma by directly interacting with specificity protein 1

Shufeng Li et al. Cancer Sci. 2018 May.

Abstract

Metastasis-associated lung adenocarcinoma transcript 1 (malat1) is an oncogenic long non-coding RNA (lncRNA) which has been proven to be associated with various types of tumors. Transcription factor specificity protein 1 (SP1) is overexpressed in many types of cancers. Previously, we observed that malat1 expression level is regulated by SP1 in lung cancer. In the present study, we found that transfection of expression construct of malat1 5' end fragment M5 enhances stability and transcriptional activity of SP1. Various SP1 target genes are also upregulated following overexpression of malat1 M5 in lung adenocarcinoma cells. We also showed that malat1 M5 interacts with the C-terminal domain of SP1 by RNA immunoprecipitation (RIP) assay coupled with UV cross-linking. Malat1-SP1 association results in increase of SP1 stability. In turn, SP1 promotes malat1 transcription, thus forming a positive feedback loop. In conclusion, our data show that in lung adenocarcinoma cells, malat1 interacts with SP1 protein and promotes SP1-mediated transcriptional regulation of SP1 target genes.

Keywords: SP1; lncRNA; lung cancer; malat1; transcription.

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Figures

Figure 1
Figure 1
Metastasis‐associated lung adenocarcinoma transcript 1 (malat1) 5′ end fragment M5 promoted cell proliferation. A, Schematic diagram of malat1 regions M1‐M5. B, Effects of malat1 fragments M1‐M5 on viability of cells were detected using CCK‐8. Values are means of 3 independent experiments ± SD,*P < .05. C, CCK‐8 test was carried out after cell transfection with si‐control, or si‐malat1. P < .01. D, Tumor growth curves indicate that malat1 siRNA injection treatment significantly inhibited A549 xenograft tumor growth whereas A549 cells transfected with pcDNA encoding the malat1 M5 fragment grew faster. *P < .01
Figure 2
Figure 2
Metastasis‐associated lung adenocarcinoma transcript 1 (malat1) fragment M5 has an important function in the migration and invasion of lung cancer A549 cells and H1299 cells. A, malat1 M5 fragment promoted cell invasion and migration, whereas knockdown of malat1 resulted in a marked inhibition of cell invasion and migration as determined by Transwell assays. A549 and H1299 cells were transfected with plasmid pcDNA as a control or pcDNA encoding the malat1 M5 fragment or control siRNA or malat1 siRNA. A‐1, Representative membranes stained with Giemsa are shown. Scale bar 100 μm. A‐2, Quantitative analysis of the number of the cells that migrated to the lower side of the membrane. Data are mean ± SD of 3 independent experiments.***P < .01 vs control. B, Wound‐healing assay was used to detect A549 cell motility changes after silencing malat1 by malat1 RNAi or overexpressing malat1 M5 fragment, then wound‐healing scratch motility assays were carried out. B‐1, Cell migration was assessed at 0 and 24 h. Representative images are shown. B‐2, Statistical analysis of wound closure. Gap size at 0 h was set to 100% and percentage of closed wound was calculated at 24 h (n = 3; ***P < .01)
Figure 3
Figure 3
Metastasis‐associated lung adenocarcinoma transcript 1 (malat1) M5 regulated the expression of specificity protein 1 (SP1) and SP1 target genes. A, malat1 siRNA significantly downregulated SP1 in A549 and H1299 cells, whereas overexpression of malat1 M5 caused accumulation of SP1 determined by immunofluorescence staining. Scale bar, 10 μm. B, After 48 h of transfection with pcDNA‐M5, blank vector pcDNA, si‐malat1 or si‐control in A549 and H1299 cells, total RNAs and protein, respectively, were collected, then (B‐1) RT‐PCR and (B‐2) western blotting were carried out to detect the levels of SP1,vascular endothelial growth factor (VEGF) and urokinase‐type plasminogen activator receptor (uPAR). GAPDH was used as control. B‐3, Relative density of SP1, VEGF and uPAR was plotted (relative to corresponding control) normalized to GAPDH ± SD from 3 independent experiments. ***P < .01. C, Analysis of luciferase intensity in cells cotransfected with pcDNA‐M5, blank vector pcDNA, si‐malat1 or si‐control, then with SP1‐Luc and the Renilla luciferase reporter plasmid. After 24 h of transfection, cells were assayed using a Dual‐Luciferase Reporter Assay System kit (Promega, Madison, WI, USA). All results are from 3 independent experiments. Values are mean ± SD, ***P < .01 compared with values from cells transfected with control
Figure 4
Figure 4
Analysis of the effect of metastasis‐associated lung adenocarcinoma transcript 1 (malat1) M5 overexpression or si‐malat1 on the half‐life of specificity protein 1 (SP1). A, A549 cells were transfected with pcDNA‐M5 or si‐malat1 for 24 h, then treated with the protein synthesis inhibitor cycloheximide (CHX, 10 μmol/L) for 0, 12, 16 or 24 h. Left panels represent quantified western blots, right panels represent mean densitometry of percentage SP1 (relative to 0 h) normalized to GAPDH ± SD from 3 independent repeats. Overexpression of M5 inhibited degradation of SP1 protein in cells treated with CHX, whereas si‐malat1 increased the degradation of SP1 protein. B, Similar experiments were carried out as above on H1299 cells. These results indicated that malat1 M5 stabilized SP1
Figure 5
Figure 5
Metastasis‐associated lung adenocarcinoma transcript 1 (malat1) M5 selectively interacted with full‐length specificity protein 1 (SP1) and SP1 C‐terminal DNA‐binding domain, but not with SP1 N‐terminus. A, Schematic diagram of SP1 protein structure and SP1 full‐length construct SP1‐FL and deletion mutant constructs SP1‐N and SP1‐C. B, In vitro transcribed digoxin‐labeled malat1 M5 RNA was incubated with A549 cell extract transfected with flag‐SP1‐FL vector or SP1 deletion mutant flag‐SP1‐N and association was detected by western blot of anti‐flag. C, In vitro direct binding of long non‐coding RNA (lncRNA) malat1 M5 with purified His‐SP1‐C protein tested by UV cross‐linking and gel‐shift assay. D, RNA‐immunoprecipitation (RNA‐IP) showed that malat1 lncRNA forms malat1/SP1 protein complexes in vivo. RIP experiments were carried out using antibody against SP1 on extracts from cells. Purified RNA was used for RT‐qPCR. Data are relative to mock‐IP (IgG). Graph depicts strong interaction with SP1 protein (n = 3)
Figure 6
Figure 6
Specificity protein 1 (SP1) and metastasis‐associated lung adenocarcinoma transcript 1 (malat1) were remarkably increased in lung adenocarcinoma cancer tissues. A, Total RNA was extracted from human lung adenocarcinoma cancer tissues and adjacent normal tissues with Trizol. Expression of long non‐coding RNA (lncRNA) malat1 and sp1 was examined by qRT‐PCR and normalized to GAPDH. ***P < .01. B, Immunohistochemistry of SP1 in adenocarcinoma cancer tissues and paired adjacent normal tissues (×400 magnification,). Scale bar, 100 μm. Immunohistochemistry analysis shows that SP1 expression was distributed in the nucleus and significantly increased in cancer tissues compared with normal tissues. C, Detection of malat1 (FISH) and SP1 (immunofluorescence) expression in the same cancer and corresponding normal tissue. Cell nuclei were stained with DAPI (blue). Malat1 was labeled by FITC (green). Tissue sections were observed at 200× magnification. Scale bar, 20 μm
Figure 7
Figure 7
Schematic diagram illustrating that metastasis‐associated lung adenocarcinoma transcript 1 (malat1) and specificity protein 1 (SP1) form a complex, ultimately affecting transcription of SP1 target genes. uPAR, urokinase‐type plasminogen activator receptor; VEGF, vascular endothelial growth factor
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