Dataset Information

Negative regulation of lncRNA GAS5 by miR-21.

ABSTRACT: In addition to protein-coding genes, the human genome makes a large amount of noncoding RNAs, including microRNAs and long noncoding RNAs (lncRNAs). Both microRNAs and lncRNAs have been shown to have a critical role in the regulation of cellular processes such as cell growth and apoptosis, as well as cancer progression and metastasis. Although it is well known that microRNAs can target a large number of protein-coding genes, little is known whether microRNAs can also target lncRNAs. In the present study, we determine whether miR-21 can regulate lncRNA expression. Using the lncRNA RT-PCR (reverse transcription-polymerase chain reaction) array carrying 83 human disease-related lncRNAs, we show that miR-21 is capable of suppressing the lncRNA growth arrest-specific 5 (GAS5). This negative correlation between miR-21 and GAS5 is also seen in breast tumor specimens. Of interest, GAS5 can also repress miR-21 expression. Whereas ectopic expression of GAS5 suppresses, GAS5-siRNA increases miR-21 expression. Importantly, there is a putative miR-21-binding site in exon 4 of GAS5; deletion of the miR-21-binding site abolishes this activity. Experiments with in vitro cell culture and xenograft mouse model suggest that GAS5 functions as a tumor suppressor. We further show that the biotin-labeled GAS5-RNA probe is able to pull down the key component (AGO2) of the RNA-induced silencing complex (RISC) and we subsequently identify miR-21 in this GAS5-RISC complex, implying that miR-21 and GAS5 may regulate each other in a way similar to the microRNA-mediated silencing of target mRNAs. Together, these results suggest that miR-21 targets not only tumor-suppressive protein-coding genes but also lncRNA GAS5.

SUBMITTER: Zhang Z

PROVIDER: S-EPMC3792431 | biostudies-other | 2013 Nov

REPOSITORIES: biostudies-other

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Negative regulation of lncRNA GAS5 by miR-21.

Zhang Z Z Zhu Z Z Watabe K K Zhang X X Bai C C Xu M M Wu F F Mo Y-Y YY

Cell death and differentiation 20130809 11

In addition to protein-coding genes, the human genome makes a large amount of noncoding RNAs, including microRNAs and long noncoding RNAs (lncRNAs). Both microRNAs and lncRNAs have been shown to have a critical role in the regulation of cellular processes such as cell growth and apoptosis, as well as cancer progression and metastasis. Although it is well known that microRNAs can target a large number of protein-coding genes, little is known whether microRNAs can also target lncRNAs. In the prese ...[more]

PMID: 23933812

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Project description:Porphyromonas gingivalis (P. gingivalis) is a major periodontal pathogen that can induce an immune response leading to a destructive inflammatory process. During the inflammatory process, interleukin-12 (IL-12) is secreted, correlating with bacterial clearance by macrophages. Bacterial sialidase has recently been shown to influence the synthesis and modification of the macromolecules on its surface, and is associated with the interaction between bacteria and host cells. We have previously constructed a P. gingivalis sialidase gene mutant strain in P. gingivalis W83 (ΔPG0352) and found that ΔPG0352 showed less pathogenicity than the wild-type strain. In this study, U937-differentiated macrophages were stimulated by P. gingivalis W83, ΔPG0352, or PG0352 complemented strain (comΔPG0352). Transmission electron microscopy showed that P. gingivalis caused a loss of membrane integrity in macrophages and the intracellular bacteria were enclosed within endocytic vacuoles. The expression of both IL-12p35 and IL-12p40 genes and the levels of IL-12p70 were significantly higher in U937 stimulated by ΔPG0352 than in those with P. gingivalis W83 and comΔPG0352. In order to explain why ΔPG0352 induced more IL-12 in macrophages, immunofluorescence assays, PCR arrays, and gene silence or overexpression experiments were carried out. Immunofluorescence assays showed that ΔPG0352 induced lower expression of CR3 in macrophages. After CR3 was suppressed, there were no significant differences in the IL-12p70 levels between macrophages stimulated by P. gingivalis W83, ΔPG0352 or comΔPG0352. PCR array experiments showed that miR-21 and lncRNA GAS5 were differentially expressed between macrophages stimulated by P. gingivalis W83 and ΔPG0352, which had been identified by real-time PCR. The results of CR3 blocking and lncRNA GAS5 gene silence or overexpression showed that the difference in IL-12 levels between P. gingivalis W83 and ΔPG0352 groups was associated with CR3, lncRNA GAS5 and miR-21. Thus it can be concluded that the sialidase-deficient strain is more easily cleared by attenuating CR3 activation, reducing the inhibition of lncRNA GAS5, inducing less miR-21 and more IL-12 in macrophages. These results indicate that inhibiting the activity of sialidase in P. gingivalis will cause rapid clearing by macrophages.

Project description:Background: Vascular aging is considered a special risk factor for cardiovascular diseases, and vascular smooth muscle cells (VSMCs) play a major role in aging-related vascular remodeling and in the pathological process of atherosclerosis. Recent research has reported that long non-coding RNA/microRNA (lncRNA/miRNA) is a critical regulator of cellular senescence. However, the role and mechanism of lncRNA GAS5/miR-665 axis in VSMC senescence remain incompletely understood. Methods: Cellular senescence was evaluated using senescence-associated β-gal activity, the NAD+/NADH ratio, and by immunofluorescence staining of γH2AX immunofluorescence. Differentially expressed miRNAs (DEMs) were identified by miRNA microarray assays and subsequently validated by quantitative real-time PCR (qRT-PCR). A dual luciferase reporter assay was conducted to confirm the binding of lncRNA GAS5 and miR-665 as well as miR-665 and syndecan 1 (SDC1). Serum levels of miR-665, lncRNA GAS5, and SDC1 in 93 subjects were detected by qRT-PCR. The participants were subdivided into control, aging, and early vascular aging (EVA) groups, and their brachial-ankle pulse wave velocity (baPWV) was measured. Results: A total of 20 overlapping DEMs were identified in young and old VSMCs via microarray analysis. MiR-665 showed a significant alteration and, therefore, was selected for further analysis. Upregulation of miR-665 was found in aging VSMCs, and downregulation of miR-665 caused an inhibition of VSMCs senescence. Subsequently, the dual luciferase reporter assay determined the binding site of miR-665 with the 3'-UTR of lncRNA GAS5 and SDC1. Increased expression of lncRNA GAS5 expression inhibited the miR-665 level and VSMC senescence. However, as shown in rescue experiment results, either miR-665 overexpression or SDC1 knockdown significantly reversed the effects of lncRNA GAS5 on VSMC senescence. Finally, compared with that of the control group, miR-665 was highly expressed in serum samples in the aging and EVA groups, especially in the EVA groups. On the contrary, serum levels of lncRNA GAS5 and SDC1 were lower in these two groups. Collectively, in the aging and EVA groups, miR-665 expression was negatively correlated with lncRNA GAS5 and SDC1 expression. Conclusion: miR-665 inhibition functions as a vital modulator of VSMC senescence by negatively regulating SDC1, which is achieved by lncRNA GAS5 that sponges miR-665. Our findings may provide a new treatment strategy for aging-related cardiovascular diseases.

Dataset Information

Negative regulation of lncRNA GAS5 by miR-21.

Publications

Negative regulation of lncRNA GAS5 by miR-21.

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