Target gene expression levels and competition between transfected and endogenous microRNAs are strong confounding factors in microRNA high-throughput experiments

Saito, Takaya; Sætrom, Pål

doi:10.1186/1758-907x-3-3

Cited by 33 publications

(24 citation statements)

References 38 publications

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“…Among exo-miRNA target genes, the genes with short 3′ UTRs were more efficiently downregulated than those with long 3′ UTRs by the transfection of exo-miRNAs (Figure 5A), consistent with the previous report [31]. In this analysis, we used the fold change values of exo-miRNA targets with 3′ UTRs complementary to seed sequence of either one strand of exo-miRNA duplex: let-7b-5p, miR-1, miR-21-5p, miR-22-5p, miR-28-5p, miR-30c-5p, miR-186-5p, miR-199b-5p, miR-200b-3p, miR-330-5p, miR-335-5p, miR-346, miR-466, miR-574-5p and miR-3126-5p.…”

Section: Resultssupporting

confidence: 93%

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Fluctuation of Global Gene Expression by Endogenous miRNA Response to the Introduction of an Exogenous miRNA

Nagata

Shimizu

Hibio

et al. 2013

IJMS

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Most of the intracellular endogenous microRNAs (endo-miRNAs) are considered to be saturated in Argonaute (Ago) proteins in the RNA-induced silencing complexes (RISCs). When exogenous miRNAs (exo-miRNAs) are introduced into cells, endo-miRNAs in the RISC may be replaced with exo-miRNAs or exo-miRNAs, and endo-miRNAs might also compete for the position in the newly synthesized RISC with each other. This would lead to the fluctuation of global gene expression not only by repression of exo-miRNA target gene expression, but also by the increase of the endo-miRNA target gene expression. In the present study, we quantified the changes in the expression levels of target genes of exo-miRNA and endo-miRNA in the cells transfected with fifteen different exo-miRNAs by microarray experiments. Different exo-miRNAs increased ratios of expression levels of target genes of a given endo-miRNA to different extents, suggesting that the replacement efficiencies might differ according to the exo-miRNA types. However, the increased ratios in the expression levels of each endo-miRNA target genes by the transfection of any particular exo-miRNA were mostly equivalent, suggesting that the endo-miRNAs present in the RISC might be replaced with excessive exo-miRNAs at similar levels, probably because they exist in single-stranded forms in the RISC.

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Section: Resultssupporting

confidence: 93%

“…The results showed good agreement with the previous study [31]. In contrast, the endo-miRNA targets with low expression levels in the normal conditions were uncovered to be intensively upregulated by the exo-miRNA transfection, and those with high expression levels were not upregulated efficiently (Figure 6B).…”

Section: Resultssupporting

confidence: 92%

Fluctuation of Global Gene Expression by Endogenous miRNA Response to the Introduction of an Exogenous miRNA

Nagata

Shimizu

Hibio

et al. 2013

IJMS

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“…Possible mechanisms could include the following: other miRs, such as miR-20b 58 and 519c, 59 triggering reverse effects upon HIF-1a may contribute to the hypoxia response in a complicated network involving the HIF-1a/Fhl-1 pathway; miR-206 may switch its regulating function in a quantitydependent manner in response to hypoxia; and when miR-206 mimics were applied, competition and saturation effects from endogenous miR-206 could have been involved in the process. 60,61 These effects would be expected to relieve repression of the HIF-1a from other endogenous miRs. The fact that miR-206 is involved in hypoxia-induced PH and that its expression changes frequently in different microenvironments suggests that multiple modes of transcriptional regulation exist for this miR.…”

Section: Discussionmentioning

confidence: 99%

MicroRNA-206 is involved in hypoxia-induced pulmonary hypertension through targeting of the HIF-1α/Fhl-1 pathway

Yue

Guan

Wang

et al. 2013

Laboratory Investigation

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Hypoxia-induced pulmonary hypertension (PH), which is characterized by vasoconstriction and subsequent structural remodeling of blood vessels, is an important event in chronic obstructive pulmonary disease patients and in people living at high altitudes. Hypoxia-inducible factor-1a (HIF-1a) and its regulator four-and-a-half LIM (Lin-11, Isl-1 and Mec-3) domain 1 (Fhl-1) have important roles in hypoxia-induced PH. is critical for myogenesis and related diseases; however, the role of miR-206 in hypoxia-induced PH is unknown. miR-206 expression was evaluated in a hypoxic rat model and in cultured hypoxic pulmonary artery smooth muscle cells (PASMCs) using real-time quantitative PCR (RT-qPCR). HIF-1a and Fhl-1 expression were evaluated using RT-qPCR, western blotting, immunohistochemistry and immunofluorescence. The function of miR-206 was assessed by transfecting miR-206 mimics and inhibitors. Dualluciferase reporter gene assays and western blotting were performed to validate the target genes of miR-206. siRNA targeted against Fhl-1 was used to investigate the effect of Fhl-1 on miR-206. Flow cytometry was used to detect the cell cycle phase distribution in each group of PASMCs. Significant downregulation of miR-206 in hypoxic lung tissue and PASMCs was identified, whereas HIF-1a and Fhl-1 were upregulated in these samples. The expression of miR-206 in the serum was different from that in the lung tissue. Transfection of pre-miR miR-206 in hypoxic conditions led to increased expression of HIF-1a and Fhl-1 rather than abolishing hypoxia-induced HIF-1a and Fhl-1, as was expected, and promoted the entry of cells into the S phase and enhanced PASMC proliferation. Fhl-1-targeted siRNA in PASMC prevented cell proliferation and led to an increased proportion of cells in the G1 phase without altering miR-206 expression. Bioinformatic analysis and dual-luciferase reporter gene assays revealed direct evidence for miR-206 targeting of HIF-1a. In conclusion, hypoxia-induced downregulation of miR-206 promotes PH by targeting the HIF-1a/Fhl-1 pathway in PASMCs. miR-206 could be a triggering factor of early stage of hypoxia-induced PH. Hypoxia-induced pulmonary hypertension (PH) is a serious disorder characterized by pulmonary vasoconstriction and enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs) that lead to structural remodeling of blood vessel walls. PH is a common clinical manifestation of chronic obstructive pulmonary disease (COPD) and in people who live at high altitude. 1 Hypoxia-inducible factor-1a (HIF-1a) has a key role in the pathophysiology of hypoxia-induced PH.The four-and-a-half LIM domain 1 (Fhl-1) protein, a member of the Fhl family, has also been implicated in the biological pathways of muscle growth and differentiation in PH 2-4 and is regulated by HIF-1a in a feedback loop that serves to limit HIF-1a activity under conditions of prolonged hypoxia. 5 MicroRNAs (miRs) are a class of B23-nucleotide, noncoding single-stranded RNAs that regulate gene function by identifying the 3 0 -untransl...

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“…In addition, Sinha et al reported that genes promoters with high CpG content were more often targeted by miRNAs 12 . Saito and Saetrom also discussed the relationships between miRNAmediated gene regulation and various features of target genes, but they did not consider the methylation status of target gene promoters 13 . Although the study of Su et al represents the first evidence of a direct link between promoter methylation and miRNA regulation, the biological significance of their findings is not clear.…”

Section: Introductionmentioning

confidence: 99%

Correlation between miRNA-targeted-gene promoter methylation and miRNA regulation of target genes

Taguchi

2013

F1000Res

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Science and Technology Norway Discuss this article AbstractmiRNA regulation of target genes and promoter methylation are Background known to be the primary mechanisms underlying the epigenetic regulation of gene expression. However, how these two processes cooperatively regulate gene expression has not been extensively studied.Gene expression and promoter methylation profiles of 271 distinct Methods human cell lines were obtained from gene expression omnibus. P-values that describe both miRNA-targeted-gene promoter methylaion and miRNA regulation of target genes were computed using the MiRaGE method proposed recently by the author.Significant changes in promoter methylation were associated with Results miRNA targeting. It was also found that miRNA-targeted-gene promoter hypomethylation was related to differential target gene expression; the genes with miRNA-targeted-gene promoter hypomethylation were downregulated during cell senescence and upregulated during cellular differentiation. Promoter hypomethylation was especially enhanced for genes targeted by miR-548 miRNAs, which are non-conserved, primate-specific miRNAs that are typically expressed at lower levels than the frequently investigated conserved miRNAs.It was found that promoter methylation was affected by miRNA Conclusions targeting. Furthermore, miRNA-targeted-gene promoter hypomethylation is suggested to facilitate gene regulation by miRNAs that are not strongly expressed (e.g., miR-548 miRNAs).

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Target gene expression levels and competition between transfected and endogenous microRNAs are strong confounding factors in microRNA high-throughput experiments

Cited by 33 publications

References 38 publications

Fluctuation of Global Gene Expression by Endogenous miRNA Response to the Introduction of an Exogenous miRNA

Fluctuation of Global Gene Expression by Endogenous miRNA Response to the Introduction of an Exogenous miRNA

MicroRNA-206 is involved in hypoxia-induced pulmonary hypertension through targeting of the HIF-1α/Fhl-1 pathway

Correlation between miRNA-targeted-gene promoter methylation and miRNA regulation of target genes

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