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Feedback regulations of miR-21 and MAPKs via Pdcd4 and Spry1 are involved in arsenite-induced cell malignant transformation.

Shen L, Ling M, Li Y, Xu Y, Zhou Y, Ye J, Pang Y, Zhao Y, Jiang R, Zhang J, Liu Q - PLoS ONE (2013)

Bottom Line: MiR-21 was highly expressed in arsenite-transformed HELF cells and normal HELF cells acutely treated with arsenite, an effect that was concomitant with activation of JNK/c-Jun and ERK/NF-κB and down-regulation of Pdcd4 and Spry1 protein levels.In arsenite-transformed HELF cells, blockages of JNK/c-Jun or ERK/NF-κB with inhibitors or with siRNAs prevented the increases of miR-21and the decreases of the protein levels but not the mRNA levels of Pdcd4 and Spry1.Moreover, in arsenite-transformed HELF cells, inhibition of miR-21 promoted cell apoptosis, inhibited clonogenicity, and reduced migration.

View Article: PubMed Central - PubMed

Affiliation: Institute of Toxicology, Ministry of Education, School of Public Health, Nanjing, Jiangsu, People's Republic of China.

ABSTRACT

Objective: To establish the functions of miR-21 and the roles of two feedback regulation loops, miR-21-Spry1-ERK/NF-κB and miR-21-Pdcd4-JNK/c-Jun, in arsenite-transformed human embryo lung fibroblast (HELF) cells.

Methods: For arsenite-transformed HELF cells, apoptosis, clonogenicity, and capacity for migration were determined by Hoechst staining, assessment of their capacity for anchorage-independent growth, and wound-healing, respectively, after blockage, with inhibitors or with siRNAs, of signal pathways for JNK/c-Jun or ERK/NF-κB. Decreases of miR-21 levels were determined with anti-miR-21, and the up-regulation of Pdcd4 and Spry1 was assessed in transfected cells; these cells were molecularly characterized by RT-PCR, qRT-PCR, Western blots, and immunofluorescence assays.

Results: MiR-21 was highly expressed in arsenite-transformed HELF cells and normal HELF cells acutely treated with arsenite, an effect that was concomitant with activation of JNK/c-Jun and ERK/NF-κB and down-regulation of Pdcd4 and Spry1 protein levels. However, there were no significant changes in mRNA levels for Pdcd4 and Spry1, which suggested that miR-21 regulates the expressions of Pdcd4 and Spry1 through translational repression. In arsenite-transformed HELF cells, blockages of JNK/c-Jun or ERK/NF-κB with inhibitors or with siRNAs prevented the increases of miR-21and the decreases of the protein levels but not the mRNA levels of Pdcd4 and Spry1. Down-regulation of miR-21 and up-regulations of Pdcd44 or Spry1 blocked the arsenite-induced activations of JNK/c-Jun or ERK/NF-κB, indicating that knockdown of miR-21 inhibits feedback of ERK activation and JNK activation via increases of Pdcd4 and Spry1 protein levels, respectively. Moreover, in arsenite-transformed HELF cells, inhibition of miR-21 promoted cell apoptosis, inhibited clonogenicity, and reduced migration.

Conclusion: The results indicate that miR-21 is both a target and a regulator of ERK/NF-κB and JNK/c-Jun and the feedback regulations of miR-21 and MAPKs via Pdcd4 and Spry1, respectively, are involved in arsenite-induced malignant transformation of HELF cells.

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The level of miR-21 is up-regulated, and the protein levels of Pdcd4 and Spry1 are decreased in arsenite-transformed HELF cells.Abbreviations: HELF-C, normal HELF cells; HELF-30C, passage-control HELF cells; HELF-30T, arsenite-transformed HELF cells. Densities of bands were quantified by Eagle Eye II software. GAPDH levels, measured in parallel, served as controls. HELF cells were exposed to 0.0 or 1.0 µM arsenite for 30 passages. (A) The levels of miR-21 were determined by qRT-PCR assays (means ± SD, n = 3). **P<0.01 different from HELF-30C cells. (B) The protein levels (upper) and mRNA levels (lower) of Pdcd4 and Spry1 (target proteins of miR-21) were analyzed by Western blots and RT-PCR, respectively. (C) The relative protein levels of Pdcd4 and Spry1 (means ± SD, n = 3). **P<0.01 different from HELF-30C cells.
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pone-0057652-g002: The level of miR-21 is up-regulated, and the protein levels of Pdcd4 and Spry1 are decreased in arsenite-transformed HELF cells.Abbreviations: HELF-C, normal HELF cells; HELF-30C, passage-control HELF cells; HELF-30T, arsenite-transformed HELF cells. Densities of bands were quantified by Eagle Eye II software. GAPDH levels, measured in parallel, served as controls. HELF cells were exposed to 0.0 or 1.0 µM arsenite for 30 passages. (A) The levels of miR-21 were determined by qRT-PCR assays (means ± SD, n = 3). **P<0.01 different from HELF-30C cells. (B) The protein levels (upper) and mRNA levels (lower) of Pdcd4 and Spry1 (target proteins of miR-21) were analyzed by Western blots and RT-PCR, respectively. (C) The relative protein levels of Pdcd4 and Spry1 (means ± SD, n = 3). **P<0.01 different from HELF-30C cells.

Mentions: Over-regulation of miR-21 is implicated in malignancy-related processes, including cell proliferation, apoptosis, invasion, and metastasis [3], [11]. To determine if miR-21 is involved in arsenite-induced transformation of HELF cells, miR-21 expression and the protein levels and miRNA levels of Pdcd4 and Spry1, target genes of miR-21, were investigated in normal HELF cells, in passage-control HELF cells, in arsenite-transformed HELF cells, and in normal HELF cells treated with 1.0 µM arsenite for 0, 6, or 24 h. The level of miR-21 was 8.8-fold higher in arsenite-transformed cells relative to passage-control cells (Figure 2A). In arsenite-transformed HELF cells, the protein levels of Pdcd4 and Spry1 were lower than those in passage control cells; however, there were no significant differences for mRNA levels of Pdcd4 and Spry1 in two groups of cells (Figure 2B and 2C). The results were the same for normal HELF cells treated acutely with 1.0 µM arsenite (Figure S2). Thus, over-expression of miR-21 and the decreases of target proteins are associated with arsenite-induced malignant transformation.


Feedback regulations of miR-21 and MAPKs via Pdcd4 and Spry1 are involved in arsenite-induced cell malignant transformation.

Shen L, Ling M, Li Y, Xu Y, Zhou Y, Ye J, Pang Y, Zhao Y, Jiang R, Zhang J, Liu Q - PLoS ONE (2013)

The level of miR-21 is up-regulated, and the protein levels of Pdcd4 and Spry1 are decreased in arsenite-transformed HELF cells.Abbreviations: HELF-C, normal HELF cells; HELF-30C, passage-control HELF cells; HELF-30T, arsenite-transformed HELF cells. Densities of bands were quantified by Eagle Eye II software. GAPDH levels, measured in parallel, served as controls. HELF cells were exposed to 0.0 or 1.0 µM arsenite for 30 passages. (A) The levels of miR-21 were determined by qRT-PCR assays (means ± SD, n = 3). **P<0.01 different from HELF-30C cells. (B) The protein levels (upper) and mRNA levels (lower) of Pdcd4 and Spry1 (target proteins of miR-21) were analyzed by Western blots and RT-PCR, respectively. (C) The relative protein levels of Pdcd4 and Spry1 (means ± SD, n = 3). **P<0.01 different from HELF-30C cells.
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getmorefigures.php?uid=PMC3585869&req=5

pone-0057652-g002: The level of miR-21 is up-regulated, and the protein levels of Pdcd4 and Spry1 are decreased in arsenite-transformed HELF cells.Abbreviations: HELF-C, normal HELF cells; HELF-30C, passage-control HELF cells; HELF-30T, arsenite-transformed HELF cells. Densities of bands were quantified by Eagle Eye II software. GAPDH levels, measured in parallel, served as controls. HELF cells were exposed to 0.0 or 1.0 µM arsenite for 30 passages. (A) The levels of miR-21 were determined by qRT-PCR assays (means ± SD, n = 3). **P<0.01 different from HELF-30C cells. (B) The protein levels (upper) and mRNA levels (lower) of Pdcd4 and Spry1 (target proteins of miR-21) were analyzed by Western blots and RT-PCR, respectively. (C) The relative protein levels of Pdcd4 and Spry1 (means ± SD, n = 3). **P<0.01 different from HELF-30C cells.
Mentions: Over-regulation of miR-21 is implicated in malignancy-related processes, including cell proliferation, apoptosis, invasion, and metastasis [3], [11]. To determine if miR-21 is involved in arsenite-induced transformation of HELF cells, miR-21 expression and the protein levels and miRNA levels of Pdcd4 and Spry1, target genes of miR-21, were investigated in normal HELF cells, in passage-control HELF cells, in arsenite-transformed HELF cells, and in normal HELF cells treated with 1.0 µM arsenite for 0, 6, or 24 h. The level of miR-21 was 8.8-fold higher in arsenite-transformed cells relative to passage-control cells (Figure 2A). In arsenite-transformed HELF cells, the protein levels of Pdcd4 and Spry1 were lower than those in passage control cells; however, there were no significant differences for mRNA levels of Pdcd4 and Spry1 in two groups of cells (Figure 2B and 2C). The results were the same for normal HELF cells treated acutely with 1.0 µM arsenite (Figure S2). Thus, over-expression of miR-21 and the decreases of target proteins are associated with arsenite-induced malignant transformation.

Bottom Line: MiR-21 was highly expressed in arsenite-transformed HELF cells and normal HELF cells acutely treated with arsenite, an effect that was concomitant with activation of JNK/c-Jun and ERK/NF-κB and down-regulation of Pdcd4 and Spry1 protein levels.In arsenite-transformed HELF cells, blockages of JNK/c-Jun or ERK/NF-κB with inhibitors or with siRNAs prevented the increases of miR-21and the decreases of the protein levels but not the mRNA levels of Pdcd4 and Spry1.Moreover, in arsenite-transformed HELF cells, inhibition of miR-21 promoted cell apoptosis, inhibited clonogenicity, and reduced migration.

View Article: PubMed Central - PubMed

Affiliation: Institute of Toxicology, Ministry of Education, School of Public Health, Nanjing, Jiangsu, People's Republic of China.

ABSTRACT

Objective: To establish the functions of miR-21 and the roles of two feedback regulation loops, miR-21-Spry1-ERK/NF-κB and miR-21-Pdcd4-JNK/c-Jun, in arsenite-transformed human embryo lung fibroblast (HELF) cells.

Methods: For arsenite-transformed HELF cells, apoptosis, clonogenicity, and capacity for migration were determined by Hoechst staining, assessment of their capacity for anchorage-independent growth, and wound-healing, respectively, after blockage, with inhibitors or with siRNAs, of signal pathways for JNK/c-Jun or ERK/NF-κB. Decreases of miR-21 levels were determined with anti-miR-21, and the up-regulation of Pdcd4 and Spry1 was assessed in transfected cells; these cells were molecularly characterized by RT-PCR, qRT-PCR, Western blots, and immunofluorescence assays.

Results: MiR-21 was highly expressed in arsenite-transformed HELF cells and normal HELF cells acutely treated with arsenite, an effect that was concomitant with activation of JNK/c-Jun and ERK/NF-κB and down-regulation of Pdcd4 and Spry1 protein levels. However, there were no significant changes in mRNA levels for Pdcd4 and Spry1, which suggested that miR-21 regulates the expressions of Pdcd4 and Spry1 through translational repression. In arsenite-transformed HELF cells, blockages of JNK/c-Jun or ERK/NF-κB with inhibitors or with siRNAs prevented the increases of miR-21and the decreases of the protein levels but not the mRNA levels of Pdcd4 and Spry1. Down-regulation of miR-21 and up-regulations of Pdcd44 or Spry1 blocked the arsenite-induced activations of JNK/c-Jun or ERK/NF-κB, indicating that knockdown of miR-21 inhibits feedback of ERK activation and JNK activation via increases of Pdcd4 and Spry1 protein levels, respectively. Moreover, in arsenite-transformed HELF cells, inhibition of miR-21 promoted cell apoptosis, inhibited clonogenicity, and reduced migration.

Conclusion: The results indicate that miR-21 is both a target and a regulator of ERK/NF-κB and JNK/c-Jun and the feedback regulations of miR-21 and MAPKs via Pdcd4 and Spry1, respectively, are involved in arsenite-induced malignant transformation of HELF cells.

Show MeSH
Related in: MedlinePlus