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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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Inhibition of miR-21 increases cell apoptosis of arsenite-transformed HELF cells.Densities of bands were quantified by Eagle Eye II software. GAPDH levels, measured in parallel, served as controls. Arsenite-transformed HELF cells were transfected with 150 nM of anti-miR-nc or anti-miR-21 for 24 h. (A) Western blots and (B) relative protein levels of cleaved caspase-3 and caspase-3 (means ± SD, n = 3). **P<0.01 different from NC group. (C) The rate of cell apoptosis was analyzed by the Hoechst 33258 assay (means ± SD, n = 3). **P<0.01 different from NC group.
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pone-0057652-g007: Inhibition of miR-21 increases cell apoptosis of arsenite-transformed HELF cells.Densities of bands were quantified by Eagle Eye II software. GAPDH levels, measured in parallel, served as controls. Arsenite-transformed HELF cells were transfected with 150 nM of anti-miR-nc or anti-miR-21 for 24 h. (A) Western blots and (B) relative protein levels of cleaved caspase-3 and caspase-3 (means ± SD, n = 3). **P<0.01 different from NC group. (C) The rate of cell apoptosis was analyzed by the Hoechst 33258 assay (means ± SD, n = 3). **P<0.01 different from NC group.

Mentions: To determine if miR-21 functions as an onco-miRNA or as anti-onco-miRNA, the effect of miR-21 was assessed on apoptosis of arsenite-transformed HELF cells. After arsenite-transformed cells were transfected with anti-miR-21, there were increases of cleaved-caspase-3 and decreases of caspase-3 levels (Figure 7A and 7B). In arsenite-transformed HELF cells, the rate of apoptosis of the anti-miR-21 group (16.81±1.50%) was higher than that of the anti-miR-nc group (3.56±0.89%) (Figure 7C). This infers that miR-21 has an essential role in the antiapoptotic capacity of these cells.


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)

Inhibition of miR-21 increases cell apoptosis of arsenite-transformed HELF cells.Densities of bands were quantified by Eagle Eye II software. GAPDH levels, measured in parallel, served as controls. Arsenite-transformed HELF cells were transfected with 150 nM of anti-miR-nc or anti-miR-21 for 24 h. (A) Western blots and (B) relative protein levels of cleaved caspase-3 and caspase-3 (means ± SD, n = 3). **P<0.01 different from NC group. (C) The rate of cell apoptosis was analyzed by the Hoechst 33258 assay (means ± SD, n = 3). **P<0.01 different from NC group.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585869&req=5

pone-0057652-g007: Inhibition of miR-21 increases cell apoptosis of arsenite-transformed HELF cells.Densities of bands were quantified by Eagle Eye II software. GAPDH levels, measured in parallel, served as controls. Arsenite-transformed HELF cells were transfected with 150 nM of anti-miR-nc or anti-miR-21 for 24 h. (A) Western blots and (B) relative protein levels of cleaved caspase-3 and caspase-3 (means ± SD, n = 3). **P<0.01 different from NC group. (C) The rate of cell apoptosis was analyzed by the Hoechst 33258 assay (means ± SD, n = 3). **P<0.01 different from NC group.
Mentions: To determine if miR-21 functions as an onco-miRNA or as anti-onco-miRNA, the effect of miR-21 was assessed on apoptosis of arsenite-transformed HELF cells. After arsenite-transformed cells were transfected with anti-miR-21, there were increases of cleaved-caspase-3 and decreases of caspase-3 levels (Figure 7A and 7B). In arsenite-transformed HELF cells, the rate of apoptosis of the anti-miR-21 group (16.81±1.50%) was higher than that of the anti-miR-nc group (3.56±0.89%) (Figure 7C). This infers that miR-21 has an essential role in the antiapoptotic capacity of these cells.

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