Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition of miR-21 decreases the neoplastic capacity and motility of arsenite-transformed HELF cells.After arsenite-transformed HELF cells were transfected with or without anti-miR-21 (150 nM) for 24 h, the neoplastic capacity and motility of cells were determined by anchorage-independent growth and scratch wound healing assays, respectively, bars = 100 µm. (A) Cell colony and (B) their numbers (means ± SD, n = 3) in soft agar. **P<0.01 difference from NC group. (C) Movement of cells into the wound and (D) the percentages of open space areas covered (means ± SD, n = 3). **P<0.01 difference from NC group.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585869&req=5

pone-0057652-g008: Inhibition of miR-21 decreases the neoplastic capacity and motility of arsenite-transformed HELF cells.After arsenite-transformed HELF cells were transfected with or without anti-miR-21 (150 nM) for 24 h, the neoplastic capacity and motility of cells were determined by anchorage-independent growth and scratch wound healing assays, respectively, bars = 100 µm. (A) Cell colony and (B) their numbers (means ± SD, n = 3) in soft agar. **P<0.01 difference from NC group. (C) Movement of cells into the wound and (D) the percentages of open space areas covered (means ± SD, n = 3). **P<0.01 difference from NC group.

Mentions: The capacity for colony formation by arsenite-transformed HELF cells with or without transfection of anti-miR-21 was examined. The cells transfected with anti-miR-21 displayed fewer colonies (30±4) compared with the NC inhibitor group (78±3) (Figure 8A and 8B). An assay for wound-scratch healing, which is used to detect cell motility, was used to characterize the function of miR-21 on cell motility. Arsenite-transformed cells transfected with anti-miR-21 showed lower motility than cells transfected with anti-miR-nc (86.7±6.5%, in comparison with 0 h), and the relative rate was 59.5±7.7% (in comparison with 0 h) (Figure 8C and 8D). These results indicate that miR-21 is involved in cell clonogenicity and motility of arsenite-transformed HELF 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 decreases the neoplastic capacity and motility of arsenite-transformed HELF cells.After arsenite-transformed HELF cells were transfected with or without anti-miR-21 (150 nM) for 24 h, the neoplastic capacity and motility of cells were determined by anchorage-independent growth and scratch wound healing assays, respectively, bars = 100 µm. (A) Cell colony and (B) their numbers (means ± SD, n = 3) in soft agar. **P<0.01 difference from NC group. (C) Movement of cells into the wound and (D) the percentages of open space areas covered (means ± SD, n = 3). **P<0.01 difference from NC group.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057652-g008: Inhibition of miR-21 decreases the neoplastic capacity and motility of arsenite-transformed HELF cells.After arsenite-transformed HELF cells were transfected with or without anti-miR-21 (150 nM) for 24 h, the neoplastic capacity and motility of cells were determined by anchorage-independent growth and scratch wound healing assays, respectively, bars = 100 µm. (A) Cell colony and (B) their numbers (means ± SD, n = 3) in soft agar. **P<0.01 difference from NC group. (C) Movement of cells into the wound and (D) the percentages of open space areas covered (means ± SD, n = 3). **P<0.01 difference from NC group.
Mentions: The capacity for colony formation by arsenite-transformed HELF cells with or without transfection of anti-miR-21 was examined. The cells transfected with anti-miR-21 displayed fewer colonies (30±4) compared with the NC inhibitor group (78±3) (Figure 8A and 8B). An assay for wound-scratch healing, which is used to detect cell motility, was used to characterize the function of miR-21 on cell motility. Arsenite-transformed cells transfected with anti-miR-21 showed lower motility than cells transfected with anti-miR-nc (86.7±6.5%, in comparison with 0 h), and the relative rate was 59.5±7.7% (in comparison with 0 h) (Figure 8C and 8D). These results indicate that miR-21 is involved in cell clonogenicity and motility of arsenite-transformed HELF 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