Abstract
ObjectiveTo 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.MethodsFor 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.ResultsMiR-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.ConclusionThe 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.
Highlights
Chronic exposure to arsenite induces cellular transformation characterized by increased proliferation and anchorage-independent growth [1,2]
The Activations of extracellular signalregulated kinase (ERK)/nuclear factor-kB (NF-kB), Jun N-terminal kinase (JNK)/c-Jun, and Akt are Induced by Arsenite in human embryo lung fibroblast (HELF) Cells
To determine whether the mitogen-activated protein kinases (MAPKs) and PI-3Ks signal pathways are involved in arsenite-induced malignant transformation of HELF cells, the levels of p-ERK, p-JNK, and p-p38; the levels of p-NF-kB p65 and pc-Jun; and the level of p-Akt were determined in normal HELF cells, in passage-control HELF cells, in arsenitetransformed HELF cells, and in normal HELF cells treated with 1.0 mM arsenite for 0, 6, or 24 h
Summary
Chronic exposure to arsenite induces cellular transformation characterized by increased proliferation and anchorage-independent growth [1,2]. Skin is thought to be the most sensitive tissue for arsenic toxicity, lung is recognized as a target as well [5,6]. Even though multiple hypotheses have been proposed to explain arsenite-induced carcinogenesis, the exact mechanism remains elusive. MicroRNAs (miRNAs), small, non-coding RNA molecules of 21 to 23 nucleotides, have the capacity to inhibit translation and induce mRNA degradation, predominantly through the 39untranslated regions (39-UTR) of mRNAs [7]. The involvement of miRNAs in lung carcinogenesis has yet to be explored [8]. MicroRNA-21 (miR-21) is over-expressed in carcinomas of lung, prostate, breast, pancreas, colon, head and neck, stomach, esophagus, and liver, relative to adjacent normal tissues, support-
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
