Rongrong Jiang

Regulation of miRNA-21 by reactive oxygen species-activated ERK/NF-κB in arsenite-induced cell transformation.

After acute exposure of cells to arsenic, reactive oxygen species mediate changes in cell behavior, including activation of proliferative signaling. For chronic exposure to arsenic, however, the function of reactive oxygen species in cell transformation remains poorly understood. Although microRNA-21 (miR-21) has been implicated in various aspects of carcinogenesis, its functions and molecular mechanisms in carcinogen-induced tumorigenesis are unclear. The purpose of this study was to determine if miR-21 is involved in arsenite-induced malignant transformation and to characterize the associated signaling pathways. During arsenite-induced transformation of human embryo lung fibroblast (HELF) cells, miR-21 was upregulated, and the extracellular signal-regulated kinase (ERK)/nuclear factor-κB (NF-κB) signal pathway was activated. Moreover, superoxide radical dismutase (a scavenger of superoxide) and catalase (a scavenger of hydroperoxides) blocked the arsenite-induced effects in HELF cells and mouse embryonic fibroblasts. Blockage of ERK by the inhibitor U0126 or inhibition of NF-κB p65 by siRNA or Bay 11-7082 prevented the increases in miR-21 and the decreases in Spry1, Pten, and Pdcd4, the target proteins of miR-21, induced by arsenite. As determined by a ChIP-qPCR assay, NF-κB p65 regulated miR-21 expression by binding directly to the promoter of miR-21. Further, anti-miR-21 downregulated miR-21 expression and prevented the arsenite-induced activation of ERK via the increase in Spry1, indicating that miR-21 has a feedback effect in regulating ERK activation. Overexpression of miR-21 with an miR-21 mimic and feedback activation of ERK and NF-κB via the decrease in Spry1 promoted the malignancy of HELF cells exposed to arsenite, but knockdown of miR-21 with anti-miR-21 and feedback blockage of ERK and NF-κB activation through an increase in Spry1 decreased anchorage-independent growth of arsenite-transformed cells. Thus, the transformation of HELF cells induced by chronic exposure to arsenite is mediated by increased miR-21 expression, which, in turn, is mediated by reactive oxygen species activation of the ERK/NF-κB pathway.

Arsenite evokes IL-6 secretion, autocrine regulation of STAT3 signaling, and miR-21 expression, processes involved in the EMT and malignant transformation of human bronchial epithelial cells

Arsenite is an established human carcinogen, and arsenite-induced inflammation contributes to malignant transformation of cells, but the molecular mechanisms by which cancers are produced remain to be established. The present results showed that, evoked by arsenite, secretion of interleukin-6 (IL-6), a pro-inflammatory cytokine, led to the activation of STAT3, a transcription activator, and to increased levels of a microRNA, miR-21. Blocking IL-6 with anti-IL-6 antibody and inhibiting STAT3 activation reduced miR-21 expression. For human bronchial epithelial cells, cultured in the presence of anti-IL-6 antibody for 3days, the arsenite-induced EMT and malignant transformation were reversed. Thus, IL-6, acting on STAT3 signaling, which up-regulates miR-21in an autocrine manner, contributes to the EMT induced by arsenite. These data define a link from inflammation to EMT in the arsenite-induced malignant transformation of HBE cells. This link, mediated through miRNAs, establishes a mechanism for arsenite-induced lung carcinogenesis.

MicroRNA-191, by promoting the EMT and increasing CSC-like properties, is involved in neoplastic and metastatic properties of transformed human bronchial epithelial cells

Lung cancer is the leading cause of cancer mortality worldwide. A common interest in lung cancer research is the identification of biomarkers for early diagnosis and accurate prognosis. There is increasing evidence that microRNAs (miRNAs) are involved in lung cancer. To explore new biomarkers of chemical exposure in risk assessment of chemical carcinogenesis and lung cancer, we analyzed miRNA expression profiles of human bronchial epithelial (HBE) cells malignantly transformed by arsenite. High‐throughput microarray analysis showed that 51 miRNAs were differentially expressed in transformed HBE cells relative to normal HBE cells. In particular, miR‐191 was up‐regulated in transformed cells. In HBE cells, arsenite induced increases of miR‐191 and WT1 levels, decreased BASP1 expression, and activated the Wnt/β‐catenin pathway, effects that were blocked by miR‐191 knockdown. In addition, a luciferase reporter assay indicated that BASP1 is a direct target of miR‐191. By inhibiting the expression of BASP1, miR‐191 increased the expression of WT1 to promote activation of Wnt/β‐catenin pathway. In transformed cells, inhibition of miR‐191 expression blocked the epithelial‐mesenchymal transition (EMT) and cancer stem cell (CSC)‐like properties of cells and decreased their migratory capacity and neoplastic properties. Thus, these results demonstrate that miR‐191 modulates the EMT and the CSC‐like properties of transformed cells and indicate that it is an onco‐miR involved in the neoplastic and metastatic properties of transformed cells.

MicroRNA-491 is Involved in Metastasis of Hepatocellular Carcinoma by Inhibitions of Matrix Metalloproteinase and Epithelial to Mesenchymal Transition

Hepatocellular carcinoma (HCC) is the third leading cause of cancer‐related mortality worldwide; the prognosis of HCC patient remains poor owing to intrahepatic and extrahepatic metastasis and postsurgical recurrence. The aim of the present study is to determine the molecular mechanisms underlying the metastasis of HCC.

Blockade of p53 by HIF-2α, but not HIF-1α, is involved in arsenite-induced malignant transformation of human bronchial epithelial cells

Hypoxia-inducible factors (HIFs), which consist of α and β subunits, are transcription factors involved in regulation of a variety of cellular functions. By blocking the function of the tumor suppressor p53, over-expressions of HIFs are linked to carcinogenesis and tumor progression. Inorganic arsenic, a ubiquitous environmental contaminant, is associated with an increased risk of cancer. Although there are several hypotheses regarding arsenic-induced carcinogenesis, the mechanism of action remains obscure. We have shown that long-term exposure of human bronchial epithelial (HBE) cells to a low level of arsenite increases their proliferation rate and anchorage-independent growth. When introduced into nude mice, the transformed cells are tumorigenic. The present report demonstrates that, with increased time of exposure to arsenite, there is more increased expression of HIF-2α, but not HIF-1α. These factors are known to have different functions, and, in some cases, opposite effects. Arsenite induces accumulation of HIF-2α by inhibiting its degradation through the ubiquitin-mediated proteasome pathway. HIF-2α knockdown, but not HIF-1α knockdown, increases the activation of p53. Finally, inhibition of HIF-2α blocks arsenite-induced proliferation and malignant transformation. Thus, our studies show that blockade of p53 function by inhibiting the ubiquitin-mediated proteasome degradation of HIF-2α, but not that of HIF-1α, is involved in arsenite-induced proliferation and neoplastic transformation of HBE cells.

Feedback Regulations of miR-21 and MAPKs via Pdcd4 and Spry1 Are Involved in Arsenite-Induced Cell Malignant Transformation

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.

Opposed arsenite-mediated regulation of p53-survivin is involved in neoplastic transformation, DNA damage, or apoptosis in human keratinocytes

Biphasic dose-response relationship induced by environmental agents is often characterized with the effect of low-dose stimulation and high dose inhibition. Some studies showed that arsenite may induce cell proliferation and apoptosis via biphasic dose-response relationship in human cells; however, mechanisms underlying this phenomenon are not well understood. Our present study shows that, for human keratinocytes (HaCaT) cells, a low concentration of arsenite activates extracellular signal-regulated kinases (ERKs), which leads to up-regulation of nuclear factor κB (NF-κB) binding to DNA and to elevated, NF-κB-dependent expression of mot-2 (a p53 inhibitor) and survivin (an inhibitor of apoptosis). Activation of p53 is blocked, and neoplastic transformation is enhanced. Inhibition of ERKs reduces cell proliferation and neoplastic transformation. In contrast, a high concentration of arsenite activates c-Jun N-terminal kinases (JNKs), positive regulators of p53, by binding to p53 and preventing its murine double minute 2 (mdm2)-mediated degradation. The elevated levels of p53 lead to repair of DNA damage and apoptosis. Inhibition of JNKs increases DNA damage but decreases apoptosis. By identifying a mechanism whereby ERKs and JNKs-mediated regulation of the p53-survivin signal pathway is involved in the biphasic effects of arsenite on human keratinocytes, our data expand understanding of arsenite-induced cell proliferation, neoplastic transformation, DNA damage, and apoptosis.

The accumulations of HIF-1α and HIF-2α by JNK and ERK are involved in biphasic effects induced by different levels of arsenite in human bronchial epithelial cells.

The biphasic effects of arsenite, in which low levels of arsenite induce cell proliferation and high levels of arsenite induce DNA damage and apoptosis, apparently contribute to arsenite-induced carcinogenesis. However, the mechanisms underlying this phenomenon are not well understood. In this study, we investigated the effects of different levels of arsenite on cell proliferation, DNA damage and apoptosis as well as on signal transduction pathways in human bronchial epithelial (HBE) cells. Our results show that a low level of arsenite activates extracellular signal-regulated kinases (ERK), which probably mediate arsenite-inhibited degradation of ubiquitinated hypoxia-inducible factor-2α (HIF-2α) in HBE cells. ERK inhibition blocks cell proliferation induced by a low level of arsenite, in part via HIF-2α. In contrast, a high level of arsenite activates c-Jun N-terminal kinases (JNK), which provoke a response to suppress ubiquitinated HIF-1α degradation. Down-regulation of HIF-1α by inhibiting JNK, however, increases the DNA damage but decreases the apoptosis induced by a high level of arsenite. Thus, data in the present study suggest that the accumulations of HIF-1α and HIF-2α by JNK and ERK are involved in different levels of arsenite-induced biphasic effects, with low levels of arsenite inducing cell proliferation and high levels of arsenite inducing DNA damage and apoptosis in HBE cells.

DNA-PKcs-mediated stabilization of p53 by JNK2 is involved in arsenite-induced DNA damage and apoptosis in human embryo lung fibroblast cells.

When cells encounter genotoxic stress, sensors for DNA lesions stabilize and activate p53; the signals involved, however, are largely unclear. Inorganic arsenite is a ubiquitous environmental contaminant associated with an increased risk of lung and skin damage and cancer. Although DNA double-strand breaks and apoptosis may relate to arsenite-induced damage and carcinogenesis, the mechanism of action remains obscure. Here, we find that, in human embryo lung fibroblast (HELF) cells, arsenite induces the activation of dependent protein kinase catalytic subunit (DNA-PKcs), which then phosphorylates and activates c-Jun N-terminal kinases 2 (JNK2), but not JNK1. As a positive regulator of p53, JNK2 binds to p53 and prevents p53 from murine double minute 2 (mdm2)-mediated, ubiquitin-proteasome-dependent degradation. Knockdown of DNA-PKcs/JNK2 signal pathway or p53 reduces apoptosis but elevates the DNA damage induced by a high level of arsenite. These results suggest that DNA-PKcs-mediated stabilization of p53 by JNK2 is involved in arsenite-induced DNA damage and apoptosis.

The inhibition of HIF-2α on the ATM/Chk-2 pathway is involved in the promotion effect of arsenite on benzo(a)pyrene-induced cell transformation.

Both arsenite and benzo(a)pyrene (BaP) are known human carcinogens. Studies on the mode-of-action of arsenite indicate that it can also act as co-carcinogen or as a cancer promoter, and that it can facilitate progression of cancers. Some studies on development of lung cancers have suggested a synergism between arsenite exposure and cigarette smoking. The mechanism of action for such an effect, however, remains obscure. In the present study, we investigated the effects of HIF-2α on arsenite- and BaP-induced cell malignant transformation as well as on signal transduction pathways in human bronchial epithelial (HBE) cells. The results show that arsenite accelerates the neoplastic transformation and migration of cells and enhances chromosomal aberrations induced by BaP. HIF-2α is involved in blocking the effects of arsenite in activating the ATM/Chk-2 pathway and in repair of DNA damage induced by BaP. Moreover, blocking of HIF-2α prevents the effects of arsenite on the neoplastic transformation, cell migration, and chromosomal aberrations caused by BaP. These results indicate that the repressive effect of HIF-2α on the ATM/Chk-2 pathway leads to genomic instability, which is involved in arsenite-accelerated, BaP-induced malignant transformation of HBE cells.