Arsenite-transformed Cells Research Articles

CircRNA-100284 activates aurora kinase B by inducing methylation of HSP70 via microRNA-217 to promote proliferation of bladder cancer cells.

The malignant transformation of normal bladder cells (SV-HUC-1) was induced by arsenite to explore the possible mechanism of circRNA-100284 influencing bladder cancer cell proliferation. Normal bladder SV-HUC-1 cells were cultured with 2μM arsenite to induce malignant transformation. After 0, 3, 6, 12, and 24h of culture, the expression level of circRNA-100284 in cells was detected by quantitative real-time PCR. Western blotting assays were used to detect the expression levels of EZH2 and cyclin-D1 proteins in cells treated with different media. Cell cycle was analyzed by flow cytometry. In addition, through cell transfection and CCK-8 experiments, the effect and mechanism of circRNA-100284 targeting microRNA-217 on proliferation was determined. The interaction between HSP70 methylation and Aurora-B was determined by Western blotting and immunoprecipitation experiments. With prolonged contact time with arsenite, the expression level of circRNA-100284 in cells increased continuously (P < 0.05). Western blotting assays showed that the expression levels of EZH2 and cyclin-D1 proteins in arsenite-transformed cells increased. Flow cytometry and CCK-8 showed that circRNA-100284 accelerated cell cycle transition and cell proliferation through miR-217. Finally, after culturing human bladder cancer T24 cells, combined with immunoprecipitation and in vitro kinase experiments, it was found that K561- dimethyl HSP70 activated Aurora-B, thus promoting the proliferation of bladder cancer cells. CircRNA-100284 activates aurora kinase B by inducing methylation of HSP70 via microRNA-217 to promote the proliferation of bladder cancer cells.

N6-methyladenosine mediates arsenite-induced human keratinocyte transformation by suppressing p53 activation

N6-methyladenosine (m6A), the most abundant and reversible RNA modification, plays critical a role in tumorigenesis. However, whether m6A can regulate p53, a leading antitumor protein remains poorly understood. In this study, we explored the regulatory role of m6A on p53 activation using an arsenite-transformed keratinocyte model, the HaCaT-T cell line. We created the cell line by exposing human keratinocyte HaCaT cells to 1 μM arsenite for 5 months. We found that the cells exhibited an increased m6A level along with an aberrant expression of the methyltransferases, demethylase, and readers of m6A. Moreover, the cells exhibited decreased p53 activity and reduced p53 phosphorylation, acetylation, and transactivation with a high nucleus export rate of p53. Knockdown of the m6A methyltransferase, METTL3 significantly decreased m6A level, restoring p53 activation and inhibiting cellular transformation phenotypes in the arsenite-transformed cells. Further, using both a bioinformatics analysis and experimental approaches, we demonstrated that m6A downregulated the expression of the positive p53 regulator, PRDM2, through the YTHDF2-promoted decay of PRDM2 mRNAs. We showed that m6A upregulated the expression of the negative p53 regulator, YY1 and MDM2 through YTHDF1-stimulated translation of YY1 and MDM2 mRNA. Taken together, our study revealed the novel role of m6A in mediating arsenite-induced human keratinocyte transformation by suppressing p53 activation. This study further sheds light on the mechanisms of arsenic carcinogenesis via RNA epigenetics.

Enhanced p62-NRF2 Feedback Loop due to Impaired Autophagic Flux Contributes to Arsenic-Induced Malignant Transformation of Human Keratinocytes.

Chronic exposure to arsenic induces a variety of cancers, particularly in the skin. Autophagy is a highly conserved process which plays a dual role in tumorigenesis. In the present study, we found that chronic exposure to an environmentally relevant dose of arsenite induced malignant transformation of human keratinocytes (HaCaT) with dysregulated autophagy as indicated by an increased number of autophagosomes, activation of mTORC1 pathway, and elevated protein levels of p62 and LC3II. Meanwhile, arsenite-transformed cells showed lower intracellular levels of reactive oxygen species compared with control. Silencing p62 ameliorated elevation in mRNA levels of NRF2 downstream genes (AKR1C1 and NQO1) and malignant phenotypes (acquired invasiveness and anchor-independent growth) induced by chronic arsenite exposure. On the other hand, silencing NRF2 abrogated the increase in mRNA and protein levels of p62 and malignant phenotypes induced by arsenite. In response to acute arsenite exposure, impaired autophagic flux with an increase in p62 protein level and interrupted autophagosome-lysosome fusion was observed. The increase in p62 protein levels in response to arsenite was not completely dependent on NRF2 activation and at least partially attributed to protein degradation. Our data indicate that accumulation of p62 by impaired autophagic flux is involved in the activation of NRF2 and contributes to skin tumorigenesis due to chronic arsenite exposure.

N6-methyladenosine mediates the cellular proliferation and apoptosis via microRNAs in arsenite-transformed cells

N6-methyladenosine (m6A) modification is implicated to play an important role in cellular biological processes, but its regulatory mechanisms in arsenite-induced carcinogenesis are largely unknown. Here, human bronchial epithelial (HBE) cells were chronically treated with 2.5 μM arsenite sodium (NaAsO2) for about 13 weeks and these cells were identified with malignant phenotype which was demonstrated by increased levels of cellular proliferation, percentages of plate colony formation and soft agar clone formation, and high potential of resistance to apoptotic induction. Our results firstly demonstrated that m6A modification on RNA was significantly increased in arsenite-transformed cells and this modification may be synergistically regulated by methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), Wilms tumor 1-associated protein (WTAP) and Fat mass and obesity-associated protein (FTO). In addition, knocking down of METTL3 in arsenite-transformed cells can dramatically reverse the malignant phenotype, which was manifested by lower percentages of clone and colony formation as well as higher rates of apoptotic induction. Given the critical roles of miRNAs in cellular proliferation and apoptosis, miRNAs regulated by m6A in arsenite-transformed cells were analyzed by Venn diagram and KEGG pathway in this study. The results showed that these m6A-mediated miRNAs can regulate pathways which are closely associated with cellular proliferation and apoptosis, implicating that these miRNAs may be the critical bridge by which m6A mediates dysregulation of cell survival and apoptosis in arsenite-transformed cells. Taken together, our results firstly demonstrated the significant role of m6A in the prevention of tumor occurrence and progression induced by arsenite.

Exosomal circRNA_100284 from arsenite-transformed cells, via microRNA-217 regulation of EZH2, is involved in the malignant transformation of human hepatic cells by accelerating the cell cycle and promoting cell proliferation

Intercellular communication between malignant cells and neighboring nonmalignant cells is involved in carcinogenesis. In the progression of carcinogenesis, exosomes are messengers for intercellular communication. Circular RNAs (circRNAs) are noncoding RNAs with functions that include regulation of the cell cycle and proliferation. However, the functions of exosomal circRNAs are not clear. The present research aimed to determine whether circRNAs secreted from arsenite-transformed human hepatic epithelial (L-02) cells are transferred into normal L-02 cells and become functionally active in the normal cells. The results showed that circRNA_100284 is involved in the malignant transformation of L-02 cells induced by arsenite. The medium from transformed L-02 cells induced upregulation of circRNA_100284, accelerated the cell cycle, and promoted proliferation of normal L-02 cells. Transformed cells transferred circRNA_100284 into normal L-02 cells via exosomes and led to the malignant transformation of the non-transformed cells. Knockdown of circRNA_100284, which reduced circRNA_100284 levels in exosomes derived from transformed L-02 cells, blocked the accelerated cell cycle and reduced proliferation and malignancy. In addition, in normal L-02 cells, exosomal circRNA_100284 derived from arsenite-transformed L-02 cells induced acceleration of the cell cycle and promoted proliferation via acting as a sponge of microRNA-217. Further, exosomal circRNA_100284 was upregulated in the sera of people exposed to arsenite. Thus, exosomes derived from transformed L-02 cells transferred circRNA_100284 to surrounding cells, which induced an accelerated cell cycle and promoted proliferation of normal liver cells and led to the malignant transformation of the non-transformed cells. The findings support the concept that exosomal circRNAs are involved in cell–cell communication during carcinogenesis induced by arsenite.

NF-kB-regulated exosomal miR-155 promotes the inflammation associated with arsenite carcinogenesis

In the cancer microenvironment, extracellular communication allows various types of cells to coordinate and execute biological functions. Emerging evidence indicates that exosomes, as mediators of cell communication, are involved in tumor progression. Little is known, however, about the mechanism by which exosomal miRNAs regulate inflammatory infiltration in arsenite-induced liver cancer. The present research aimed to determine if miRNAs secreted from arsenite-transformed human hepatic epithelial (L-02) cells are transferred into normal L-02 and THLE-3 cells, which are functionally active in the recipient cells. The results show that the exosomes from arsenite-transformed L-02 cells enhance miR-155 expression and the pro-inflammatory properties of normal L-02 and THLE-3 cells. Transformed cells transfer miR-155 into normal L-02 cells via exosomes. The inhibition of NF-κB by siRNA and inhibitor, which reduces miR-155 levels in exosomes derived from transformed L-02 cells, blocks inflammation. Arsenite-transformed cells secrete exosomes to enhance inflammation, but the inhibition of the synthesis of exosomes fails to stimulate inflammation. miR-155 is involved in exosome-mediated intercellular communication between neoplastic and normal liver cells. In addition, miR-155, IL-6, and IL-8 were over-expressed in the serum of arsenite exposure group. And there was a positive correlation between miR-155 and IL-6 or IL-8 levels. Further, exosomal miR-155 was up-regulated in the serum of arsenite exposure group. Thus, these results show that exosomes derived from transformed L-02 cells transfer miR-155 to surrounding cells, which induces pro-inflammatory activity of normal liver cells. The findings support the concept that exosomal miRNAs are involved in cell–cell communication during carcinogenesis induced by environmental chemicals.

Hypermethylation of the Keap1 gene inactivates its function, promotes Nrf2 nuclear accumulation, and is involved in arsenite-induced human keratinocyte transformation

It is well known that long-term exposure to arsenite leads to human skin cancer, but the underlying mechanisms of carcinogenesis remain obscure. The transcription factor Nrf2-mediated antioxidant response represents a critical cellular defense mechanism; however, emerging data suggest that constitutive activation of Nrf2 is associated with cancer development and chemotherapy resistance. The reasons Nrf2 continuously accumulates in cancer cells remain to be fully understood. By establishing transformed human keratinocyte cells via chronic arsenite treatment, we observed a continuous reduction in reactive oxygen species levels and enhanced levels of Nrf2 and its target antioxidant enzymes in the later stage of arsenite-induced cell transformation. We also revealed that hypermethylation of the Keap1 gene promoter region induced by DNA methyltransferase-3 leading to inactivation of its function was responsible for constitutive activation of Nrf2 and its target enzymes. To validate these observations, the expression of Keap1 protein was restored in arsenite-transformed cells by treatment with a DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-Aza-dC), and protein levels of Nrf2 and colony formation were then determined after these treatments. Results showed that enhancement of Keap1 expression by 5-Aza-dC significantly reduced Nrf2 and its target antioxidant enzyme levels, and that in turn suppressed cell proliferation and colony formation of the transformed cells. Taken together, the present study strongly suggests that loss of Keap1 function by hypermethylation of its promoter region leading to Nrf2 nuclear accumulation appears to play a role in arsenite-induced human keratinocyte transformation.

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.

Reversal and Prevention of Arsenic-Induced Human Bronchial Epithelial Cell Malignant Transformation by microRNA-200b

Arsenic is a well-recognized human carcinogen, yet the mechanism by which it causes human cancer has not been elucidated. MicroRNAs (miRNAs) are a big family of small noncoding RNAs and negatively regulate the expression of a large number of protein-coding genes. We investigated the role of miRNAs in arsenic-induced human bronchial epithelial cell malignant transformation and tumor formation. We found that prolonged exposure of immortalized p53-knocked down human bronchial epithelial cells (p53(low)HBECs) to low levels of arsenite (NaAsO₂, 2.5 μM) caused malignant transformation that was accompanied by epithelial to mesenchymal transition (EMT) and reduction in the levels of miR-200 family members. Stably reexpressing miR-200b in arsenite-transformed cells (As-p53(low)HBECs) completely reversed their transformed phenotypes, as evidenced by inhibition of colony formation in soft agar and prevention of xenograft tumor formation in nude mice. Moreover, stably expressing miR-200b alone in parental nontransformed p53(low)HBECs was sufficient to completely prevent arsenite exposure from inducing EMT and malignant transformation. Further mechanistic studies showed that depletion of miR-200 in arsenite-transformed cells involved induction of the EMT-inducing transcription factors zinc-finger E-box-binding homeobox factor 1 (ZEB1) and ZEB2 and increased methylation of miR-200 promoters. Stably expressing ZEB1 alone in parental nontransformed p53(low)HBECs was sufficient to deplete miR-200, induce EMT and cause cell transformation, phenocopying the oncogenic effect of 16-week arsenite exposure. These findings establish for the first time a causal role for depletion of miR-200b expression in human cell malignant transformation and tumor formation resulting from arsenic exposure.