Hexavalent chromium [Cr(VI)] is a well-established human carcinogen strongly linked to increased lung cancer risk. Despite extensive epidemiological evidence, the molecular mechanisms underlying Cr(VI)-induced carcinogenesis remain poorly understood due to the absence of reliable in vitro models mimicking chronic exposure. To address this gap, we developed an in vitro transformation model by chronically exposing non-tumorigenic human bronchial epithelial BEAS-2B cells to 1 µM Cr(VI) over 24 weeks. This dose reflects occupational exposure levels and exhibits minimal cytotoxicity. The resulting Cr(VI)-transformed cells (designated BEAS-Cr) displayed hallmark features of malignant transformation: enhanced proliferation, anchorage-independent growth, and tumor formation in nude mice.
Our miRNA microarray analysis revealed a dramatic downregulation of miR-143 in BEAS-Cr cells compared to parental BEAS-2B cells. TaqMan RT-qPCR confirmed a ~35-fold reduction in miR-143 expression. This repression was consistent across multiple independently isolated clones, indicating it is a stable feature of Cr(VI)-induced transformation. Furthermore, miR-143 levels were significantly lower in established lung cancer cell lines (A549, H2195) than in normal BEAS-2B cells, suggesting its potential role as a tumor suppressor in lung carcinogenesis.
To investigate the functional significance of miR-143 repression, we ectopically expressed miR-143 in BEAS-Cr cells. This intervention significantly inhibited their tumorigenic potential both in vitro and in vivo. In tube formation assays using human umbilical vein endothelial cells (HUVECs), conditioned medium from BEAS-Cr cells robustly induced angiogenesis, which was suppressed by miR-143 overexpression. Similarly, in chicken chorioallantoic membrane (CAM) assays, BEAS-Cr cells triggered strong neovascularization, an effect markedly attenuated by miR-143 expression. In a xenograft mouse model, tumors derived from miR-143-overexpressing BEAS-Cr cells were approximately 50% smaller and exhibited a 50% reduction in microvessel density, as assessed by CD31 immunohistochemistry.
Mechanistically, we identified insulin-like growth factor-1 receptor (IGF-IR) and insulin receptor substrate-1 (IRS1) as direct targets of miR-143. Bioinformatics prediction and luciferase reporter assays confirmed that miR-143 binds specifically to the 3′-UTRs of both IGF-IR and IRS1 mRNAs. Overexpression of miR-143 in BEAS-Cr cells reduced IGF-IR and IRS1 protein levels, while knockdown of IGF-IR or IRS1 using siRNAs impaired colony formation and angiogenesis, phenocopying the effects of miR-143 restoration. Critically, forced expression of IGF-IR or IRS1 without their 3′-UTRs reversed the anti-angiogenic effects of miR-143, confirming that these two proteins are key mediators downstream of miR-143.
We further demonstrated that miR-143 represses the ERK signaling pathway via IGF-IR/IRS1. Cr(VI)-transformed cells showed elevated phosphorylation of ERK, which was significantly reduced upon miR-143 overexpression or siRNA-mediated knockdown of IGF-IR/IRS1. This establishes a clear link between miR-143 loss, IGF-IR/IRS1 activation, and ERK hyperactivation in transformed cells.
Finally, we identified interleukin-8 (IL-8) as the primary angiogenic factor upregulated in response to Cr(VI).Benzil web IL-8 mRNA and protein levels were markedly elevated in BEAS-Cr cells. This induction was dependent on IGF-IR/IRS1/ERK signaling, as inhibition of any component suppressed IL-8 expression.Bicalutamide sulfide Biological Activity Recombinant IL-8 potently stimulated HUVEC tube formation, and siRNA-mediated knockdown of IL-8 reduced angiogenesis by 50%.PMID:34710552 Moreover, IL-8 induction was shown to be mediated through hypoxia-inducible factor-1 (HIF-1) and nuclear factor-kappa B (NF-κB), both of which were activated in BEAS-Cr cells and could be suppressed by miR-143 overexpression.
In conclusion, our study establishes a causal and mechanistic link between Cr(VI)-induced miR-143 repression and tumor angiogenesis. The suppression of miR-143 leads to the derepression of IGF-IR and IRS1, resulting in sustained activation of the ERK pathway and subsequent upregulation of IL-8 via HIF-1 and NF-κB. This defines a novel molecular axis—miR-143/IGF-IR/IRS1/ERK/IL-8—that drives the angiogenic switch during Cr(VI)-induced lung carcinogenesis. These findings highlight miR-143 as a potential therapeutic target for preventing or treating Cr(VI)-associated lung cancers.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com