The development of tumor angiogenesis is a critical step in cancer progression, enabling the growth and spread of malignant lesions. In this study, we investigated the role of miR-143 in regulating angiogenic signaling in human lung epithelial cells transformed by chronic exposure to hexavalent chromium [Cr(VI)]. We utilized an established in vitro model where nontumorigenic BEAS-2B cells were chronically exposed to 1 µM Cr(VI) for 24 weeks, resulting in the generation of BEAS-Cr cells that exhibit hallmark characteristics of malignancy, including enhanced proliferation, anchorage-independent growth, and tumorigenicity in nude mice.
Our initial findings revealed that miR-143 expression was profoundly downregulated in Cr(VI)-transformed BEAS-Cr cells compared to their parental counterparts. This repression was consistent across multiple independently derived clones, suggesting it is a stable and integral feature of Cr(VI)-induced transformation. To explore the functional implications of this downregulation, we performed gain-of-function experiments by ectopically overexpressing miR-143 in BEAS-Cr cells using lentiviral vectors. The results demonstrated that miR-143 restoration significantly impaired the ability of these cells to induce angiogenesis both in vitro and in vivo.
In vitro tube formation assays using human umbilical vein endothelial cells (HUVECs) showed that conditioned medium from control BEAS-Cr cells strongly promoted capillary-like structure formation. However, when BEAS-Cr cells were engineered to overexpress miR-143, the pro-angiogenic capacity of their conditioned medium was markedly reduced—by approximately 35%. Similarly, in the chicken chorioallantoic membrane (CAM) assay, BEAS-Cr cells induced robust neovascularization. Transient transfection with miR-143 precursor significantly attenuated this angiogenic response, indicating that miR-143 acts as a potent inhibitor of tumor-induced vascularization.
To confirm the role of miR-143 in tumor angiogenesis within a physiological context, we generated stable xenograft models in nude mice. BEAS-Cr cells stably overexpressing miR-143 or a negative control were implanted subcutaneously. After four weeks, tumors derived from miR-143-overexpressing cells were approximately 50% smaller than those from control cells. Immunohistochemical analysis using anti-CD31 antibodies revealed a significant reduction in microvessel density—about 50% less—within miR-143-expressing tumors, confirming its anti-angiogenic effect in vivo.
We then sought to identify the molecular targets through which miR-143 exerts its anti-angiogenic function. Using bioinformatic prediction tools, we identified insulin-like growth factor-1 receptor (IGF-IR) and insulin receptor substrate-1 (IRS1) as potential targets of miR-143 due to conserved binding sites in their 3′-UTRs. Luciferase reporter assays confirmed direct interaction: co-transfection of miR-143 mimics with wild-type 3′-UTR reporters of IGF-IR and IRS1 led to a significant decrease in luciferase activity, while mutation of the seed regions abolished this effect. These data demonstrate that miR-143 directly binds to and represses both IGF-IR and IRS1.
Functional validation showed that overexpression of miR-143 in BEAS-Cr cells resulted in a marked reduction of IGF-IR and IRS1 protein levels. Conversely, knockdown of IGF-IR or IRS1 using siRNA SMARTpools inhibited colony formation in soft agar and suppressed angiogenesis in CAM assays, mimicking the effects of miR-143 overexpression.LC3A Antibody site To test whether IGF-IR and IRS1 are necessary mediators of miR-143’s action, we generated stable BEAS-Cr cell lines that overexpressed either IGF-IR or IRS1 without their 3′-UTRs.TD-165 Epigenetics These constructs bypassed miR-143 regulation.PMID:35045949 Remarkably, forced expression of IGF-IR or IRS1 completely reversed the inhibitory effect of miR-143 on tube formation and angiogenesis, proving that IGF-IR and IRS1 are key downstream effectors of miR-143 in suppressing tumor angiogenesis.
Further investigation revealed that IGF-IR/IRS1 signaling activates the ERK pathway in Cr(VI)-transformed cells. Phosphorylation of ERK was significantly elevated in BEAS-Cr cells and was effectively suppressed by miR-143 overexpression or siRNA-mediated knockdown of IGF-IR/IRS1. This establishes a clear signaling cascade: loss of miR-143 → derepression of IGF-IR/IRS1 → activation of ERK → induction of pro-angiogenic factors.
Collectively, our data demonstrate that miR-143 functions as a critical suppressor of tumor angiogenesis in Cr(VI)-transformed lung epithelial cells. By directly targeting and repressing IGF-IR and IRS1, miR-143 interrupts a central oncogenic signaling axis that drives vascularization. This study provides strong mechanistic evidence linking miRNA dysregulation to environmental carcinogen-induced angiogenesis and identifies miR-143 as a promising candidate for therapeutic intervention in chromium-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