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LncRNA DUXAP10 Upregulation and the Hedgehog Pathway Activation Are Critically Involved in Chronic Cadmium Exposure-Induced Cancer Stem Cell-Like Property.

ABSTRACT: Cadmium (Cd) is a well-known lung carcinogen. However, the mechanism of Cd carcinogenesis remains to be clearly defined. Cd has been shown to act as a weak mutagen, suggesting that it may exert tumorigenic effect through nongenotoxic ways, such as epigenetic mechanisms. Long noncoding RNAs (lncRNAs) refer to RNA molecules that are longer than 200 nucleotides in length but lack protein-coding capacities. Regulation of gene expressions by lncRNAs is considered as one of important epigenetic mechanisms. The goal of this study is to investigate the mechanism of Cd carcinogenesis focusing on the role of lncRNA dysregulations. Cd-induced malignant transformation of human bronchial epithelia BEAS-2B cells was accomplished by a 9-month low-dose Cd (CdCl2, 2.5 µM) exposure. The Cd-exposed cells formed significantly more colonies in soft agar, displayed cancer stem cell (CSC)-like property, and formed tumors in nude mice. Mechanistically, chronic low-dose Cd exposure did not cause significant genotoxic effects but dysregulated lncRNA expressions. Further Q-PCR analysis confirmed the significant upregulation of the oncogenic lncRNA DUXAP10 in Cd-transformed cells. DUXAP10 knockdown in Cd-transformed cells significantly reduced their CSC-like property. Further mechanistic studies showed that the Hedgehog pathway is activated in Cd-transformed cells and inhibition of this pathway reduces Cd-induced CSC-like property. DUXAP10 knockdown caused the Hedgehog pathway inactivation in Cd-transformed cells. Furthermore, Pax6 expression was upregulated in Cd-transformed cells and Pax6 knockdown significantly reduced their DUXAP10 levels and CSC-like property. In summary, these findings suggest that the lncRNA DUXAP10 upregulation may play an important role in Cd carcinogenesis.

SUBMITTER: Lin HP

PROVIDER: S-EPMC8677432 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cadmium (Cd) is a toxic heavy metal and one of carcinogens that cause lung cancer. However, the exact mechanism of Cd carcinogenesis remains unclear. To investigate the mechanism of Cd carcinogenesis, we exposed human bronchial epithelial cells (BEAS-2B) to a low dose of Cd (2.5 μM, CdCl2) for 9 months, which caused cell malignant transformation and generated cancer stem cell (CSC)-like cells. The goal of this study is to investigate the underlying mechanism. The long non-coding RNA (lncRNA) microarray analysis showed that the expression level of a tumor suppressive lncRNA maternally expressed 3 (MEG3) is significantly down-regulated in Cd-transformed cells, which is confirmed by further q-PCR analysis. Mechanistically, it was found that chronic Cd exposure up-regulates the levels of DNA methyltransferases (DNMTs), which increases the methylation of the differentially methylated region (DMR) 1.5 kb upstream of MEG3 transcription start site to reduce MEG3 expression. Functional studies showed that stably overexpressing MEG3 in Cd-transformed cells significantly reduces their transformed phenotypes. Moreover, stably overexpressing MEG3 in parental non-transformed human bronchial epithelial cells significantly impaired the capability of chronic Cd exposure to induce cell transformation and CSC-like property. Further mechanistic studies revealed that the cell cycle inhibitor p21 level is reduced and retinoblastoma protein (Rb) phosphorylation is increased in Cd-transformed cells to promote cell cycle progression. In addition, Cd-transformed cells also expressed higher levels of Bcl-xL and displayed apoptosis resistance. In contrast, stably overexpressing MEG3 increased p21 levels and reduced Rb phosphorylation and Bcl-xL levels in Cd-exposed cells and reduced their cell cycle progression and apoptosis resistance. Together, these findings suggest that MEG3 down-regulation may play important roles in Cd-induced cell transformation and CSC-like property by promoting cell cycle progression and apoptosis resistance.

Project description:ObjectivePrenatal cadmium (Cd) exposure leads to immunotoxic phenotypes in the offspring affecting coding and non-coding genes. Recent studies have shown that long non-coding RNAs (lncRNAs) are integral to T cell regulation. Here, we investigated the role of long non-coding RNA small nucleolar RNA host gene 7 (lncSnhg7) in T cell proliferation.MethodsRNA sequencing was used to analyze the expression of lncRNAs in splenic CD4+ T cells with and without CD3/CD28 stimulation. Next, T cells isolated from offspring exposed to control or Cd water throughout mating and gestation were analyzed with and without stimulation with anti-CD3/CD28 beads. Quantitative qPCR and western blotting were used to detect RNA and protein levels of specific genes. Overexpression of a miR-34a mimic was achieved using nucleofection. Apoptosis was measured using flow cytometry and luminescence assays. Flow cytometry was also used to measure T cell proliferation in culture. Finally, lncSnhg7 was knocked down in splenic CD4+ T cells with lentivirus to assess its effect on proliferation.ResultsWe identified 23 lncRNAs that were differentially expressed in stimulated versus unstimulated T cells, including lncSnhg7. LncSnhg7 and a downstream protein, GALNT7, are upregulated in T cells from offspring exposed to Cd during gestation. Overexpression of miR-34a, a regulator of lncSnhg7 and GALNT7, suppresses GALNT7 protein levels in primary T cells, but not in a mouse T lymphocyte cell line. The T cells isolated from Cd-exposed offspring exhibit increased proliferation after activation in vitro, but Treg suppression and CD4+ T cell apoptosis are not affected by prenatal Cd exposure. Knockdown on lncSnhg7 inhibits proliferation of CD4+ T cells.ConclusionPrenatal Cd exposure alters the expression of lncRNAs during T cell activation. The induction of lncSnhg7 is enhanced in splenic T cells from Cd offspring resulting in the upregulation of GALNT7 protein and increased proliferation following activation. miR-34a overexpression decreased GALNT7 expression and knockdown of lncSnhg7 inhibited proliferation suggesting that the lncSnhg7/miR-34a/GALNT7 is an important pathway in primary CD4+ T cells. These data highlight the need to understand the consequences of environmental exposures on lncRNA functions in non-cancerous cells as well as the effects in utero.

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LncRNA DUXAP10 Upregulation and the Hedgehog Pathway Activation Are Critically Involved in Chronic Cadmium Exposure-Induced Cancer Stem Cell-Like Property.

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