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. 2021 Oct 27;184(1):33-45.
doi: 10.1093/toxsci/kfab099.

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

Hsuan-Pei Lin  1 , Zhishan Wang  2 , Chengfeng Yang  2
Affiliations

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

Hsuan-Pei Lin et al. Toxicol Sci. .

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.

Keywords: DUXAP10; Hedgehog pathway; Pax6; cadmium; cancer stem cell-like property; long noncoding RNA.

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Figures

Figure 1.
Figure 1.
Chronic exposure to a low dose of Cd induces CSC-like property and tumorigenesis. A, Representative images of soft agar colonies formed from the passage-paired BEAS-2B cells and the Cd transformed BEAS-2B cells (Cd-T) (means ± SD, n = 3), ***p < .001. B, Representative images of the first and the second generation of suspension spheres formed from the passage-paired BEAS-2B cells and Cd-T cells. Scale bar: 100 µm. The bar graph represents the average numbers of the spheres formed in the repeated assays (means ± SD, n = 3), ***p < .001. C, Representative images of flow cytometry analysis of CD133 positive cells in the control BEAS-2B cells and Cd-T cells. The bar graph shows the average percentage of CD133+ population in each group (means ± SD, n = 3), **p < .01. D, Representative Western blot analysis images of stemness marker levels in the control BEAS-2B and Cd-T cells. E, Nude mouse xenograft tumorigenesis study as described in Materials and Method (n = 10).
Figure 2.
Figure 2.
Chronic exposure to Cd does not induce significant DNA damage but cause long noncoding RNA (lncRNA) dysregulations. A, Representative IF staining overlaid images of γH2A.X in red fluorescence and DAPI in blue fluorescence from control BEAS-2B and Cd-T cells. Scale bar: 100 µm. B, Representative IF staining overlaid images of γH2A.X in red fluorescence and DAPI in blue fluorescence from the BEAS-2B treated with Cd 2.5 or 10 µM, respectively. Scale bar: 100 µm. C, Microarray result shows 225 lncRNA upregulated and 75 lncRNA downregulated (cut-off 2). D, Quantitative PCR analysis of the relative DUXAP10 level in control BEAS-2B and Cd-T cells. The RNA level in the Cd-T is expressed relative to control BEAS-2B cells (means ± SD, n = 3), ***p < .001.
Figure 3.
Figure 3.
Knockdown of DUXAP10 in Cd-T cells significantly reduces their CSC-like property. A, Kaplan-Meier plotter survival analysis revealed negative correlation between DUXAP10 level and the overall survival rate, relapse-free survival rate in lung cancer patients. B, Quantitative PCR analysis of the relative DUXAP10 level in Cd-T cells transfected with control siRNA and the siRNA targeting DUXAP10. The RNA level in the Cd-T with DUXAP10 knockdown is expressed relative to the control group (means ± SD, n = 3), **p < .01. C, Representative Western blot images of stemness marker levels in Cd-T cells transfected with control siRNA or the siRNA targeting DUXAP10. D, Representative images of suspension spheres formed from Cd-T cells transfected with control siRNA or DUXAP10 siRNA. Scale bar: 100 µm. The bar graph represents the average numbers of the spheres formed in the repeated assays (means ± SD, n = 3), **p < .01. E, Representative images of flow cytometry analysis of CD133 levels in the Cd-T cells transfected with control siRNA or DUXAP10 siRNA. The bar graph shows the average percentage of CD133+ population in each group (means ± SD, n = 3), **p < .01.
Figure 4.
Figure 4.
The Hedgehog pathway is highly activated in Cd-T cells and inhibition of this pathway reduces their CSC-like property. A, Quantitative PCR analysis of the relative levels of GLI1, HES1, TCF12 mRNA in control BEAS-2B and Cd-T cells. The mRNA levels in the Cd-T are expressed relative to the control group (means ± SD, n = 3), **p < .01. B, Representative Western blot images of the levels of GLI1, SHH, PTCH2, SMO, SUFU in control BEAS-2B and Cd-T cells. C, Representative IF staining overlaid images of GLI1 in red fluorescence and DAPI in blue fluorescence from control BEAS-2B and Cd-T cells. Scale bar: 100 µm. D, Representative IF staining overlaid images of GLI1 in red fluorescence and DAPI in blue fluorescence from Cd-T cells treated with DMSO (vehicle control) and cyclopamine (10 µM), respectively. Scale bar: 100 µm. E, Representative images of suspension spheres formed from Cd-T cells treated with DMSO (vehicle control) or cyclopamine (10 µM), respectively. Scale bar: 100 µm. The bar graph represents the average numbers of the spheres formed in the repeated assays (means ± SD, n = 3), ****p < .0001. F, Representative Western blot images of the levels of GLI1, KLF4, KLF5, Nanog in Cd-T cells treated with DMSO (vehicle control) or cyclopamine (10 µM).
Figure 5.
Figure 5.
Knockdown of DUXAP10 inactivated the Hedgehog pathway in Cd-T cells. A, Representative Western blot images of the levels of GLI1, SHH, PTCH2 in Cd-T cells transfected with control siRNA or DUXAP10 siRNA. The assay was repeated, and similar results were obtained. B, Representative IF staining overlaid images of GLI1 in red fluorescence and DAPI in blue fluorescence from the Cd-T cells transfected with control siRNA or DUXAP10 siRNA. Scale bar: 100 µm. C, Representative Western blot images of the levels of GLI1 and SHH in the Cd-T protein lysates applied to DUXAP10-sense and DUXAP10-antisense biotinylated RNA pull down, respectively. D, Representative Western blot images of the level of GLI1 in Cd-T cells transfected with control siRNA or DUXAP10 siRNA and treated with 10 µM MG132 or a vehicle control DMSO, respectively.
Figure 6.
Figure 6.
Chronic Cd exposure increases the expression of Pax6 and knockdown of Pax6 reduces DUXAP10 levels in Cd-T cells. A, Schematic map shows that the putative binding site of Pax6 locates 3 kb upstream of the DUXAP10 transcription start site, close to the 3′ end of the promoter region. B, Representative Western blot images of Pax6 level in control BEAS-2B and Cd-T cells. C, Kaplan-Meier plotter survival analysis revealed negative correlation between the expression level of Pax6 and the overall survival rate in lung cancer patients. D, ChIP qPCR analysis of relative level of DUXAP10 promoter bound by Pax6 in control BEAS-2B and Cd-T cells. Rabbit IgG was used as negative control. Percentage of input method was adopted to analyze the raw data. The repeated results are presented as means ± SD (n = 3), ****p < .0001. E, Quantitative PCR analysis of the relative level of DUXAP10 in Cd-T cells transfected with control siRNA or siRNA targeting Pax6, respectively. The RNA level in the Cd-T with Pax6 knockdown is expressed relative to the control group (means ± SD, n = 3), *p < .05. F, Representative Western blot images of levels of Pax6 and GLI1 in Cd-T cells transfected with control siRNA or Pax6 siRNA, respectively. G, Representative IF staining overlaid images of GLI1 in red fluorescence and DAPI in blue fluorescence from the Cd-T cells transfected with control siRNA or Pax6 siRNA. Scale bar: 100 µm. H, Representative images of suspension spheres formed from the Cd-T cells transfected with control siRNA or Pax6 siRNA. Scale bar: 100 µm. The bar graph represents the average numbers of the spheres formed in the repeated assays (means ± SD, n = 3), *p < .05.
Figure 7.
Figure 7.
A schematic summary of the mechanism of Cd-induced CSC-like property and tumorigenesis.
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