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. 2021 Jun 29:11:707032.
doi: 10.3389/fonc.2021.707032. eCollection 2021.

TCF11 Has a Potent Tumor-Repressing Effect Than Its Prototypic Nrf1α by Definition of Both Similar Yet Different Regulatory Profiles, With a Striking Disparity From Nrf2

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TCF11 Has a Potent Tumor-Repressing Effect Than Its Prototypic Nrf1α by Definition of Both Similar Yet Different Regulatory Profiles, With a Striking Disparity From Nrf2

Meng Wang et al. Front Oncol. .

Abstract

Nrf1 and Nrf2, as two principal CNC-bZIP transcription factors, regulate similar but different targets involved in a variety of biological functions for maintaining cell homeostasis and organ integrity VSports手机版. Of note, the unique topobiological behavior of Nrf1 makes its functions more complicated than Nrf2, because it is allowed for alternatively transcribing and selectively splicing to yield multiple isoforms (e. g. , TCF11, Nrf1α). In order to gain a better understanding of their similarities and differences in distinct regulatory profiles, all four distinct cell models for stably expressing TCF11, TCF11ΔN , Nrf1α or Nrf2 have been herein established by an Flp-In™ T-REx™-293 system and then identified by transcriptomic sequencing. Further analysis revealed that Nrf1α and TCF11 have similar yet different regulatory profiles, although both contribute basically to positive regulation of their co-targets, which are disparate from those regulated by Nrf2. Such disparity in those gene regulations by Nrf1 and Nrf2 was further corroborated by scrutinizing comprehensive functional annotation of their specific and/or common target genes. Conversely, the mutant TCF11ΔN, resulting from a deletion of the N-terminal amino acids 2-156 from TCF11, resembles Nrf2 with the largely consistent structure and function. Interestingly, our further experimental evidence demonstrates that TCF11 acts as a potent tumor-repressor relative to Nrf1α, albeit both isoforms possess a congruous capability to prevent malignant growth of tumor and upregulate those genes critical for improving the survival of patients with hepatocellular carcinoma. .

Keywords: Nrf1α; Nrf2; TCF11; hepatocellular carcinoma; regulatory profiling; transcriptomic sequencing. V体育安卓版.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Establishment of four distinct model cell lines to stabilize expression of TCF11, TCF11ΔN, Nrf1α and Nrf2. (A) A schematic diagram of the Flp-In™ T-REx™-293 system. The system allows the Flp recombinase-mediated homologous recombination of each indicated pcDNA5/FRT/TO-V5 expression constructs (for TCF11, TCF11ΔN, Nrf1α or Nrf2) with the Flp-In™ T-REx™-293 host cells through the FRT sites. (B–D) After incubation of TCF11, Nrf1α, Nrf2 or TCF11ΔN, as well as control cell lines with 1 μg/ml Tet for 12 h, total RNAs were isolated and then reversely transcribed into the first strand of cDNA. Subsequently, quantitative real-time PCR was employed to identify the mRNA expression levels of Nrf1α (B), TCF11 (C), TCF11ΔN (C) and Nrf2 (D) in each of indicated cell lines. The data are shown as mean ± SEM (n = 3 × 3, $$, p <0.01, when compared to the Control). (E–G) Total lysates of each cell line that had been treated with 1 μg/ml Tet (+) or not (−) were subjected to protein separation by SDS-PAGE gels, and then visualized by immunoblotting with distinct primary antibodies against V5, Nrf1 or Nrf2 to identify the protein levels of TCF11, Nrf1α, Nrf2 and TCF11ΔN. (H–K) Total lysates of experimental cells, which had been induced with 1 μg/ml Tet for 12 h before being treated with CHX (50 μg/mL) alone or in combination with MG132 (10 μmol/L) for distinct times as indicated, were resolved by SDS-PAGE and then analyzed by Wester blotting with V5 antibody to identify the stability of TCF11 (H), Nrf1α (I), TCF11ΔN (J) and Nrf2 (K) respectively.
Figure 2
Figure 2
Statistical analysis of the data obtained from transcriptome sequencing. (A) Differentially expressed genes (DEGs) in distinct cell lines were analyzed by transcriptome sequencing, and relevant differences in the number of those increased or decreased DEGs are shown in the histogram. The DEGs were selected according to the following criteria: fold change ≥2 or ≤0.5 and FDR ≤0.001 (as compared to the Control cells). (B) The specific DEGs in each cell line and their common DEGs between every two cell lines were also counted as indicated in the chart, and the number of increased and decreased DEGs in each group is shown separately in black font, and the total is shown in white. In addition, the change trends of DEGs in each group were indicated in red or green, which represent up-regulated or down-regulated in the cells in the first row, respectively. (C) The heatmap with hierarchical clustering of 90 DEGs shared in all four cells lines. (D) Distinct groupings of the subsequent functional annotation and also the Venn diagram of DEGs between every two cell lines.
Figure 3
Figure 3
Functional annotation of specific or common DEGs in Nrf1α and Nrf2 cells. (A) The top 10 of significant biological process terms and pathways enriched by DEGs in Groups A, B, and C were exhibited in histograms and scatterplots, respectively. (B) After Nrf1α, Nrf2 and Control cell lines were incubated with 1 μg/ml Tet for 12 h, total RNAs were isolated and reversely transcribed into the first strand of cDNA. Subsequently, the mRNA levels of DEGs that were associated with more functions, along with high expression levels and well significance in Groups A to C, were determined by quantitative real-time PCR analysis of Nrf1α, Nrf2 and Control cell lines. The data are shown as mean ± SEM (n = 3 × 3, *p 0.05; **p < 0.01; $ p < 0.05; $$ p < 0.01, when compared to the Control values).
Figure 4
Figure 4
Functional annotation of specific or common DEGs in Nrf2 and TCF11ΔN cells. (A) Top 10 of significant biological process terms and pathways enriched by DEGs in Groups D, E, and F were exhibited in histograms and scatterplots, respectively. (B) After induced with 1 μg/ml Tet for 12 h, total RNAs were isolated from Nrf2, TCF11ΔN or Control cell lines and then reversely transcribed into the first strand of cDNA. Subsequently, the mRNA levels of DEGs that were associated with more functions as annotated, along with high expression levels and well significance in Groups D to F, were determined by quantitative real-time PCR analysis of Nrf2, TCF11ΔN and Control cells. The data are shown as mean ± SEM (n = 3 × 3, *p < 0.05; **p < 0.01; $ p < 0.05; $$ p < 0.01, when compared to the Control values).
Figure 5
Figure 5
Functional annotation of specific or common DEGs in TCF11ΔN and TCF11 cells. (A) Top 10 of significant biological process terms and pathways enriched by DEGs in Groups G, H, and I were exhibited in histograms and scatterplots, respectively. (B) After induced with 1 μg/ml Tet for 12 h, total RNAs were isolated from Control, TCF11ΔN or TCF11 cell lines before being reversely transcribed into the first strand of cDNA. Subsequently, relative mRNA levels of DEGs that were associated with more functions, along with high expression levels and well significance in Groups G to I, were determined by quantitative real-time PCR in Control, TCF11ΔN and TCF11 cells. The data are shown as mean ± SEM (n = 3 × 3, *p < 0.05; $ p < 0.05, when compared to the Control values).
Figure 6
Figure 6
Functional annotation of specific or common DEGs in TCF11 and Nrf1α cells. (A) Top 10 of significant biological process terms and pathways enriched by DEGs in Groups J, K, and L were exhibited in histograms and scatterplots, respectively. (B) After induced with 1 μg/ml Tet for 12 h, total RNAs were isolated from Control, TCF11 or Nrf1α cell lines and then reversely transcribed into the first strand of cDNA. Subsequently, relevant mRNA levels of DEGs that were associated with more functions as annotated, along with high expression levels and well significance in Groups J to L, were determined by quantitative real-time PCR in Control, TCF11 and Nrf1α cells. The data are shown as mean ± SEM (n = 3 × 3, **p <0.01; $ p <0.05; $$ p <0.01, when compared to the Control values).
Figure 7
Figure 7
Malgrowth of Nrf1α−/−-derived hepatoma cells were significantly suppressed by restoration of Nrf1α and TCF11. (A) Both quantitative real-time PCR (up) and Western blotting (down) were employed to identify the protein and mRNA levels of Nrf1α and TCF11 in Nrf1α- and TCF11-Restored hepatoma cells. The experimental cells had been treated with or without 10 μmol/L MG132 for 4 h before being harvested for Western blotting. The data are shown as mean ± SEM (n = 3 × 3, **p < 0.01; $$ p < 0.01). (B) Differences in mouse subcutaneous xenograft tumors derived from wild type HepG2 (WT), Nrf1α−/−, Nrf1α-Restored and TCF11-Restored cells were measured in size every two days, before being sacrificed on the 42nd day. The data are shown as mean ± SD (n = 7 per group, *p < 0.05; **p < 0.01; $ p < 0.05; $$ p <0.01, when compared with the WT group). (C–H) The soft agar colony formation (C, D), as well as migration (E, F) and invasion (G, H), of wild type HepG2 (WT), Nrf1α−/−, Nrf1α-Restored and TCF11-Restored cells were examined as described in Materials and Methods. The data are shown as mean ± SD (n = 9, *p < 0.05, **p < 0.01).
Figure 8
Figure 8
Diverse effects of Nrf1 and Nrf2 on those HCC-relevant proteins that are significantly correlated with the overall survival (OS). (A) The correlation analysis between of hepatocellular carcinoma (HCC) associated genes and the relevant OS rates of patients with HCC. (B) The protein expression levels of HCC-associated proteins in Nrf1α, TCF11, TCF11ΔN and Nrf2 cell models were examined by Western blotting analysis of these experimental cell lines that had been induced with or without 1 μg/ml Tet for 12 h before being harvested. (C) Both wild type HepG2 and its derived Nrf1α−/− cells were transfected with either Nrf1α or TCF11 expression plasmids, and then allowed for 24-recovery from transfection in the fresh medium before being subjected to Western blotting, to identify the protein levels of HCC-associated proteins. (D) Both wild type HepG2 and its derived Nrf2−/− cells were transfected with an Nrf2 expression plasmid, and then allowed for 24-recovery from transfection in the fresh medium before being subjected to Western blotting. (E) MHCC97L or MHCC97H cells were transfected with each of Nrf1α, TCF11 and Nrf2 expression constructs, and then allowed for 24-recovery from transfection in the fresh medium before being examined by Western blotting to identify abundances of HCC-associated proteins as described above. The intensity of all the immunoblots was calculated and shown on the bottom (B–E).
Figure 9
Figure 9
Relationship and difference between the regulation patterns of Nrf1 and Nrf2 on their targets. (A) The functional protein association networks of targets regulated by Nrf1 or Nrf2. Of note, the protein-protein associations are determined by various ways, which are thus represented by different colored edges as indicated. (B) The heatmap of the sequencing expression of genes, which are composed of the network, with distinct expression levels in TCF11, Nrf1α, Nrf2 and TCF11ΔN cell lines. The color of the nodes in the heatmap represents the value of log2 (fold change) as shown in the color bars, indicating the gene expression trend as compared with the control group (upregulation or downregulation, were marked in red or green, respectively). (C) The heatmap of the sequencing expression of genes in Nrf1α−/− and Nrf2−/− cell lines. The color of the nodes in the heatmap represents the value of log2 (fold change) as shown in the color bars, indicating the gene expression trend as compared with the wild-type HepG2 cells. (D) A comprehensive regulatory model is proposed to reveal the different effects of Nrf1 and Nrf2 on hepatoma (right panel). In addition, the Hox hub abutting the distinct Nrf loci was also indicated (at the lower left corner).

"V体育ios版" References

    1. Lambert SA, Jolma A, Campitelli LF, Das PK, Yin Y, Albu M, et al. . The Human Transcription Factors. Cell (2018) 175:598–9. 10.1016/j.cell.2018.09.045 - DOI - PubMed
    1. Sykiotis GP, Bohmann D. Stress-Activated Cap’n’collar Transcription Factors in Aging and Human Disease. Sci Signal (2010) 3:re3. 10.1126/scisignal.3112re3 - DOI - PMC - PubMed
    1. Zhang Y, Xiang Y. Molecular and Cellular Basis for the Unique Functioning of Nrf1, an Indispensable Transcription Factor for Maintaining Cell Homoeostasis and Organ Integrity. Biochem J (2016) 473:961–1000. 10.1042/BJ20151182 - DOI - PubMed
    1. Zhu YP, Wang M, Xiang Y, Qiu L, Hu S, Zhang Z, et al. . Nach Is a Novel Subgroup at an Early Evolutionary Stage of the CNC-bZIP Subfamily Transcription Factors From the Marine Bacteria to Humans. Int J Mol Sci (2018) 19. 10.3390/ijms19102927 - DOI - PMC - PubMed
    1. Kim HM, Han JW, Chan JY. Nuclear Factor Erythroid-2 Like 1 (NFE2L1): Structure, Function and Regulation. Gene (2016) 584:17–25. 10.1016/j.gene.2016.03.002 - DOI - PMC - PubMed

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