Thyroid hormone negatively regulates tumorigenesis through suppression of BC200

in Endocrine-Related Cancer
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Thyroid hormone (T3) and its receptor (TR) are involved in cancer progression. While deregulation of long non-coding RNA (lncRNA) expression has been detected in many tumor types, the mechanisms underlying specific involvement of lncRNAs in tumorigenicity remain unclear. Experiments from the current study revealed negative regulation of BC200 expression by T3/TR. BC200 was highly expressed in hepatocellular carcinoma (HCC) and effective as an independent prognostic marker. BC200 promoted cell growth and tumor sphere formation, which was mediated via regulation of cell cycle-related genes and stemness markers. Moreover, BC200 protected cyclin E2 mRNA from degradation. Cell growth ability was repressed by T3, but partially enhanced upon BC200 overexpression. Mechanistically, BC200 directly interacted with cyclin E2 and promoted CDK2–cyclin E2 complex formation. Upregulation of cell cycle-related genes in hepatoma samples was positively correlated with BC200 expression. Our collective findings support the utility of a potential therapeutic strategy involving targeting of BC200 for the treatment of HCC.

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  • Supplementary Table 1. The primers were used for qRT-PCR analysis.
  • Supplementary Table 2. Correlation among BC200, cyclin E2 and CDK2 in HCC specimens.
  • Supplementary Figure 1
  • Supplementary Figure 2
  • Supplementary Figure 3
  • Supplementary Figure 4
  • Supplementary Figure 5
  • Supplementary Figure 6

 

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    BC200 is downregulated by T3/TR and highly expressed in HCC. (A and B) HepG2-TR, HepG2-neo, J7-TR and Huh-7 cells were treated with T3 for 24–72 h, and BC200 levels measured using qRT-PCR. 18S rRNA was used as the internal control. (C) qRT-PCR analysis of BC200 expression in human HCC. 18S rRNA was used as the internal control. (D) ISH analysis of BC200 expression in human HCC. (E) Kaplan–Meier analysis of overall survival based on BC200 expression in HCC specimens. Mean expression of BC200 was used as the cutoff. Overall survival was analyzed using the log-rank test.

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    BC200 promotes cell growth and transformation ability. Effects of BC200 on cell growth (A) and soft agar colony formation (B). (C) Comparison of tumor volumes between hepatoma xenografts in BC200 stable cell lines. Xenografts were derived from Hep3B (5 × 106, n = 3 per group) and SK-Hep1 cells (2 × 106, n = 4 per group). Tumor volumes and weights were measured in xenografts after subcutaneous injection of cells. (D) Sphere formation assay of BC200-silenced stable cells. (E) CD133+ HepG2 cells treated with T3 or left untreated were infected with adenovirus-TRα1 (Ad-TRα1) and adenovirus-TRβ1 (Ad-TRβ1), and the sphere formation assay performed.

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    BC200 regulates cell cycle-related genes and is involved in T3/TR-mediated cell growth function. (A) qRT-PCR analysis of total RNA in the indicated cell lines using 18S rRNA as a loading control to determine the mRNA expression levels of cell cycle-related genes. (B and C) Western blot analysis of cyclin E, CDK2, p21 and p27 expression in the indicated cell lines and tissues. (D) Immunoblot analysis of the expression patterns of CD44, Nanog, Sox2 and cell cycle-related genes in the indicated cell lines. GAPDH was used as the loading control. (E) Left panel: Western blot analysis of expression of cell cycle-related genes in the presence and absence of T3. Right panel: Vector control (vc) and BC200 cell lines stably expressing in HepG2-TRβ1 were grown in 6 cm dishes, and cell growth detected in the presence and absence of T3.

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    BC200 promotes cyclin E2-CDK2 complex formation. (A and B) Half-life of cyclin E mRNA was calculated in the indicated cell lines after adding actinomycin D (ActD), normalizing to 18S rRNA. (C) Lysates from indicated cells were immunoprecipitated with IgG, anti-cyclin E1 and anti-cyclin E2 antibody, respectively. The immunoprecipitates were analyzed by Western blotting with an anti-CDK2 antibody. (D) RIP experiments were performed in J7 and SK-Hep1 cells and the coprecipitated RNA was subjected to qRT-PCR for BC200. GAPDH was used as negative controls. (E) Immunoblot analysis of the expression patterns of p27 and p21 in the indicated cell lines.

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    The BC200/cyclin E2/CDK2 axis is correlated with poor prognosis in HCC patients. QRT-PCR (A) and immunoblot analysis (B) of hepatic levels of BC200, cyclin E2 and CDK2 in the numbers of mice receiving DEN treatment (n = 8 per group). The quantitative results of western blot were shown. (C) qRT-PCR analysis of cyclin E2 and CDK2 expression in 140 pairs of human HCC (T) and adjacent normal (N) tissues using 18S rRNA as the loading control. (D) Kaplan–Meier overall survival analysis curve for high- or low-risk survival groups were measured in 140 paired HCC patients. High BC200 and simultaneous high the other genes were significantly associated with poorest overall survival. P values were determined via the log-rank test. (E) Proposed model for the T3/TR/BC200/stemness marker/cell cycle-related gene pathway in regulation of tumor formation and self-renewal of hepatoma.

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