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14-3-3σ induces heat shock protein 70 expression in hepatocellular carcinoma.

Liu CC, Jan YJ, Ko BS, Wu YM, Liang SM, Chen SC, Lee YM, Liu TA, Chang TC, Wang J, Shyue SK, Sung LY, Liou JY - BMC Cancer (2014)

Bottom Line: Moreover, expression of 14-3-3σ significantly correlates with HSF-1α/HSP70 in HCC tumors and both 14-3-3σ and HSP70 overexpression are associated with micro-vascular thrombi in HCC patients, suggesting that 14-3-3σ/HSP70 expression is potentially involved in cell migration/invasion.Finally, 14-3-3σ-induced HSF-1α/HSP70 expression is abolished by the knockdown of β-catenin or activation of GSK-3β.Thus, 14-3-3σ alone or combined with HSP70 are potential prognostic biomarkers for HCC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan. liyingsung@ntu.edu.tw.

ABSTRACT

Background: 14-3-3σ is implicated in promoting tumor development of various malignancies. However, the clinical relevance of 14-3-3σ in hepatocellular carcinoma (HCC) tumor progression and modulation and pathway elucidation remain unclear.

Methods: We investigated 14-3-3σ expression in 109 HCC tissues by immunohistochemistry. Overexpression and knockdown experiments were performed by transfection with cDNA or siRNA. Protein expression and cell migration were determined by Western blot and Boyden chamber assay.

Results: In this study, we found that 14-3-3σ is abundantly expressed in HCC tumors. Stable or transient overexpression of 14-3-3σ induces the expression of heat shock factor-1α (HSF-1α) and heat shock protein 70 (HSP70) in HCC cells. Moreover, expression of 14-3-3σ significantly correlates with HSF-1α/HSP70 in HCC tumors and both 14-3-3σ and HSP70 overexpression are associated with micro-vascular thrombi in HCC patients, suggesting that 14-3-3σ/HSP70 expression is potentially involved in cell migration/invasion. Results of an in vitro migration assay indicate that 14-3-3σ promotes cell migration and that 14-3-3σ-induced cell migration is impaired by siRNA knockdown of HSP70. Finally, 14-3-3σ-induced HSF-1α/HSP70 expression is abolished by the knockdown of β-catenin or activation of GSK-3β.

Conclusions: Our findings indicate that 14-3-3σ participates in promoting HCC cell migration and tumor development via β-catenin/HSF-1α/HSP70 pathway regulation. Thus, 14-3-3σ alone or combined with HSP70 are potential prognostic biomarkers for HCC.

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14-3-3σ induces HSF-1 and HSP70 expression. (A) Increased expression of HSF-1 and HSP70 induced by 14-3-3σ stable overexpression was determined by Western blotting and (B) real-time PCR analysis. Lanes 1–4 indicate as 4 different stable clones selected from the single colonies. Scale bars: mean ± SD. (C) Transient transfection of 14-3-3σ overexpression induced HSF-1 and HSP70 expression. (D) Knockdown of 14-3-3σ with siRNA suppressed HSF-1 and HSP70 expression in 14-3-3σ stable cells and (E) SK-Hep1 cells. Expression of HSF-1, HSP70, 14-3-3σ and Flag was determined by Western blot analysis. Actin was used as a loading control.
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Figure 2: 14-3-3σ induces HSF-1 and HSP70 expression. (A) Increased expression of HSF-1 and HSP70 induced by 14-3-3σ stable overexpression was determined by Western blotting and (B) real-time PCR analysis. Lanes 1–4 indicate as 4 different stable clones selected from the single colonies. Scale bars: mean ± SD. (C) Transient transfection of 14-3-3σ overexpression induced HSF-1 and HSP70 expression. (D) Knockdown of 14-3-3σ with siRNA suppressed HSF-1 and HSP70 expression in 14-3-3σ stable cells and (E) SK-Hep1 cells. Expression of HSF-1, HSP70, 14-3-3σ and Flag was determined by Western blot analysis. Actin was used as a loading control.

Mentions: To explore the downstream factors of 14-3-3σ which are involved in modulating HCC tumor progression, we performed a gene expression profile analysis of 14-3-3σ overexpresion (Additional file1: Table S3 and Table S4). Among the genes altered by 14-3-3σ overexpression, we identified HSP70 as one of the potential targets. Earlier studies have suggested that HSP70 serves as a potential biomarker for early detection of HCC[18-23]. To validate whether HSP70 expression is induced by 14-3-3σ, we determined HSP70 and its transcriptional activator, HSF-1α levels by Western blot analysis. Stable cells with increased 14-3-3σ expression significantly induced HSF-1α as well as HSP70 expression levels (Figure 2A). Induction of HSF-1α/HSP70 was further validated on the RNA level by quantitative real-time PCR analysis (Figure 2B). We next performed transient transfection experiments of 14-3-3σ overexpression in HCC cells. Transient overexpression of 14-3-3σ significantly induced HSF-1α/HSP70 expression in Huh-7 cells (Figure 2C). Furthermore, 14-3-3σ-induced HSF-1α and HSP70 expressions were attenuated by siRNA knockdown of 14-3-3σ (Figure 2D) and HSF-1α (Additional file1: Figure S3). In addition, transfection of 14-3-3σ siRNA suppressed HSF-1α and HSP70 expressions in SK-Hep1 cells (Figure 2E). These results indicate that HSF-1α/HSP70 expressions are induced by 14-3-3σ in HCC.


14-3-3σ induces heat shock protein 70 expression in hepatocellular carcinoma.

Liu CC, Jan YJ, Ko BS, Wu YM, Liang SM, Chen SC, Lee YM, Liu TA, Chang TC, Wang J, Shyue SK, Sung LY, Liou JY - BMC Cancer (2014)

14-3-3σ induces HSF-1 and HSP70 expression. (A) Increased expression of HSF-1 and HSP70 induced by 14-3-3σ stable overexpression was determined by Western blotting and (B) real-time PCR analysis. Lanes 1–4 indicate as 4 different stable clones selected from the single colonies. Scale bars: mean ± SD. (C) Transient transfection of 14-3-3σ overexpression induced HSF-1 and HSP70 expression. (D) Knockdown of 14-3-3σ with siRNA suppressed HSF-1 and HSP70 expression in 14-3-3σ stable cells and (E) SK-Hep1 cells. Expression of HSF-1, HSP70, 14-3-3σ and Flag was determined by Western blot analysis. Actin was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061114&req=5

Figure 2: 14-3-3σ induces HSF-1 and HSP70 expression. (A) Increased expression of HSF-1 and HSP70 induced by 14-3-3σ stable overexpression was determined by Western blotting and (B) real-time PCR analysis. Lanes 1–4 indicate as 4 different stable clones selected from the single colonies. Scale bars: mean ± SD. (C) Transient transfection of 14-3-3σ overexpression induced HSF-1 and HSP70 expression. (D) Knockdown of 14-3-3σ with siRNA suppressed HSF-1 and HSP70 expression in 14-3-3σ stable cells and (E) SK-Hep1 cells. Expression of HSF-1, HSP70, 14-3-3σ and Flag was determined by Western blot analysis. Actin was used as a loading control.
Mentions: To explore the downstream factors of 14-3-3σ which are involved in modulating HCC tumor progression, we performed a gene expression profile analysis of 14-3-3σ overexpresion (Additional file1: Table S3 and Table S4). Among the genes altered by 14-3-3σ overexpression, we identified HSP70 as one of the potential targets. Earlier studies have suggested that HSP70 serves as a potential biomarker for early detection of HCC[18-23]. To validate whether HSP70 expression is induced by 14-3-3σ, we determined HSP70 and its transcriptional activator, HSF-1α levels by Western blot analysis. Stable cells with increased 14-3-3σ expression significantly induced HSF-1α as well as HSP70 expression levels (Figure 2A). Induction of HSF-1α/HSP70 was further validated on the RNA level by quantitative real-time PCR analysis (Figure 2B). We next performed transient transfection experiments of 14-3-3σ overexpression in HCC cells. Transient overexpression of 14-3-3σ significantly induced HSF-1α/HSP70 expression in Huh-7 cells (Figure 2C). Furthermore, 14-3-3σ-induced HSF-1α and HSP70 expressions were attenuated by siRNA knockdown of 14-3-3σ (Figure 2D) and HSF-1α (Additional file1: Figure S3). In addition, transfection of 14-3-3σ siRNA suppressed HSF-1α and HSP70 expressions in SK-Hep1 cells (Figure 2E). These results indicate that HSF-1α/HSP70 expressions are induced by 14-3-3σ in HCC.

Bottom Line: Moreover, expression of 14-3-3σ significantly correlates with HSF-1α/HSP70 in HCC tumors and both 14-3-3σ and HSP70 overexpression are associated with micro-vascular thrombi in HCC patients, suggesting that 14-3-3σ/HSP70 expression is potentially involved in cell migration/invasion.Finally, 14-3-3σ-induced HSF-1α/HSP70 expression is abolished by the knockdown of β-catenin or activation of GSK-3β.Thus, 14-3-3σ alone or combined with HSP70 are potential prognostic biomarkers for HCC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan. liyingsung@ntu.edu.tw.

ABSTRACT

Background: 14-3-3σ is implicated in promoting tumor development of various malignancies. However, the clinical relevance of 14-3-3σ in hepatocellular carcinoma (HCC) tumor progression and modulation and pathway elucidation remain unclear.

Methods: We investigated 14-3-3σ expression in 109 HCC tissues by immunohistochemistry. Overexpression and knockdown experiments were performed by transfection with cDNA or siRNA. Protein expression and cell migration were determined by Western blot and Boyden chamber assay.

Results: In this study, we found that 14-3-3σ is abundantly expressed in HCC tumors. Stable or transient overexpression of 14-3-3σ induces the expression of heat shock factor-1α (HSF-1α) and heat shock protein 70 (HSP70) in HCC cells. Moreover, expression of 14-3-3σ significantly correlates with HSF-1α/HSP70 in HCC tumors and both 14-3-3σ and HSP70 overexpression are associated with micro-vascular thrombi in HCC patients, suggesting that 14-3-3σ/HSP70 expression is potentially involved in cell migration/invasion. Results of an in vitro migration assay indicate that 14-3-3σ promotes cell migration and that 14-3-3σ-induced cell migration is impaired by siRNA knockdown of HSP70. Finally, 14-3-3σ-induced HSF-1α/HSP70 expression is abolished by the knockdown of β-catenin or activation of GSK-3β.

Conclusions: Our findings indicate that 14-3-3σ participates in promoting HCC cell migration and tumor development via β-catenin/HSF-1α/HSP70 pathway regulation. Thus, 14-3-3σ alone or combined with HSP70 are potential prognostic biomarkers for HCC.

Show MeSH
Related in: MedlinePlus