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TGFβ1 secreted by cancer-associated fibroblasts induces epithelial-mesenchymal transition of bladder cancer cells through lncRNA-ZEB2NAT.

Zhuang J, Lu Q, Shen B, Huang X, Shen L, Zheng X, Huang R, Yan J, Guo H - Sci Rep (2015)

Bottom Line: CAF-CM induced the epithelial-mesenchymal transition (EMT) by regulating expression levels of EMT-associated markers in UBC cells.Additionally, inhibition of TGFβ1 signaling decreased the EMT-associated gene expression, and cancer cell invasion.ZEB2NAT depletion reversed CAF-CM-induced EMT and invasion of cancer cells, as well as reduced the ZEB2 protein level.

View Article: PubMed Central - PubMed

Affiliation: Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, Jiangsu 210008, China.

ABSTRACT
Urinary bladder cancer (UBC) patients at muscle invasive stage have poor clinical outcome, due to high propensity for metastasis. Cancer-associated fibroblasts (CAFs), one of the principal constituents of the tumor stroma, play an important role in tumor development. However, it is unclear whether CAFs from UBC induce cell invasion and which signaling pathway is involved. Herein, we found that conditional medium from UBC CAFs (CAF-CM) enhanced the invasion of UBC cells. CAF-CM induced the epithelial-mesenchymal transition (EMT) by regulating expression levels of EMT-associated markers in UBC cells. Higher concentration of TGFβ1 in CAF-CM, comparing with the CM from adjacent normal fibroblast, led to phosphorylation of Smad2 in UBC cells. Additionally, inhibition of TGFβ1 signaling decreased the EMT-associated gene expression, and cancer cell invasion. Interestingly, a long non-coding RNA, ZEB2NAT, was demonstrated to be essential for this TGFβ1-dependent process. ZEB2NAT depletion reversed CAF-CM-induced EMT and invasion of cancer cells, as well as reduced the ZEB2 protein level. Consistently, TGFβ1 mRNA expression is positively correlated with ZEB2NAT transcript and ZEB2 protein levels in human bladder cancer specimens. Our data revealed a novel mechanism that CAFs induces EMT and invasion of human UBC cells through the TGFβ1-ZEB2NAT-ZEB2 axis.

No MeSH data available.


Related in: MedlinePlus

Correlations of TGFβ1, ZEB2NAT, and ZEB2 in clinical UBC samples.The expression levels of ZEB2NAT (A) and TGFβ1 transcripts (B), and ZEB2 protein (C) in 30 human bladder cancer tissues (Tumor or T) and the paired normal tissues (Normal or N) from the same patient. Relative ZEB2NAT and TGFβ1 mRNA levels were assessed by qRT-PCR and normalized by β-actin gene. D–F: The correlations among ZEB2NAT, TGFβ1 and ZEB2 were decided by Pearson correlation analysis. ΔCT values in qRT-PCR were used as the expression levels of ZEB2NAT and TGFβ1 transcripts. ZEB2 protein bands on Western blot were quantified by Image J software. P < 0.05 was considered statistically significant.
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f7: Correlations of TGFβ1, ZEB2NAT, and ZEB2 in clinical UBC samples.The expression levels of ZEB2NAT (A) and TGFβ1 transcripts (B), and ZEB2 protein (C) in 30 human bladder cancer tissues (Tumor or T) and the paired normal tissues (Normal or N) from the same patient. Relative ZEB2NAT and TGFβ1 mRNA levels were assessed by qRT-PCR and normalized by β-actin gene. D–F: The correlations among ZEB2NAT, TGFβ1 and ZEB2 were decided by Pearson correlation analysis. ΔCT values in qRT-PCR were used as the expression levels of ZEB2NAT and TGFβ1 transcripts. ZEB2 protein bands on Western blot were quantified by Image J software. P < 0.05 was considered statistically significant.

Mentions: The correlations among TGFβ1, ZEB2 and ZEB2NAT were investigated in 30 human UBC samples. Quantitative RT-PCR revealed that ZEB2NAT and TGFβ1 are significantly increased by 1.5 folds (P = 0.010) and 1.4 folds (P = 0.001) in human UBC samples, respectively (Fig. 7A,B). Western blotting data also demonstrated that ZEB2 protein level significantly increased in 26 out of 30 human UBC samples (Fig. 7C). Interestingly, we observed the significant increased levels of TGFβ1 mRNA (2.3 folds, P = 0.019), ZEB2NAT transcript (2.12 folds, P = 0.026) and ZEB2 protein (1.45 folds, P = 0.012) in muscle invasive group (MI), compared to non-muscle invasive group (NMI; Suppl Fig. 6). The correlations between the mRNA levels of ZEB2NAT and TGFβ1 by qRT-PCR assay plus the ZEB2 protein levels by Western blotting assay were also analyzed by Pearson’s correlation analysis. ZEB2NAT mRNA level was highly correlated with TGFβ1 mRNA level (r = 0.521, P = 0.003, Fig. 7D) and ZEB2 protein level (r = 0.692, P < 0.001, Fig. 7E). TGFβ1 mRNA level is also associated with ZEB2 protein level (r = 0.497, P = 0.005, Fig. 7F). These data indicate the high incidence of activation of TGFβ1-ZEB2NAT-ZEB2 axis in human bladder cancer patients, rendering high migration and invasion capabilities to the bladder cancer cells.


TGFβ1 secreted by cancer-associated fibroblasts induces epithelial-mesenchymal transition of bladder cancer cells through lncRNA-ZEB2NAT.

Zhuang J, Lu Q, Shen B, Huang X, Shen L, Zheng X, Huang R, Yan J, Guo H - Sci Rep (2015)

Correlations of TGFβ1, ZEB2NAT, and ZEB2 in clinical UBC samples.The expression levels of ZEB2NAT (A) and TGFβ1 transcripts (B), and ZEB2 protein (C) in 30 human bladder cancer tissues (Tumor or T) and the paired normal tissues (Normal or N) from the same patient. Relative ZEB2NAT and TGFβ1 mRNA levels were assessed by qRT-PCR and normalized by β-actin gene. D–F: The correlations among ZEB2NAT, TGFβ1 and ZEB2 were decided by Pearson correlation analysis. ΔCT values in qRT-PCR were used as the expression levels of ZEB2NAT and TGFβ1 transcripts. ZEB2 protein bands on Western blot were quantified by Image J software. P < 0.05 was considered statistically significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495469&req=5

f7: Correlations of TGFβ1, ZEB2NAT, and ZEB2 in clinical UBC samples.The expression levels of ZEB2NAT (A) and TGFβ1 transcripts (B), and ZEB2 protein (C) in 30 human bladder cancer tissues (Tumor or T) and the paired normal tissues (Normal or N) from the same patient. Relative ZEB2NAT and TGFβ1 mRNA levels were assessed by qRT-PCR and normalized by β-actin gene. D–F: The correlations among ZEB2NAT, TGFβ1 and ZEB2 were decided by Pearson correlation analysis. ΔCT values in qRT-PCR were used as the expression levels of ZEB2NAT and TGFβ1 transcripts. ZEB2 protein bands on Western blot were quantified by Image J software. P < 0.05 was considered statistically significant.
Mentions: The correlations among TGFβ1, ZEB2 and ZEB2NAT were investigated in 30 human UBC samples. Quantitative RT-PCR revealed that ZEB2NAT and TGFβ1 are significantly increased by 1.5 folds (P = 0.010) and 1.4 folds (P = 0.001) in human UBC samples, respectively (Fig. 7A,B). Western blotting data also demonstrated that ZEB2 protein level significantly increased in 26 out of 30 human UBC samples (Fig. 7C). Interestingly, we observed the significant increased levels of TGFβ1 mRNA (2.3 folds, P = 0.019), ZEB2NAT transcript (2.12 folds, P = 0.026) and ZEB2 protein (1.45 folds, P = 0.012) in muscle invasive group (MI), compared to non-muscle invasive group (NMI; Suppl Fig. 6). The correlations between the mRNA levels of ZEB2NAT and TGFβ1 by qRT-PCR assay plus the ZEB2 protein levels by Western blotting assay were also analyzed by Pearson’s correlation analysis. ZEB2NAT mRNA level was highly correlated with TGFβ1 mRNA level (r = 0.521, P = 0.003, Fig. 7D) and ZEB2 protein level (r = 0.692, P < 0.001, Fig. 7E). TGFβ1 mRNA level is also associated with ZEB2 protein level (r = 0.497, P = 0.005, Fig. 7F). These data indicate the high incidence of activation of TGFβ1-ZEB2NAT-ZEB2 axis in human bladder cancer patients, rendering high migration and invasion capabilities to the bladder cancer cells.

Bottom Line: CAF-CM induced the epithelial-mesenchymal transition (EMT) by regulating expression levels of EMT-associated markers in UBC cells.Additionally, inhibition of TGFβ1 signaling decreased the EMT-associated gene expression, and cancer cell invasion.ZEB2NAT depletion reversed CAF-CM-induced EMT and invasion of cancer cells, as well as reduced the ZEB2 protein level.

View Article: PubMed Central - PubMed

Affiliation: Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, Jiangsu 210008, China.

ABSTRACT
Urinary bladder cancer (UBC) patients at muscle invasive stage have poor clinical outcome, due to high propensity for metastasis. Cancer-associated fibroblasts (CAFs), one of the principal constituents of the tumor stroma, play an important role in tumor development. However, it is unclear whether CAFs from UBC induce cell invasion and which signaling pathway is involved. Herein, we found that conditional medium from UBC CAFs (CAF-CM) enhanced the invasion of UBC cells. CAF-CM induced the epithelial-mesenchymal transition (EMT) by regulating expression levels of EMT-associated markers in UBC cells. Higher concentration of TGFβ1 in CAF-CM, comparing with the CM from adjacent normal fibroblast, led to phosphorylation of Smad2 in UBC cells. Additionally, inhibition of TGFβ1 signaling decreased the EMT-associated gene expression, and cancer cell invasion. Interestingly, a long non-coding RNA, ZEB2NAT, was demonstrated to be essential for this TGFβ1-dependent process. ZEB2NAT depletion reversed CAF-CM-induced EMT and invasion of cancer cells, as well as reduced the ZEB2 protein level. Consistently, TGFβ1 mRNA expression is positively correlated with ZEB2NAT transcript and ZEB2 protein levels in human bladder cancer specimens. Our data revealed a novel mechanism that CAFs induces EMT and invasion of human UBC cells through the TGFβ1-ZEB2NAT-ZEB2 axis.

No MeSH data available.


Related in: MedlinePlus