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TGFβR2 is a major target of miR-93 in nasopharyngeal carcinoma aggressiveness.

Lyu X, Fang W, Cai L, Zheng H, Ye Y, Zhang L, Li J, Peng H, Cho WC, Wang E, Marincola FM, Yao K, Cai H, Li J, Li X - Mol. Cancer (2014)

Bottom Line: A cluster set of 4 TGFβR2-associated miRNAs was identified; they are all from miR-17-92 cluster and its paralogues, of which miR-93 was one of the most significant miRNAs, directly targeting TGFβR2, promoting cell proliferation, invasion and metastasis in vitro and in vivo.Moreover, miR-93 resulted in the attenuation of Smad-dependent TGF-β signaling and the activation of PI3K/Akt pathway by suppressing TGFβR2, further promoting NPC cell uncontrolled growth, invasion, metastasis and EMT-like process.Impressively, the knockdown of TGFβR2 by siRNA displayed a consentaneous phenocopy with the effect of miR-93 in NPC cells, supporting TGFβR2 is a major target of miR-93.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Research Institute and the Provincial Key Laboratory of Functional Proteomics, Southern Medical University, Guangzhou, China. chbing2008@126.com.

ABSTRACT

Background: MiR-17-92 cluster and its paralogues have emerged as crucial regulators of many oncogenes and tumor suppressors. Transforming growth factor-β receptor II (TGFβR2), as an important tumor suppressor, is involved in various cancer types. However, it is in cancer that only two miRNAs of this cluster and its paralogues have been reported so far to regulate TGFβR2. MiR-93 is oncogenic, but its targetome in cancer has not been fully defined. The role of miR-93 in nasopharyngeal carcinoma (NPC) still remains largely unknown.

Methods: We firstly evaluated the clinical signature of TGFβR2 down-regulation in clinical samples, and next used a miRNA expression profiling analysis followed by multi-validations, including Luciferase reporter assay, to identify miRNAs targeting TGFβR2 in NPC. In vitro and in vivo studies were performed to further investigate the effects of miRNA-mediated TGFβR2 down-regulation on NPC aggressiveness. Finally, mechanism studies were conducted to explore the associated pathway and genes influenced by this miRNA-mediated TGFβR2 down-regulation.

Results: TGFβR2 was down-regulated in more than 50% of NPC patients. It is an unfavorable prognosis factor contributing to clinical NPC aggressiveness. A cluster set of 4 TGFβR2-associated miRNAs was identified; they are all from miR-17-92 cluster and its paralogues, of which miR-93 was one of the most significant miRNAs, directly targeting TGFβR2, promoting cell proliferation, invasion and metastasis in vitro and in vivo. Moreover, miR-93 resulted in the attenuation of Smad-dependent TGF-β signaling and the activation of PI3K/Akt pathway by suppressing TGFβR2, further promoting NPC cell uncontrolled growth, invasion, metastasis and EMT-like process. Impressively, the knockdown of TGFβR2 by siRNA displayed a consentaneous phenocopy with the effect of miR-93 in NPC cells, supporting TGFβR2 is a major target of miR-93. Our findings were also substantiated by investigation of the clinical signatures of miR-93 and TGFβR2 in NPC.

Conclusion: The present study reports an involvement of miR-93-mediated TGFβR2 down-regulation in NPC aggressiveness, thus giving extended insights into molecular mechanisms underlying cancer aggressiveness. Approaches aimed at blocking miR-93 may serve as a promising therapeutic strategy for treating NPC patients.

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TGFβR2 down-regulation is associated with NPC aggressiveness. (A) TGFβR2 mRNA expression was detected by qRT-PCR in 21 NP tissues and 35 NPC tissues. Values represent mean ± SD, ***P < 0.001. (B) Representative TGFβR2 IHC images (400×). a. Normal nasopharynx epithelium, b. NPC with high TGFβR2 expression, c. NPC with low TGFβR2 expression. (C) Kaplan-Meier survival analysis in NPC patients according to TGFβR2 protein expression levels. The log-rank test was used to calculate p values (p < 0.001).
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Figure 1: TGFβR2 down-regulation is associated with NPC aggressiveness. (A) TGFβR2 mRNA expression was detected by qRT-PCR in 21 NP tissues and 35 NPC tissues. Values represent mean ± SD, ***P < 0.001. (B) Representative TGFβR2 IHC images (400×). a. Normal nasopharynx epithelium, b. NPC with high TGFβR2 expression, c. NPC with low TGFβR2 expression. (C) Kaplan-Meier survival analysis in NPC patients according to TGFβR2 protein expression levels. The log-rank test was used to calculate p values (p < 0.001).

Mentions: Our previous study reported a down-regulated TGFβR2 expression in NPC [40], so we initially confirmed it in the present study. TGFβR2 expression was indeed observed to be significantly reduced in NPC patients relative to non-cancerous nasopharyngeal (NP) (Figure 1A). Next, we investigated the clinical signature of TGFβR2 down- regulation using IHC in additional 300 clinical NPC samples. TGFβR2 protein was down-regulated in 51.9% (108/208) of NPC, 38.5% (5/13) of atypical hyperplasia, 9.1% (2/22) of normal squamous epithelium, and 5.3% (3/57) of normal epithelium, displaying a gradual reduction trend from normal epithelium to NPC (Table 1). Pathological analysis showed that the expression level of TGFβR2 is negatively correlated with T classification (the size of the primary tumor and whether it has invaded nearby tissue), N classification (the degree of spread to regional lymph nodes), and clinical stage of NPC patients (Additional file 1: Table S1). Kaplan-Meier survival analysis revealed that TGFβR2 expression was significantly correlated with patient overall survival (Figure 1B, C and Additional file 2: Figure S1). Multivariate survival analysis using the Cox's proportional hazards model showed a close correlation of low TGFβR2 protein expression with clinical prognosis (Additional file 1: Table S2).


TGFβR2 is a major target of miR-93 in nasopharyngeal carcinoma aggressiveness.

Lyu X, Fang W, Cai L, Zheng H, Ye Y, Zhang L, Li J, Peng H, Cho WC, Wang E, Marincola FM, Yao K, Cai H, Li J, Li X - Mol. Cancer (2014)

TGFβR2 down-regulation is associated with NPC aggressiveness. (A) TGFβR2 mRNA expression was detected by qRT-PCR in 21 NP tissues and 35 NPC tissues. Values represent mean ± SD, ***P < 0.001. (B) Representative TGFβR2 IHC images (400×). a. Normal nasopharynx epithelium, b. NPC with high TGFβR2 expression, c. NPC with low TGFβR2 expression. (C) Kaplan-Meier survival analysis in NPC patients according to TGFβR2 protein expression levels. The log-rank test was used to calculate p values (p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4016586&req=5

Figure 1: TGFβR2 down-regulation is associated with NPC aggressiveness. (A) TGFβR2 mRNA expression was detected by qRT-PCR in 21 NP tissues and 35 NPC tissues. Values represent mean ± SD, ***P < 0.001. (B) Representative TGFβR2 IHC images (400×). a. Normal nasopharynx epithelium, b. NPC with high TGFβR2 expression, c. NPC with low TGFβR2 expression. (C) Kaplan-Meier survival analysis in NPC patients according to TGFβR2 protein expression levels. The log-rank test was used to calculate p values (p < 0.001).
Mentions: Our previous study reported a down-regulated TGFβR2 expression in NPC [40], so we initially confirmed it in the present study. TGFβR2 expression was indeed observed to be significantly reduced in NPC patients relative to non-cancerous nasopharyngeal (NP) (Figure 1A). Next, we investigated the clinical signature of TGFβR2 down- regulation using IHC in additional 300 clinical NPC samples. TGFβR2 protein was down-regulated in 51.9% (108/208) of NPC, 38.5% (5/13) of atypical hyperplasia, 9.1% (2/22) of normal squamous epithelium, and 5.3% (3/57) of normal epithelium, displaying a gradual reduction trend from normal epithelium to NPC (Table 1). Pathological analysis showed that the expression level of TGFβR2 is negatively correlated with T classification (the size of the primary tumor and whether it has invaded nearby tissue), N classification (the degree of spread to regional lymph nodes), and clinical stage of NPC patients (Additional file 1: Table S1). Kaplan-Meier survival analysis revealed that TGFβR2 expression was significantly correlated with patient overall survival (Figure 1B, C and Additional file 2: Figure S1). Multivariate survival analysis using the Cox's proportional hazards model showed a close correlation of low TGFβR2 protein expression with clinical prognosis (Additional file 1: Table S2).

Bottom Line: A cluster set of 4 TGFβR2-associated miRNAs was identified; they are all from miR-17-92 cluster and its paralogues, of which miR-93 was one of the most significant miRNAs, directly targeting TGFβR2, promoting cell proliferation, invasion and metastasis in vitro and in vivo.Moreover, miR-93 resulted in the attenuation of Smad-dependent TGF-β signaling and the activation of PI3K/Akt pathway by suppressing TGFβR2, further promoting NPC cell uncontrolled growth, invasion, metastasis and EMT-like process.Impressively, the knockdown of TGFβR2 by siRNA displayed a consentaneous phenocopy with the effect of miR-93 in NPC cells, supporting TGFβR2 is a major target of miR-93.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Research Institute and the Provincial Key Laboratory of Functional Proteomics, Southern Medical University, Guangzhou, China. chbing2008@126.com.

ABSTRACT

Background: MiR-17-92 cluster and its paralogues have emerged as crucial regulators of many oncogenes and tumor suppressors. Transforming growth factor-β receptor II (TGFβR2), as an important tumor suppressor, is involved in various cancer types. However, it is in cancer that only two miRNAs of this cluster and its paralogues have been reported so far to regulate TGFβR2. MiR-93 is oncogenic, but its targetome in cancer has not been fully defined. The role of miR-93 in nasopharyngeal carcinoma (NPC) still remains largely unknown.

Methods: We firstly evaluated the clinical signature of TGFβR2 down-regulation in clinical samples, and next used a miRNA expression profiling analysis followed by multi-validations, including Luciferase reporter assay, to identify miRNAs targeting TGFβR2 in NPC. In vitro and in vivo studies were performed to further investigate the effects of miRNA-mediated TGFβR2 down-regulation on NPC aggressiveness. Finally, mechanism studies were conducted to explore the associated pathway and genes influenced by this miRNA-mediated TGFβR2 down-regulation.

Results: TGFβR2 was down-regulated in more than 50% of NPC patients. It is an unfavorable prognosis factor contributing to clinical NPC aggressiveness. A cluster set of 4 TGFβR2-associated miRNAs was identified; they are all from miR-17-92 cluster and its paralogues, of which miR-93 was one of the most significant miRNAs, directly targeting TGFβR2, promoting cell proliferation, invasion and metastasis in vitro and in vivo. Moreover, miR-93 resulted in the attenuation of Smad-dependent TGF-β signaling and the activation of PI3K/Akt pathway by suppressing TGFβR2, further promoting NPC cell uncontrolled growth, invasion, metastasis and EMT-like process. Impressively, the knockdown of TGFβR2 by siRNA displayed a consentaneous phenocopy with the effect of miR-93 in NPC cells, supporting TGFβR2 is a major target of miR-93. Our findings were also substantiated by investigation of the clinical signatures of miR-93 and TGFβR2 in NPC.

Conclusion: The present study reports an involvement of miR-93-mediated TGFβR2 down-regulation in NPC aggressiveness, thus giving extended insights into molecular mechanisms underlying cancer aggressiveness. Approaches aimed at blocking miR-93 may serve as a promising therapeutic strategy for treating NPC patients.

Show MeSH
Related in: MedlinePlus