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Activation of miR-21 by STAT3 induces proliferation and suppresses apoptosis in nasopharyngeal carcinoma by targeting PTEN gene.

Ou H, Li Y, Kang M - PLoS ONE (2014)

Bottom Line: To test the effect of miR-21 on the cell growth and apoptosis of NPC cells in vitro, transfection of CNE1 and CNE2 cell lines and flow cytometry were performed.STAT3, a transcription factor activated by IL-6, directly activated miR-21 in transformed NPC cell lines.Furthermore, miR-21 markedly inhibited PTEN tumor suppressor, leading to increased AKT activity.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Guangxi Medical University, Nanning City, Guangxi Province, P.R. China.

ABSTRACT
The present study is to investigate the role of microRNA-21 (miR-21) in nasopharyngeal carcinoma (NPC) and the mechanisms of regulation of PTEN by miR-21. Fifty-four tissue samples were collected from 42 patients with NPC and 12 healthy controls. Human NPC cell lines CNE-1, CNE-2, TWO3 and C666-1 were used for cell assays. To investigate the expression of miR-21, RT-PCR was employed. RT-PCR, Western blotting, and immunohistochemistry were used to measure the expression of STAT3 mRNA and STAT3 protein. To test the effect of miR-21 on the cell growth and apoptosis of NPC cells in vitro, transfection of CNE1 and CNE2 cell lines and flow cytometry were performed. TUNEL assay was used to detect DNA fragmentation. To validate whether miR-21 directly recognizes the 3'-UTRs of PTEN mRNA, luciferase reporter assay was employed. miR-21 expression was increased in NPC tissues compared with control and the same result was found in NPC cell lines. Notably, increased expression of miR-21 was directly related to advanced clinical stage and lymph node metastasis. STAT3, a transcription factor activated by IL-6, directly activated miR-21 in transformed NPC cell lines. Furthermore, miR-21 markedly inhibited PTEN tumor suppressor, leading to increased AKT activity. Both in vitro and in vivo assays revealed that miR-21 enhanced NPC cell proliferation and suppressed apoptosis. miR-21, activated by STAT3, induced proliferation and suppressed apoptosis in NPC by targeting PTEN-AKT pathway.

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The effect of miR-21 on NPC cell growth and apoptosis in vitro.(A) Relative expression of miR-21 in CNE1 and CNE2 cells stably transfected by empty vector (NC) or vectors expressing miR-21 and anti-miR-21 detected by RT-PCR. The results were normalized to U6 expression and expressed as fold change relative to the corresponding negative control. anti-miR-21 is the inhibitor of miR-21. Data are means ± SD. Asterisks indicate values that are significantly different from the NC group (n = 3). (B) Proliferation curve of CNE1 and CNE2 cells stably transfected by empty vector (NC) or vectors expressing miR-21 and anti-miR-21. Data are means ± SD. Asterisks indicate values that are significantly different from the NC group (n = 3). (C) Representative results of colony formation of CNE1 and CNE2 cells transfected with miR-21 mimics and miR control (NC). Data are presented as means ± SD. Asterisks indicate values that are significantly different from the NC group. (D) The apoptosis of CNE1 and CNE2 cells transfected with empty vector (NC) or vector expressing miR-21 detected by flow cytometry. Data are means ± SD. Asterisks indicate values that are significantly different from the NC group.
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pone-0109929-g004: The effect of miR-21 on NPC cell growth and apoptosis in vitro.(A) Relative expression of miR-21 in CNE1 and CNE2 cells stably transfected by empty vector (NC) or vectors expressing miR-21 and anti-miR-21 detected by RT-PCR. The results were normalized to U6 expression and expressed as fold change relative to the corresponding negative control. anti-miR-21 is the inhibitor of miR-21. Data are means ± SD. Asterisks indicate values that are significantly different from the NC group (n = 3). (B) Proliferation curve of CNE1 and CNE2 cells stably transfected by empty vector (NC) or vectors expressing miR-21 and anti-miR-21. Data are means ± SD. Asterisks indicate values that are significantly different from the NC group (n = 3). (C) Representative results of colony formation of CNE1 and CNE2 cells transfected with miR-21 mimics and miR control (NC). Data are presented as means ± SD. Asterisks indicate values that are significantly different from the NC group. (D) The apoptosis of CNE1 and CNE2 cells transfected with empty vector (NC) or vector expressing miR-21 detected by flow cytometry. Data are means ± SD. Asterisks indicate values that are significantly different from the NC group.

Mentions: To test the effect of miR-21 on the cell growth and apoptosis of NPC cells in vitro, transfection of CNE1 and CNE2 cell lines and flow cytometry were performed. After transfection, the miR-21 levels was increased by 90% in CNE1 cells, and 78% in CNE2 cells (P<0.01), while knockdown of miR-21 by anti-miRNA reduced miR-21 levels by 88% in CNE1 cells, and 73% in CNE2 cells (P<0.01) (Fig. 4A). miR-21 led to an increase in CNE1 and CNE2 cell growth and proliferation (P<0.05, Fig. 4B and C). By contrast, anti-miR-21 decreased CNE1 and CNE2 cell growth (P<0.01) (Fig. 4B). Flow cytometry data showed that overexpression of miR-21 significantly inhibited CNE1 and CNE2 apoptosis compared with the control (P<0.05) (Fig. 4D). These data demonstrate miR-21 stimulated NPC cell growth and inhibited apoptosis in vitro.


Activation of miR-21 by STAT3 induces proliferation and suppresses apoptosis in nasopharyngeal carcinoma by targeting PTEN gene.

Ou H, Li Y, Kang M - PLoS ONE (2014)

The effect of miR-21 on NPC cell growth and apoptosis in vitro.(A) Relative expression of miR-21 in CNE1 and CNE2 cells stably transfected by empty vector (NC) or vectors expressing miR-21 and anti-miR-21 detected by RT-PCR. The results were normalized to U6 expression and expressed as fold change relative to the corresponding negative control. anti-miR-21 is the inhibitor of miR-21. Data are means ± SD. Asterisks indicate values that are significantly different from the NC group (n = 3). (B) Proliferation curve of CNE1 and CNE2 cells stably transfected by empty vector (NC) or vectors expressing miR-21 and anti-miR-21. Data are means ± SD. Asterisks indicate values that are significantly different from the NC group (n = 3). (C) Representative results of colony formation of CNE1 and CNE2 cells transfected with miR-21 mimics and miR control (NC). Data are presented as means ± SD. Asterisks indicate values that are significantly different from the NC group. (D) The apoptosis of CNE1 and CNE2 cells transfected with empty vector (NC) or vector expressing miR-21 detected by flow cytometry. Data are means ± SD. Asterisks indicate values that are significantly different from the NC group.
© Copyright Policy
Related In: Results  -  Collection

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pone-0109929-g004: The effect of miR-21 on NPC cell growth and apoptosis in vitro.(A) Relative expression of miR-21 in CNE1 and CNE2 cells stably transfected by empty vector (NC) or vectors expressing miR-21 and anti-miR-21 detected by RT-PCR. The results were normalized to U6 expression and expressed as fold change relative to the corresponding negative control. anti-miR-21 is the inhibitor of miR-21. Data are means ± SD. Asterisks indicate values that are significantly different from the NC group (n = 3). (B) Proliferation curve of CNE1 and CNE2 cells stably transfected by empty vector (NC) or vectors expressing miR-21 and anti-miR-21. Data are means ± SD. Asterisks indicate values that are significantly different from the NC group (n = 3). (C) Representative results of colony formation of CNE1 and CNE2 cells transfected with miR-21 mimics and miR control (NC). Data are presented as means ± SD. Asterisks indicate values that are significantly different from the NC group. (D) The apoptosis of CNE1 and CNE2 cells transfected with empty vector (NC) or vector expressing miR-21 detected by flow cytometry. Data are means ± SD. Asterisks indicate values that are significantly different from the NC group.
Mentions: To test the effect of miR-21 on the cell growth and apoptosis of NPC cells in vitro, transfection of CNE1 and CNE2 cell lines and flow cytometry were performed. After transfection, the miR-21 levels was increased by 90% in CNE1 cells, and 78% in CNE2 cells (P<0.01), while knockdown of miR-21 by anti-miRNA reduced miR-21 levels by 88% in CNE1 cells, and 73% in CNE2 cells (P<0.01) (Fig. 4A). miR-21 led to an increase in CNE1 and CNE2 cell growth and proliferation (P<0.05, Fig. 4B and C). By contrast, anti-miR-21 decreased CNE1 and CNE2 cell growth (P<0.01) (Fig. 4B). Flow cytometry data showed that overexpression of miR-21 significantly inhibited CNE1 and CNE2 apoptosis compared with the control (P<0.05) (Fig. 4D). These data demonstrate miR-21 stimulated NPC cell growth and inhibited apoptosis in vitro.

Bottom Line: To test the effect of miR-21 on the cell growth and apoptosis of NPC cells in vitro, transfection of CNE1 and CNE2 cell lines and flow cytometry were performed.STAT3, a transcription factor activated by IL-6, directly activated miR-21 in transformed NPC cell lines.Furthermore, miR-21 markedly inhibited PTEN tumor suppressor, leading to increased AKT activity.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Guangxi Medical University, Nanning City, Guangxi Province, P.R. China.

ABSTRACT
The present study is to investigate the role of microRNA-21 (miR-21) in nasopharyngeal carcinoma (NPC) and the mechanisms of regulation of PTEN by miR-21. Fifty-four tissue samples were collected from 42 patients with NPC and 12 healthy controls. Human NPC cell lines CNE-1, CNE-2, TWO3 and C666-1 were used for cell assays. To investigate the expression of miR-21, RT-PCR was employed. RT-PCR, Western blotting, and immunohistochemistry were used to measure the expression of STAT3 mRNA and STAT3 protein. To test the effect of miR-21 on the cell growth and apoptosis of NPC cells in vitro, transfection of CNE1 and CNE2 cell lines and flow cytometry were performed. TUNEL assay was used to detect DNA fragmentation. To validate whether miR-21 directly recognizes the 3'-UTRs of PTEN mRNA, luciferase reporter assay was employed. miR-21 expression was increased in NPC tissues compared with control and the same result was found in NPC cell lines. Notably, increased expression of miR-21 was directly related to advanced clinical stage and lymph node metastasis. STAT3, a transcription factor activated by IL-6, directly activated miR-21 in transformed NPC cell lines. Furthermore, miR-21 markedly inhibited PTEN tumor suppressor, leading to increased AKT activity. Both in vitro and in vivo assays revealed that miR-21 enhanced NPC cell proliferation and suppressed apoptosis. miR-21, activated by STAT3, induced proliferation and suppressed apoptosis in NPC by targeting PTEN-AKT pathway.

Show MeSH
Related in: MedlinePlus