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MicroRNA-24 inhibits growth, induces apoptosis, and reverses radioresistance in laryngeal squamous cell carcinoma by targeting X-linked inhibitor of apoptosis protein.

Xu L, Chen Z, Xue F, Chen W, Ma R, Cheng S, Cui P - Cancer Cell Int. (2015)

Bottom Line: Functional analyses indicated that re-expression of miR-24 inhibits growth, reduces colony formation, and enhances apoptosis in LSCC cells.Upregulation of miR-24 inhibits XIAP protein expression in LSCC cells, and silencing of XIAP mimics the effects of miR-24 upregulation on LSCC cells.In addition, XIAP mRNA expression significantly increases in LSCC tissues and is inversely correlated with miR-24 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology-Head and Neck Surgery, Tangdu Hospital and Laboratory for Laryngotracheal Reconstruction, Fourth Military Medical University, Xi'an, Shaanxi 710038 PR China ; Department of Otolaryngology-Head and Neck Surgery, Nanjing General Hospital of Nanjing Military Command, Nanjing, Jiangsu 210002 PR China.

ABSTRACT

Background: Increasing evidence indicates that dysregulation of microRNAs is involved in tumor progression and development. The aim of this study was to investigate the expression of microRNA-24 (miR-24) and its function in laryngeal squamous cell carcinoma (LSCC).

Methods: Quantitative RT-PCR (qRT-PCR) was used to detect miR-24 expression in LSCC cell lines and tissue samples. MTT, colony formation, and flow cytometry was performed to analyze the effects of miR-24 expression on growth, apoptosis, and radiosensitivity of LSCC cells. Dual-luciferase reporter assays were performed to examine regulation of putative miR-24 targets. Expression of X-linked inhibitor of apoptosis protein (XIAP) mRNA and protein, cleaved or total caspase-3, and cleaved or total PARP protein were detected by qRT-PCR and western blotting assays, respectively.

Results: miR-24 expression levels in LSCC cell lines or tissue were significantly lower than in a normal human keratinocyte cell line or adjacent normal tissues. Functional analyses indicated that re-expression of miR-24 inhibits growth, reduces colony formation, and enhances apoptosis in LSCC cells. In addition, miR-24 upregulation increases LSCC sensitivity to irradiation by enhancing irradiation-induced apoptosis, and luciferase activity indicated that miR-24 binds to the 3'-untranslated region (3'-UTR) of XIAP mRNA. Upregulation of miR-24 inhibits XIAP protein expression in LSCC cells, and silencing of XIAP mimics the effects of miR-24 upregulation on LSCC cells. In addition, XIAP mRNA expression significantly increases in LSCC tissues and is inversely correlated with miR-24 expression.

Conclusions: Our data suggest that miR-24 inhibits growth, increases apoptosis, and enhances radiosensitivity in LSCC cells by targeting XIAP. Therefore, miR-24 may be a potential molecular target for the treatment of human LSCC.

No MeSH data available.


Related in: MedlinePlus

Effects of miR-24 expression on growth, colony formation, and apoptosis in LSCC. a qRT-PCR of miR-24 expression in mock or stably transfected Hep-2 and AMC-HN-8 cells. U6 was used as an internal control. b MTT analysis of Hep-2 and AMC-HN-8 growth following stable transfection with pGCMV/miR-NC or pGVMV/miR-24, respectively. c Colony formation assay was performed as described in Methods. d Flow cytometric analysis of apoptosis in Hep-2 and AMC-HN-8 stably transfected with pGCMV/miR-NC or pGVMV/miR-24, respectively. e Western blot detection of c-caspase-3, total caspase-3, c-PARP, and total PARP in the stably transfected Hep-2 and AMC-HN-8 cells. GAPDH was used as an internal control. Each experiment was performed at least in triplicate. *P < 0.05, **P < 0.01 vs control
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Fig2: Effects of miR-24 expression on growth, colony formation, and apoptosis in LSCC. a qRT-PCR of miR-24 expression in mock or stably transfected Hep-2 and AMC-HN-8 cells. U6 was used as an internal control. b MTT analysis of Hep-2 and AMC-HN-8 growth following stable transfection with pGCMV/miR-NC or pGVMV/miR-24, respectively. c Colony formation assay was performed as described in Methods. d Flow cytometric analysis of apoptosis in Hep-2 and AMC-HN-8 stably transfected with pGCMV/miR-NC or pGVMV/miR-24, respectively. e Western blot detection of c-caspase-3, total caspase-3, c-PARP, and total PARP in the stably transfected Hep-2 and AMC-HN-8 cells. GAPDH was used as an internal control. Each experiment was performed at least in triplicate. *P < 0.05, **P < 0.01 vs control

Mentions: To determine the effects of miR-24 expression on LSCC cells, pGCMV/miR-224 or pGCMV/miR-NC was stably transfected into Hep-2 and AMC-HN-8, and qRT-PCR used to confirm upregulation of miR-24 (Fig. 2a). The effects of miR-24 expression on LSCC growth were then examined by MTT and colony formation assays, indicating reduced growth (Fig. 2b). Similarly, colony formation capacity in Hep-2/miR-24 and AMC-HN-8/miR-24 cells was significantly reduced compared to controls (Fig. 2c). Flow cytometric analysis showed that miR-24 re-expression enhanced apoptosis in LSCC cells. Furthermore, upregulation of miR-24 increased expression levels of cleaved caspase-3 (c-caspase-3), cleaved PARP (c-PARP), and decreased expression levels of total caspase-3 and PARP (Fig. 2d). Therefore, re-expression of miR-24 appears to inhibit growth of LSCC cells by inducing caspase-3-dependent apoptosis.Fig. 2


MicroRNA-24 inhibits growth, induces apoptosis, and reverses radioresistance in laryngeal squamous cell carcinoma by targeting X-linked inhibitor of apoptosis protein.

Xu L, Chen Z, Xue F, Chen W, Ma R, Cheng S, Cui P - Cancer Cell Int. (2015)

Effects of miR-24 expression on growth, colony formation, and apoptosis in LSCC. a qRT-PCR of miR-24 expression in mock or stably transfected Hep-2 and AMC-HN-8 cells. U6 was used as an internal control. b MTT analysis of Hep-2 and AMC-HN-8 growth following stable transfection with pGCMV/miR-NC or pGVMV/miR-24, respectively. c Colony formation assay was performed as described in Methods. d Flow cytometric analysis of apoptosis in Hep-2 and AMC-HN-8 stably transfected with pGCMV/miR-NC or pGVMV/miR-24, respectively. e Western blot detection of c-caspase-3, total caspase-3, c-PARP, and total PARP in the stably transfected Hep-2 and AMC-HN-8 cells. GAPDH was used as an internal control. Each experiment was performed at least in triplicate. *P < 0.05, **P < 0.01 vs control
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4477309&req=5

Fig2: Effects of miR-24 expression on growth, colony formation, and apoptosis in LSCC. a qRT-PCR of miR-24 expression in mock or stably transfected Hep-2 and AMC-HN-8 cells. U6 was used as an internal control. b MTT analysis of Hep-2 and AMC-HN-8 growth following stable transfection with pGCMV/miR-NC or pGVMV/miR-24, respectively. c Colony formation assay was performed as described in Methods. d Flow cytometric analysis of apoptosis in Hep-2 and AMC-HN-8 stably transfected with pGCMV/miR-NC or pGVMV/miR-24, respectively. e Western blot detection of c-caspase-3, total caspase-3, c-PARP, and total PARP in the stably transfected Hep-2 and AMC-HN-8 cells. GAPDH was used as an internal control. Each experiment was performed at least in triplicate. *P < 0.05, **P < 0.01 vs control
Mentions: To determine the effects of miR-24 expression on LSCC cells, pGCMV/miR-224 or pGCMV/miR-NC was stably transfected into Hep-2 and AMC-HN-8, and qRT-PCR used to confirm upregulation of miR-24 (Fig. 2a). The effects of miR-24 expression on LSCC growth were then examined by MTT and colony formation assays, indicating reduced growth (Fig. 2b). Similarly, colony formation capacity in Hep-2/miR-24 and AMC-HN-8/miR-24 cells was significantly reduced compared to controls (Fig. 2c). Flow cytometric analysis showed that miR-24 re-expression enhanced apoptosis in LSCC cells. Furthermore, upregulation of miR-24 increased expression levels of cleaved caspase-3 (c-caspase-3), cleaved PARP (c-PARP), and decreased expression levels of total caspase-3 and PARP (Fig. 2d). Therefore, re-expression of miR-24 appears to inhibit growth of LSCC cells by inducing caspase-3-dependent apoptosis.Fig. 2

Bottom Line: Functional analyses indicated that re-expression of miR-24 inhibits growth, reduces colony formation, and enhances apoptosis in LSCC cells.Upregulation of miR-24 inhibits XIAP protein expression in LSCC cells, and silencing of XIAP mimics the effects of miR-24 upregulation on LSCC cells.In addition, XIAP mRNA expression significantly increases in LSCC tissues and is inversely correlated with miR-24 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology-Head and Neck Surgery, Tangdu Hospital and Laboratory for Laryngotracheal Reconstruction, Fourth Military Medical University, Xi'an, Shaanxi 710038 PR China ; Department of Otolaryngology-Head and Neck Surgery, Nanjing General Hospital of Nanjing Military Command, Nanjing, Jiangsu 210002 PR China.

ABSTRACT

Background: Increasing evidence indicates that dysregulation of microRNAs is involved in tumor progression and development. The aim of this study was to investigate the expression of microRNA-24 (miR-24) and its function in laryngeal squamous cell carcinoma (LSCC).

Methods: Quantitative RT-PCR (qRT-PCR) was used to detect miR-24 expression in LSCC cell lines and tissue samples. MTT, colony formation, and flow cytometry was performed to analyze the effects of miR-24 expression on growth, apoptosis, and radiosensitivity of LSCC cells. Dual-luciferase reporter assays were performed to examine regulation of putative miR-24 targets. Expression of X-linked inhibitor of apoptosis protein (XIAP) mRNA and protein, cleaved or total caspase-3, and cleaved or total PARP protein were detected by qRT-PCR and western blotting assays, respectively.

Results: miR-24 expression levels in LSCC cell lines or tissue were significantly lower than in a normal human keratinocyte cell line or adjacent normal tissues. Functional analyses indicated that re-expression of miR-24 inhibits growth, reduces colony formation, and enhances apoptosis in LSCC cells. In addition, miR-24 upregulation increases LSCC sensitivity to irradiation by enhancing irradiation-induced apoptosis, and luciferase activity indicated that miR-24 binds to the 3'-untranslated region (3'-UTR) of XIAP mRNA. Upregulation of miR-24 inhibits XIAP protein expression in LSCC cells, and silencing of XIAP mimics the effects of miR-24 upregulation on LSCC cells. In addition, XIAP mRNA expression significantly increases in LSCC tissues and is inversely correlated with miR-24 expression.

Conclusions: Our data suggest that miR-24 inhibits growth, increases apoptosis, and enhances radiosensitivity in LSCC cells by targeting XIAP. Therefore, miR-24 may be a potential molecular target for the treatment of human LSCC.

No MeSH data available.


Related in: MedlinePlus