Limits...
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 XIAP knockdown on growth, colony formation, and apoptosis in LSCC cells. a Western blot of XIAP protein expression in Hep-2 and AMC-HN-8 cells stably transfected with pSil/shXIAP or pSil/shcontrol, respectively. GAPDH was used as an internal control. b MTT analysis of growth in Hep-2 and AMC-HN-8 cells stably transfected with pSil/shXIAP or pSil/shcontrol, respectively. c Colony formation assay was performed as described in Methods. d Flow cytometric detection of apoptosis in Hep-2 and AMC-HN-8 cells stably transfected with pSil/shcontrol or pSil/shXIAP, respectively. e Western blot detection of c-caspase-3, total caspase-3, c-PARP, and total PARP proteins in the stably transfected Hep-2 and AMC-HN-8. 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
getmorefigures.php?uid=PMC4477309&req=5

Fig5: Effects of XIAP knockdown on growth, colony formation, and apoptosis in LSCC cells. a Western blot of XIAP protein expression in Hep-2 and AMC-HN-8 cells stably transfected with pSil/shXIAP or pSil/shcontrol, respectively. GAPDH was used as an internal control. b MTT analysis of growth in Hep-2 and AMC-HN-8 cells stably transfected with pSil/shXIAP or pSil/shcontrol, respectively. c Colony formation assay was performed as described in Methods. d Flow cytometric detection of apoptosis in Hep-2 and AMC-HN-8 cells stably transfected with pSil/shcontrol or pSil/shXIAP, respectively. e Western blot detection of c-caspase-3, total caspase-3, c-PARP, and total PARP proteins in the stably transfected Hep-2 and AMC-HN-8. 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 validate that miR-24-mediated effects in LSCC cells resulted from targeting XIAP, pSil/shXIAP or pSil/shcontrol was stably transfected into Hep-2 (Hep-2/shXIAP or Hep-2/shcontrol) and AMC-HN-8 (AMC-HN-8/shXIAP or AMC-HN-8/shcontrol) cells. Western blot confirmed the decreased expression of XIAP protein in Hep-2/shXIAP or AMC-HN-8/shXIAP cells compared with mock Hep-2 or AMC-HN-8, and Hep-2/shcontrol or AMC-HN-8/sh controls (Fig. 5a). MTT and colony formation assays revealed that siRNA-mediated XIAP downregulation decreases growth and colony formation in LSCC cells (Fig. 5b, c). Flow cytometric analysis further indicated that silencing of XIAP increases caspase-3-dependent apoptosis in LSCC cells (Fig. 5d, e). Furthermore, silencing of XIAP increased the sensitivity of LSCC cells to irradiation by enhancing irradiation-induced caspase-3-dependent apoptosis (Fig. 6a-c). Therefore, silencing of XIAP mimics the effects of miR-24 upregulation on LSCC cells.Fig. 5


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 XIAP knockdown on growth, colony formation, and apoptosis in LSCC cells. a Western blot of XIAP protein expression in Hep-2 and AMC-HN-8 cells stably transfected with pSil/shXIAP or pSil/shcontrol, respectively. GAPDH was used as an internal control. b MTT analysis of growth in Hep-2 and AMC-HN-8 cells stably transfected with pSil/shXIAP or pSil/shcontrol, respectively. c Colony formation assay was performed as described in Methods. d Flow cytometric detection of apoptosis in Hep-2 and AMC-HN-8 cells stably transfected with pSil/shcontrol or pSil/shXIAP, respectively. e Western blot detection of c-caspase-3, total caspase-3, c-PARP, and total PARP proteins in the stably transfected Hep-2 and AMC-HN-8. 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

Fig5: Effects of XIAP knockdown on growth, colony formation, and apoptosis in LSCC cells. a Western blot of XIAP protein expression in Hep-2 and AMC-HN-8 cells stably transfected with pSil/shXIAP or pSil/shcontrol, respectively. GAPDH was used as an internal control. b MTT analysis of growth in Hep-2 and AMC-HN-8 cells stably transfected with pSil/shXIAP or pSil/shcontrol, respectively. c Colony formation assay was performed as described in Methods. d Flow cytometric detection of apoptosis in Hep-2 and AMC-HN-8 cells stably transfected with pSil/shcontrol or pSil/shXIAP, respectively. e Western blot detection of c-caspase-3, total caspase-3, c-PARP, and total PARP proteins in the stably transfected Hep-2 and AMC-HN-8. 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 validate that miR-24-mediated effects in LSCC cells resulted from targeting XIAP, pSil/shXIAP or pSil/shcontrol was stably transfected into Hep-2 (Hep-2/shXIAP or Hep-2/shcontrol) and AMC-HN-8 (AMC-HN-8/shXIAP or AMC-HN-8/shcontrol) cells. Western blot confirmed the decreased expression of XIAP protein in Hep-2/shXIAP or AMC-HN-8/shXIAP cells compared with mock Hep-2 or AMC-HN-8, and Hep-2/shcontrol or AMC-HN-8/sh controls (Fig. 5a). MTT and colony formation assays revealed that siRNA-mediated XIAP downregulation decreases growth and colony formation in LSCC cells (Fig. 5b, c). Flow cytometric analysis further indicated that silencing of XIAP increases caspase-3-dependent apoptosis in LSCC cells (Fig. 5d, e). Furthermore, silencing of XIAP increased the sensitivity of LSCC cells to irradiation by enhancing irradiation-induced caspase-3-dependent apoptosis (Fig. 6a-c). Therefore, silencing of XIAP mimics the effects of miR-24 upregulation on LSCC cells.Fig. 5

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