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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

miR-24 binds to the 3′-UTR of XIAP mRNA. a A human XIAP/3′-UTR fragment containing wild-type or mutant miR-24-binding sequence was cloned downstream of the luciferase reporter gene in pLUC-luc. b pLUC-luc vector contains XIAP/3′-UTR-wt or XIAP/3′-UTR-mut and pGCMV/miR-24 or pGCMV/miR-NC were co-transfected into Hep-2 cells, and cell lysates prepared at 48 h for measuring luciferase activity, which was normalized to Renilla luciferase activity. c Western blot detection of XIAP protein expression 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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Fig4: miR-24 binds to the 3′-UTR of XIAP mRNA. a A human XIAP/3′-UTR fragment containing wild-type or mutant miR-24-binding sequence was cloned downstream of the luciferase reporter gene in pLUC-luc. b pLUC-luc vector contains XIAP/3′-UTR-wt or XIAP/3′-UTR-mut and pGCMV/miR-24 or pGCMV/miR-NC were co-transfected into Hep-2 cells, and cell lysates prepared at 48 h for measuring luciferase activity, which was normalized to Renilla luciferase activity. c Western blot detection of XIAP protein expression 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 explore how miR-24 affects malignant development of LSCC, we searched for potential regulatory targets of miR-24 using three prediction tools (miRanda, PicTar, and TargetScan), and identified a putative miR-24-binding site at positions 2301–2308 (CUGAGCCA) in the 3′-UTR of XIAP mRNA. To test directly whether XIAP is a target of miR-24, we constructed a luciferase reporter (pLUC/XIAP/3′-UTR-wt) in which the XIAP/3′-UTR nucleotides complementary to miR-24 (nt 2301–2308) were inserted into the pLUC vector. We also generated a mutant reporter (pLUC/XIAP/3′-UTR-mut), in which the first six nucleotides in the miR-24 seed region complementary sites were mutated (Fig. 4a). We then co-transfected appropriate plasmids with either the negative control miR-NC or miR-24 mimics into Hep-2 cells, and measured luciferase activity. Luciferase activity indicated that miR-24 inhibited signal compared with the miR-NC negative control (Fig. 4b), but had no effect on the activity of reporter vector containing the 3′-UTR of XIAP with six point mutations in the miR-24-binding site, suggesting that miR-24 interacts directly with the 3′-UTR of XIAP mRNA. Western blot results suggest that XIAP is controlled by miR-24 in Hep-2/miR-24 (or Hep-2/miR-NC) and AMC-HN-8/miR-24 (AMC-HN-8/miR-NC) cells, as miR-24 decreased expression of XIAP protein in LSCC cells (Fig. 4c). Collectively, these results indicate that XIAP may be a direct target of miR-24 in LSCC.Fig. 4


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)

miR-24 binds to the 3′-UTR of XIAP mRNA. a A human XIAP/3′-UTR fragment containing wild-type or mutant miR-24-binding sequence was cloned downstream of the luciferase reporter gene in pLUC-luc. b pLUC-luc vector contains XIAP/3′-UTR-wt or XIAP/3′-UTR-mut and pGCMV/miR-24 or pGCMV/miR-NC were co-transfected into Hep-2 cells, and cell lysates prepared at 48 h for measuring luciferase activity, which was normalized to Renilla luciferase activity. c Western blot detection of XIAP protein expression 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

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getmorefigures.php?uid=PMC4477309&req=5

Fig4: miR-24 binds to the 3′-UTR of XIAP mRNA. a A human XIAP/3′-UTR fragment containing wild-type or mutant miR-24-binding sequence was cloned downstream of the luciferase reporter gene in pLUC-luc. b pLUC-luc vector contains XIAP/3′-UTR-wt or XIAP/3′-UTR-mut and pGCMV/miR-24 or pGCMV/miR-NC were co-transfected into Hep-2 cells, and cell lysates prepared at 48 h for measuring luciferase activity, which was normalized to Renilla luciferase activity. c Western blot detection of XIAP protein expression 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 explore how miR-24 affects malignant development of LSCC, we searched for potential regulatory targets of miR-24 using three prediction tools (miRanda, PicTar, and TargetScan), and identified a putative miR-24-binding site at positions 2301–2308 (CUGAGCCA) in the 3′-UTR of XIAP mRNA. To test directly whether XIAP is a target of miR-24, we constructed a luciferase reporter (pLUC/XIAP/3′-UTR-wt) in which the XIAP/3′-UTR nucleotides complementary to miR-24 (nt 2301–2308) were inserted into the pLUC vector. We also generated a mutant reporter (pLUC/XIAP/3′-UTR-mut), in which the first six nucleotides in the miR-24 seed region complementary sites were mutated (Fig. 4a). We then co-transfected appropriate plasmids with either the negative control miR-NC or miR-24 mimics into Hep-2 cells, and measured luciferase activity. Luciferase activity indicated that miR-24 inhibited signal compared with the miR-NC negative control (Fig. 4b), but had no effect on the activity of reporter vector containing the 3′-UTR of XIAP with six point mutations in the miR-24-binding site, suggesting that miR-24 interacts directly with the 3′-UTR of XIAP mRNA. Western blot results suggest that XIAP is controlled by miR-24 in Hep-2/miR-24 (or Hep-2/miR-NC) and AMC-HN-8/miR-24 (AMC-HN-8/miR-NC) cells, as miR-24 decreased expression of XIAP protein in LSCC cells (Fig. 4c). Collectively, these results indicate that XIAP may be a direct target of miR-24 in LSCC.Fig. 4

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