Limits...
miR-512-5p suppresses tumor growth by targeting hTERT in telomerase positive head and neck squamous cell carcinoma in vitro and in vivo.

Li J, Lei H, Xu Y, Tao ZZ - PLoS ONE (2015)

Bottom Line: Both in vitro and in vivo assays revealed that miR-512-5P mimic attenuated HNSCC cell proliferation, and tumor growth in nude mice, which exerts its tumor suppressor function through elevated apoptosis, inhibition of the telomerase activity, decrease of telomere-binding proteins and shortening of telomere length by human telomerase reverse transcriptase (hTERT) downregulation.We conclude that the frequently miR-512-5P overexpression can regulate hTERT and function as a tumor suppressor in HNSCC.Therefore, miR-512-5P may serve as a potential therapeutic agent for miR-based HNSCC therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology-Head and Neck Surgery, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China; Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, China.

ABSTRACT
Telomerase activation has very important implications for head and neck squamous cell carcinoma (HNSCC), but the regulatory mechanisms of telomerase in HNSCC remain unclear. In our present study, we found that miR-512-5P was markedly downregulated in telomerase-positive HNSCC cell lines. Both in vitro and in vivo assays revealed that miR-512-5P mimic attenuated HNSCC cell proliferation, and tumor growth in nude mice, which exerts its tumor suppressor function through elevated apoptosis, inhibition of the telomerase activity, decrease of telomere-binding proteins and shortening of telomere length by human telomerase reverse transcriptase (hTERT) downregulation. Furthermore, the dual-luciferase reporter gene assay results demonstrated that hTERT was a direct target of miR-512-5P. We conclude that the frequently miR-512-5P overexpression can regulate hTERT and function as a tumor suppressor in HNSCC. Therefore, miR-512-5P may serve as a potential therapeutic agent for miR-based HNSCC therapy.

No MeSH data available.


Related in: MedlinePlus

Validation of hTERT mRNA as a direct target of miR-512-5p in CNE cells.(A) Sequence of potential binding site of miR-512-5p in the 3’UTR of hTERT mRNA, mutations were introduced into the binding site for generation of mutated hTERT-3’UTR. (B) MiR-512-5p resulted in a significant decrease in luciferase expression in hTERT-3’UTR transfected cells compared with the scramble, while MiR-512-5p caused no statistical change in luciferase expression in mut-hTERT-3’UTR compared with the scramble(C) miR-512-5p mimic led to down-regulation of hTERT and TRF2 in CNE cells. All data are shown as mean±SD of triplicate experiments. *P<0.05.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4530866&req=5

pone.0135265.g003: Validation of hTERT mRNA as a direct target of miR-512-5p in CNE cells.(A) Sequence of potential binding site of miR-512-5p in the 3’UTR of hTERT mRNA, mutations were introduced into the binding site for generation of mutated hTERT-3’UTR. (B) MiR-512-5p resulted in a significant decrease in luciferase expression in hTERT-3’UTR transfected cells compared with the scramble, while MiR-512-5p caused no statistical change in luciferase expression in mut-hTERT-3’UTR compared with the scramble(C) miR-512-5p mimic led to down-regulation of hTERT and TRF2 in CNE cells. All data are shown as mean±SD of triplicate experiments. *P<0.05.

Mentions: We predicted potential direct targets of miR-512-5p by Targetscan Release 6.2., PicTar5 and miRanda 3.0 programs (S1 Table). The hTERT gene was predicted to have at least one potential binding site at their 3’-UTRs for miR-512-5p (Fig 3A). To validate whether miR-512-5p directly repress identified mRNA targets through 3’UTR interactions, we cloned a sequence with the predicted target sites of hTERT or a mutated sequence to downstream of the pMIR luciferase reporter gene. The above plasmids and miR-512-5p mimic or miR-512-5p-scramble were transiently transfected into 293T cells and a dual luciferase reporter assay system was used to detect luciferase expression 48 h after tranfection. The results indicated that overexpression of miR-512-5p resulted in a significant decrease in luciferase expression in hTERT-3’UTR transfected cells, compared with the scramble. However, transfection of miR-512-5p in mut-hTERT-3’UTR transfected cells did not display significant reduction of luciferase levels (Fig 3B). We further investigated the effect of miR-512-5p on the expression of hTERT and TRF2 by Western blot. Obviously, the result showed that miR-512-5p mimic led to down-regulation of hTERT and TRF2 in CNE cells (Fig 3C). These data demonstrated that miR-512-5p regulated hTERT expression at the post-transcriptional level and influenced the telomere-binding proteins.


miR-512-5p suppresses tumor growth by targeting hTERT in telomerase positive head and neck squamous cell carcinoma in vitro and in vivo.

Li J, Lei H, Xu Y, Tao ZZ - PLoS ONE (2015)

Validation of hTERT mRNA as a direct target of miR-512-5p in CNE cells.(A) Sequence of potential binding site of miR-512-5p in the 3’UTR of hTERT mRNA, mutations were introduced into the binding site for generation of mutated hTERT-3’UTR. (B) MiR-512-5p resulted in a significant decrease in luciferase expression in hTERT-3’UTR transfected cells compared with the scramble, while MiR-512-5p caused no statistical change in luciferase expression in mut-hTERT-3’UTR compared with the scramble(C) miR-512-5p mimic led to down-regulation of hTERT and TRF2 in CNE cells. All data are shown as mean±SD of triplicate experiments. *P<0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4530866&req=5

pone.0135265.g003: Validation of hTERT mRNA as a direct target of miR-512-5p in CNE cells.(A) Sequence of potential binding site of miR-512-5p in the 3’UTR of hTERT mRNA, mutations were introduced into the binding site for generation of mutated hTERT-3’UTR. (B) MiR-512-5p resulted in a significant decrease in luciferase expression in hTERT-3’UTR transfected cells compared with the scramble, while MiR-512-5p caused no statistical change in luciferase expression in mut-hTERT-3’UTR compared with the scramble(C) miR-512-5p mimic led to down-regulation of hTERT and TRF2 in CNE cells. All data are shown as mean±SD of triplicate experiments. *P<0.05.
Mentions: We predicted potential direct targets of miR-512-5p by Targetscan Release 6.2., PicTar5 and miRanda 3.0 programs (S1 Table). The hTERT gene was predicted to have at least one potential binding site at their 3’-UTRs for miR-512-5p (Fig 3A). To validate whether miR-512-5p directly repress identified mRNA targets through 3’UTR interactions, we cloned a sequence with the predicted target sites of hTERT or a mutated sequence to downstream of the pMIR luciferase reporter gene. The above plasmids and miR-512-5p mimic or miR-512-5p-scramble were transiently transfected into 293T cells and a dual luciferase reporter assay system was used to detect luciferase expression 48 h after tranfection. The results indicated that overexpression of miR-512-5p resulted in a significant decrease in luciferase expression in hTERT-3’UTR transfected cells, compared with the scramble. However, transfection of miR-512-5p in mut-hTERT-3’UTR transfected cells did not display significant reduction of luciferase levels (Fig 3B). We further investigated the effect of miR-512-5p on the expression of hTERT and TRF2 by Western blot. Obviously, the result showed that miR-512-5p mimic led to down-regulation of hTERT and TRF2 in CNE cells (Fig 3C). These data demonstrated that miR-512-5p regulated hTERT expression at the post-transcriptional level and influenced the telomere-binding proteins.

Bottom Line: Both in vitro and in vivo assays revealed that miR-512-5P mimic attenuated HNSCC cell proliferation, and tumor growth in nude mice, which exerts its tumor suppressor function through elevated apoptosis, inhibition of the telomerase activity, decrease of telomere-binding proteins and shortening of telomere length by human telomerase reverse transcriptase (hTERT) downregulation.We conclude that the frequently miR-512-5P overexpression can regulate hTERT and function as a tumor suppressor in HNSCC.Therefore, miR-512-5P may serve as a potential therapeutic agent for miR-based HNSCC therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology-Head and Neck Surgery, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China; Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, China.

ABSTRACT
Telomerase activation has very important implications for head and neck squamous cell carcinoma (HNSCC), but the regulatory mechanisms of telomerase in HNSCC remain unclear. In our present study, we found that miR-512-5P was markedly downregulated in telomerase-positive HNSCC cell lines. Both in vitro and in vivo assays revealed that miR-512-5P mimic attenuated HNSCC cell proliferation, and tumor growth in nude mice, which exerts its tumor suppressor function through elevated apoptosis, inhibition of the telomerase activity, decrease of telomere-binding proteins and shortening of telomere length by human telomerase reverse transcriptase (hTERT) downregulation. Furthermore, the dual-luciferase reporter gene assay results demonstrated that hTERT was a direct target of miR-512-5P. We conclude that the frequently miR-512-5P overexpression can regulate hTERT and function as a tumor suppressor in HNSCC. Therefore, miR-512-5P may serve as a potential therapeutic agent for miR-based HNSCC therapy.

No MeSH data available.


Related in: MedlinePlus