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Functional elucidation of miR-494 in the tumorigenesis of nasopharyngeal carcinoma.

Duan HF, Li XQ, Hu HY, Li YC, Cai Z, Mei XS, Yu P, Nie LP, Zhang W, Yu ZD, Nie GH - Tumour Biol. (2015)

Bottom Line: Nasopharyngeal carcinoma has very high incidence and high mortality worldwide.In the present study, we verify that the expression of miR-494 in NPC tissues and NPC-derived cells was down-regulated, respectively.The proliferation, colony formation, migration, and invasion of NPC-derived cells were suppressed, while the cell apoptosis was promoted, when miR-494 was over-expressed in these cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngological, Peking University Shenzhen Hospital, 518036, Shenzhen, Guangdong Province, China.

ABSTRACT
Nasopharyngeal carcinoma has very high incidence and high mortality worldwide. MiRNA is related to the tumorigenesis and metastasis of a variety of tumors. In the present study, we verify that the expression of miR-494 in NPC tissues and NPC-derived cells was down-regulated, respectively. The proliferation, colony formation, migration, and invasion of NPC-derived cells were suppressed, while the cell apoptosis was promoted, when miR-494 was over-expressed in these cells. GALNT7 and CDK16 were confirmed to be the direct targets of miR-494. These results suggested that miR-494 play an inhibitory role in the tumorigenesis of NPC.

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Related in: MedlinePlus

GALNT7 and CDK16 are two target genes of miR-494. a, c Four fragments of GALNT7 and CDK16 3′-UTR were constructed, which contained the wild-type (WT) potential binding sites of miR-494 and the mutated sequence (MU). b, d The psiCHECKTM-2 luciferase constructs containing WT or MU sequence were transfected together with miR-494 mimic, negative control, inhibitor, or inhibitor negative control into 6-10B, 9-4E, and CNE2 cells. Luciferase activity was measured through the dual luciferase assay system. Data are presented as mean ± SD (*p < 0.05, **p < 0.001)
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Fig6: GALNT7 and CDK16 are two target genes of miR-494. a, c Four fragments of GALNT7 and CDK16 3′-UTR were constructed, which contained the wild-type (WT) potential binding sites of miR-494 and the mutated sequence (MU). b, d The psiCHECKTM-2 luciferase constructs containing WT or MU sequence were transfected together with miR-494 mimic, negative control, inhibitor, or inhibitor negative control into 6-10B, 9-4E, and CNE2 cells. Luciferase activity was measured through the dual luciferase assay system. Data are presented as mean ± SD (*p < 0.05, **p < 0.001)

Mentions: The miRNAs target sequences were inserted between the XhoI–NotI restriction sites in the 3′-UTR of the hRluc gene of the psiCHECKTM-2 luciferase vector (Promega, Madison, WI, USA) to generate reporter constructs. The primer sequences for the 3′-UTR of GALNT7 mRNA (forward primer 5′-CCG CTC GAG ATT GTC CAC TGA CAT TTG GGA TTT A-3′ and reverse primer 5′-AAG GAA AAA AGC GGC CGC CAA ACT TCC TCT GGC AGT AGT TTG T-3′) and CDK16 mRNA (forward primer 5′-CCG CTC GAG TCA TAC CAG CCC CCA GGA CCA CTA C-3′ and reverse primer 5′-AAG GAA AAA AGC GGC CGC GTT CCA AAT AGG GGC TGT GTC CCT G-3′) were designed. They contain the potential binding sites to verify the binding sites of the miR-494. Meanwhile, G and T, and A and C were substituted to mutate the potential binding sites (Fig. 6a, c). All these four short fragments were cloned into psiCHECKTM-2 luciferase vectors, respectively, and all the constructs were verified by sequencing. They were transfected together with miR-494 mimic, negative control, inhibitor, or inhibitor negative control into 6-10B, 9-4E, or CNE2 cells in three replicate wells. After 48 h, the dual luciferase assay system (Promega, Madison, WI, USA) was used to detect the luciferase activity according to the manufacturer’s instructions. Normalized data were analyzed by the quotient of Renilla/firefly luciferase activities. The experiments were repeated at least three times.


Functional elucidation of miR-494 in the tumorigenesis of nasopharyngeal carcinoma.

Duan HF, Li XQ, Hu HY, Li YC, Cai Z, Mei XS, Yu P, Nie LP, Zhang W, Yu ZD, Nie GH - Tumour Biol. (2015)

GALNT7 and CDK16 are two target genes of miR-494. a, c Four fragments of GALNT7 and CDK16 3′-UTR were constructed, which contained the wild-type (WT) potential binding sites of miR-494 and the mutated sequence (MU). b, d The psiCHECKTM-2 luciferase constructs containing WT or MU sequence were transfected together with miR-494 mimic, negative control, inhibitor, or inhibitor negative control into 6-10B, 9-4E, and CNE2 cells. Luciferase activity was measured through the dual luciferase assay system. Data are presented as mean ± SD (*p < 0.05, **p < 0.001)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4644213&req=5

Fig6: GALNT7 and CDK16 are two target genes of miR-494. a, c Four fragments of GALNT7 and CDK16 3′-UTR were constructed, which contained the wild-type (WT) potential binding sites of miR-494 and the mutated sequence (MU). b, d The psiCHECKTM-2 luciferase constructs containing WT or MU sequence were transfected together with miR-494 mimic, negative control, inhibitor, or inhibitor negative control into 6-10B, 9-4E, and CNE2 cells. Luciferase activity was measured through the dual luciferase assay system. Data are presented as mean ± SD (*p < 0.05, **p < 0.001)
Mentions: The miRNAs target sequences were inserted between the XhoI–NotI restriction sites in the 3′-UTR of the hRluc gene of the psiCHECKTM-2 luciferase vector (Promega, Madison, WI, USA) to generate reporter constructs. The primer sequences for the 3′-UTR of GALNT7 mRNA (forward primer 5′-CCG CTC GAG ATT GTC CAC TGA CAT TTG GGA TTT A-3′ and reverse primer 5′-AAG GAA AAA AGC GGC CGC CAA ACT TCC TCT GGC AGT AGT TTG T-3′) and CDK16 mRNA (forward primer 5′-CCG CTC GAG TCA TAC CAG CCC CCA GGA CCA CTA C-3′ and reverse primer 5′-AAG GAA AAA AGC GGC CGC GTT CCA AAT AGG GGC TGT GTC CCT G-3′) were designed. They contain the potential binding sites to verify the binding sites of the miR-494. Meanwhile, G and T, and A and C were substituted to mutate the potential binding sites (Fig. 6a, c). All these four short fragments were cloned into psiCHECKTM-2 luciferase vectors, respectively, and all the constructs were verified by sequencing. They were transfected together with miR-494 mimic, negative control, inhibitor, or inhibitor negative control into 6-10B, 9-4E, or CNE2 cells in three replicate wells. After 48 h, the dual luciferase assay system (Promega, Madison, WI, USA) was used to detect the luciferase activity according to the manufacturer’s instructions. Normalized data were analyzed by the quotient of Renilla/firefly luciferase activities. The experiments were repeated at least three times.

Bottom Line: Nasopharyngeal carcinoma has very high incidence and high mortality worldwide.In the present study, we verify that the expression of miR-494 in NPC tissues and NPC-derived cells was down-regulated, respectively.The proliferation, colony formation, migration, and invasion of NPC-derived cells were suppressed, while the cell apoptosis was promoted, when miR-494 was over-expressed in these cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngological, Peking University Shenzhen Hospital, 518036, Shenzhen, Guangdong Province, China.

ABSTRACT
Nasopharyngeal carcinoma has very high incidence and high mortality worldwide. MiRNA is related to the tumorigenesis and metastasis of a variety of tumors. In the present study, we verify that the expression of miR-494 in NPC tissues and NPC-derived cells was down-regulated, respectively. The proliferation, colony formation, migration, and invasion of NPC-derived cells were suppressed, while the cell apoptosis was promoted, when miR-494 was over-expressed in these cells. GALNT7 and CDK16 were confirmed to be the direct targets of miR-494. These results suggested that miR-494 play an inhibitory role in the tumorigenesis of NPC.

Show MeSH
Related in: MedlinePlus