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MKK7 mediates miR-493-dependent suppression of liver metastasis of colon cancer cells.

Sakai H, Sato A, Aihara Y, Ikarashi Y, Midorikawa Y, Kracht M, Nakagama H, Okamoto K - Cancer Sci. (2014)

Bottom Line: However, major functional targets that mediate the antimetastatic activity of miR-493 remain elusive.Here, we extended our search for target genes and identified MKK7, a mitogen-activated protein kinase kinase, as a novel target of miR-493. miR-493 inhibits MKK7 expression by targeting the binding site at the 3'-UTR of the mkk7 gene.Immunohistochemical examination in human primary colon tumors revealed that the occurrence of liver metastasis is associated with elevated levels of MKK7.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Development System, National Cancer Center Research Institute, Tokyo, Japan.

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MKK7 is a novel target of miR-493. (a) Two-cycle Venn diagram that shows the overlap of predicted target genes of miR-493 (40 genes) and genes inhibited by miR-493 expression (1533 genes). (b, c) Inhibition of MKK7 by miR-493. HCT116 cells (b) or DLD-1 cells (c) were transfected with the indicated miRNA mimics, and used for Western blot analyses with the indicated antibodies. Asterisk, a star form of miR-493; cont, control. (d) Sequence comparison of MKK7 3′-UTR (positions 2052–2074; mRNA RefSeq, NM_145185.2), mutated (mut) MKK7 3′-UTR, and human (hsa) miR-493. *Mutated nucleotides. (e) Transient luciferase assays. The indicated reporter plasmid was transfected into HCT116 cells together with control oligo (Cont) or miR-493 (493), and luciferase activity was measured 2 days after transfection.
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fig01: MKK7 is a novel target of miR-493. (a) Two-cycle Venn diagram that shows the overlap of predicted target genes of miR-493 (40 genes) and genes inhibited by miR-493 expression (1533 genes). (b, c) Inhibition of MKK7 by miR-493. HCT116 cells (b) or DLD-1 cells (c) were transfected with the indicated miRNA mimics, and used for Western blot analyses with the indicated antibodies. Asterisk, a star form of miR-493; cont, control. (d) Sequence comparison of MKK7 3′-UTR (positions 2052–2074; mRNA RefSeq, NM_145185.2), mutated (mut) MKK7 3′-UTR, and human (hsa) miR-493. *Mutated nucleotides. (e) Transient luciferase assays. The indicated reporter plasmid was transfected into HCT116 cells together with control oligo (Cont) or miR-493 (493), and luciferase activity was measured 2 days after transfection.

Mentions: Our previous study showed that miR-493 directly targets IGF1R, and the inhibition of IGF1R induces cell death of metastatic cells and suppresses liver metastasis of colon cancer cells.(11) Because the extent of the suppression by IGF1R inhibition is modest in comparison to that caused by miR-493, it is likely that an unknown target gene(s) plays important roles on the inhibition of metastasis by miR-493.(11) In order to look for crucial target genes of miR-493, we used a searching criterion that differs from the one used in our previous studies: statistical significance (P < 0.05), instead of twofold cut-off, was used to select candidate genes through expression analyses of miR-493-transfected HCT116 colon cancer cells (Fig. 1a). We combined the new criterion from the expression studies with the targets predicted from a combination of in silico programs (Targetscan, PITA, miRanda).(11) The examination of overlapped genes between the criterion and the predicted targets led to identification of 16 candidates as potential targets of miR-493 (Fig. 1a), whereas only eight genes were identified if the previous criterion with a twofold cut-off was applied.(11)


MKK7 mediates miR-493-dependent suppression of liver metastasis of colon cancer cells.

Sakai H, Sato A, Aihara Y, Ikarashi Y, Midorikawa Y, Kracht M, Nakagama H, Okamoto K - Cancer Sci. (2014)

MKK7 is a novel target of miR-493. (a) Two-cycle Venn diagram that shows the overlap of predicted target genes of miR-493 (40 genes) and genes inhibited by miR-493 expression (1533 genes). (b, c) Inhibition of MKK7 by miR-493. HCT116 cells (b) or DLD-1 cells (c) were transfected with the indicated miRNA mimics, and used for Western blot analyses with the indicated antibodies. Asterisk, a star form of miR-493; cont, control. (d) Sequence comparison of MKK7 3′-UTR (positions 2052–2074; mRNA RefSeq, NM_145185.2), mutated (mut) MKK7 3′-UTR, and human (hsa) miR-493. *Mutated nucleotides. (e) Transient luciferase assays. The indicated reporter plasmid was transfected into HCT116 cells together with control oligo (Cont) or miR-493 (493), and luciferase activity was measured 2 days after transfection.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4317799&req=5

fig01: MKK7 is a novel target of miR-493. (a) Two-cycle Venn diagram that shows the overlap of predicted target genes of miR-493 (40 genes) and genes inhibited by miR-493 expression (1533 genes). (b, c) Inhibition of MKK7 by miR-493. HCT116 cells (b) or DLD-1 cells (c) were transfected with the indicated miRNA mimics, and used for Western blot analyses with the indicated antibodies. Asterisk, a star form of miR-493; cont, control. (d) Sequence comparison of MKK7 3′-UTR (positions 2052–2074; mRNA RefSeq, NM_145185.2), mutated (mut) MKK7 3′-UTR, and human (hsa) miR-493. *Mutated nucleotides. (e) Transient luciferase assays. The indicated reporter plasmid was transfected into HCT116 cells together with control oligo (Cont) or miR-493 (493), and luciferase activity was measured 2 days after transfection.
Mentions: Our previous study showed that miR-493 directly targets IGF1R, and the inhibition of IGF1R induces cell death of metastatic cells and suppresses liver metastasis of colon cancer cells.(11) Because the extent of the suppression by IGF1R inhibition is modest in comparison to that caused by miR-493, it is likely that an unknown target gene(s) plays important roles on the inhibition of metastasis by miR-493.(11) In order to look for crucial target genes of miR-493, we used a searching criterion that differs from the one used in our previous studies: statistical significance (P < 0.05), instead of twofold cut-off, was used to select candidate genes through expression analyses of miR-493-transfected HCT116 colon cancer cells (Fig. 1a). We combined the new criterion from the expression studies with the targets predicted from a combination of in silico programs (Targetscan, PITA, miRanda).(11) The examination of overlapped genes between the criterion and the predicted targets led to identification of 16 candidates as potential targets of miR-493 (Fig. 1a), whereas only eight genes were identified if the previous criterion with a twofold cut-off was applied.(11)

Bottom Line: However, major functional targets that mediate the antimetastatic activity of miR-493 remain elusive.Here, we extended our search for target genes and identified MKK7, a mitogen-activated protein kinase kinase, as a novel target of miR-493. miR-493 inhibits MKK7 expression by targeting the binding site at the 3'-UTR of the mkk7 gene.Immunohistochemical examination in human primary colon tumors revealed that the occurrence of liver metastasis is associated with elevated levels of MKK7.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Development System, National Cancer Center Research Institute, Tokyo, Japan.

Show MeSH
Related in: MedlinePlus