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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 inhibition blocks liver settlement of metastasized cells. (a) Upper panel, Western blot analyses of siRNA-transfected cells: #1, a set of siRNAs from Invitrogen; #2, a set of siRNAs from Qiagen. Bottom panel: Two or 4 days after the splenic injection of a mixture of HCT116/GFP and HCT116/RFP cells, GFP/RFP dual fluorescence images for metastasized liver were taken. The inhibitory effect was evaluated by counting the number of GFP/RFP fluorescent foci. (b) Left panel, Western blot analyses of control or miR-493-transfected HCT116/GFP cells after the lentiviral introduction of myc-tagged MKK7-β1. The control or MKK7-introduced cells were transfected with the control oligo or miR-493, and used for Western blot analyses. *A major form of endogenous MKK7, which presumably represents the γ form of MKK7 based on its molecular weight. Right panel, a partial rescue of miR-493-mediated inhibition of liver metastasis by expression of exogenous MKK7. The transfected cells were mixed with HCT116/RFP, and used to generate liver metastasis. The ratio of GFP/RFP-positive foci was calculated at day 4.
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fig04: MKK7 inhibition blocks liver settlement of metastasized cells. (a) Upper panel, Western blot analyses of siRNA-transfected cells: #1, a set of siRNAs from Invitrogen; #2, a set of siRNAs from Qiagen. Bottom panel: Two or 4 days after the splenic injection of a mixture of HCT116/GFP and HCT116/RFP cells, GFP/RFP dual fluorescence images for metastasized liver were taken. The inhibitory effect was evaluated by counting the number of GFP/RFP fluorescent foci. (b) Left panel, Western blot analyses of control or miR-493-transfected HCT116/GFP cells after the lentiviral introduction of myc-tagged MKK7-β1. The control or MKK7-introduced cells were transfected with the control oligo or miR-493, and used for Western blot analyses. *A major form of endogenous MKK7, which presumably represents the γ form of MKK7 based on its molecular weight. Right panel, a partial rescue of miR-493-mediated inhibition of liver metastasis by expression of exogenous MKK7. The transfected cells were mixed with HCT116/RFP, and used to generate liver metastasis. The ratio of GFP/RFP-positive foci was calculated at day 4.

Mentions: It was shown that miR-493 expression in HCT116 cells blocks settlement of the metastasized cells in the liver, at least in part, by inducing cell death at liver mesenchyme.(11) Hence, we examined whether the inhibition of liver metastasis is observed during the early phase of mesenchymal settlement of metastatic cells by MKK7 inhibition, as well as by miR-493 introduction. The GFP-expressing HCT116 cells were transfected with either the control or MKK7 siRNAs (Fig. 4a, upper panel), mixed with the RFP-positive cells and then used to generate metastatic foci in the liver, as we did in experiments shown in Figure 3. Two or 4 days after the splenic injection, the presence of the metastatic cells in liver mesenchyme was visualized by imaging (Fig. S2, right panel), and the GFP- and RFP-positive cells were counted to calculate the extent of the inhibition of metastasis. In accordance with the time course by miR-493 expression,(11) inhibition of MKK7 suppressed the settlement of metastatic foci by day 4 (Fig. 4a, lower panel). Thus, the inhibition of MKK7 blocks formation of metastatic foci during the early phase of the mesenchymal settlement.


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 inhibition blocks liver settlement of metastasized cells. (a) Upper panel, Western blot analyses of siRNA-transfected cells: #1, a set of siRNAs from Invitrogen; #2, a set of siRNAs from Qiagen. Bottom panel: Two or 4 days after the splenic injection of a mixture of HCT116/GFP and HCT116/RFP cells, GFP/RFP dual fluorescence images for metastasized liver were taken. The inhibitory effect was evaluated by counting the number of GFP/RFP fluorescent foci. (b) Left panel, Western blot analyses of control or miR-493-transfected HCT116/GFP cells after the lentiviral introduction of myc-tagged MKK7-β1. The control or MKK7-introduced cells were transfected with the control oligo or miR-493, and used for Western blot analyses. *A major form of endogenous MKK7, which presumably represents the γ form of MKK7 based on its molecular weight. Right panel, a partial rescue of miR-493-mediated inhibition of liver metastasis by expression of exogenous MKK7. The transfected cells were mixed with HCT116/RFP, and used to generate liver metastasis. The ratio of GFP/RFP-positive foci was calculated at day 4.
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fig04: MKK7 inhibition blocks liver settlement of metastasized cells. (a) Upper panel, Western blot analyses of siRNA-transfected cells: #1, a set of siRNAs from Invitrogen; #2, a set of siRNAs from Qiagen. Bottom panel: Two or 4 days after the splenic injection of a mixture of HCT116/GFP and HCT116/RFP cells, GFP/RFP dual fluorescence images for metastasized liver were taken. The inhibitory effect was evaluated by counting the number of GFP/RFP fluorescent foci. (b) Left panel, Western blot analyses of control or miR-493-transfected HCT116/GFP cells after the lentiviral introduction of myc-tagged MKK7-β1. The control or MKK7-introduced cells were transfected with the control oligo or miR-493, and used for Western blot analyses. *A major form of endogenous MKK7, which presumably represents the γ form of MKK7 based on its molecular weight. Right panel, a partial rescue of miR-493-mediated inhibition of liver metastasis by expression of exogenous MKK7. The transfected cells were mixed with HCT116/RFP, and used to generate liver metastasis. The ratio of GFP/RFP-positive foci was calculated at day 4.
Mentions: It was shown that miR-493 expression in HCT116 cells blocks settlement of the metastasized cells in the liver, at least in part, by inducing cell death at liver mesenchyme.(11) Hence, we examined whether the inhibition of liver metastasis is observed during the early phase of mesenchymal settlement of metastatic cells by MKK7 inhibition, as well as by miR-493 introduction. The GFP-expressing HCT116 cells were transfected with either the control or MKK7 siRNAs (Fig. 4a, upper panel), mixed with the RFP-positive cells and then used to generate metastatic foci in the liver, as we did in experiments shown in Figure 3. Two or 4 days after the splenic injection, the presence of the metastatic cells in liver mesenchyme was visualized by imaging (Fig. S2, right panel), and the GFP- and RFP-positive cells were counted to calculate the extent of the inhibition of metastasis. In accordance with the time course by miR-493 expression,(11) inhibition of MKK7 suppressed the settlement of metastatic foci by day 4 (Fig. 4a, lower panel). Thus, the inhibition of MKK7 blocks formation of metastatic foci during the early phase of the mesenchymal settlement.

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