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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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Inhibition of MKK7 suppresses liver metastasis of colon cancer cells. (a, c) Western blot analyses of siRNA-transfected cells. HCT116/GFP cells (a) or DLD-1/GFP cells (c) were transfected with control or MKK7 siRNA. #1 and #2 designate a set of siRNAs from Invitrogen and Qiagen, respectively. The transfected cells were used for Western blot analyses with anti-MKK7 or anti-actin antibody. (b, d) Inhibition of liver metastasis by MKK7 siRNA. HCT116/GFP cells or DLD-1/GFP cells were transfected with the indicated siRNA, and the mixtures of the transfected HCT116/GFP cells and HCT116/red fluorescent protein (RFP) cells were injected into the spleen. (b) Ten days after the splenic injection of the mixture of the cells, metastasized cells were isolated from the liver and the number of GFP-positive and RFP-positive cells were counted by flow cytometry. The inhibitory effect of each siRNA was evaluated by calculating the GFP/RFP ratio. (d) Ten days after the splenic injection of a mixture of DLD-1/GFP and DLD-1/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.
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fig03: Inhibition of MKK7 suppresses liver metastasis of colon cancer cells. (a, c) Western blot analyses of siRNA-transfected cells. HCT116/GFP cells (a) or DLD-1/GFP cells (c) were transfected with control or MKK7 siRNA. #1 and #2 designate a set of siRNAs from Invitrogen and Qiagen, respectively. The transfected cells were used for Western blot analyses with anti-MKK7 or anti-actin antibody. (b, d) Inhibition of liver metastasis by MKK7 siRNA. HCT116/GFP cells or DLD-1/GFP cells were transfected with the indicated siRNA, and the mixtures of the transfected HCT116/GFP cells and HCT116/red fluorescent protein (RFP) cells were injected into the spleen. (b) Ten days after the splenic injection of the mixture of the cells, metastasized cells were isolated from the liver and the number of GFP-positive and RFP-positive cells were counted by flow cytometry. The inhibitory effect of each siRNA was evaluated by calculating the GFP/RFP ratio. (d) Ten days after the splenic injection of a mixture of DLD-1/GFP and DLD-1/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.

Mentions: Identification of MKK7 as a novel target gene of miR-493 prompted us to examine whether MKK7 is involved in liver metastasis of colon cancer cells. We inhibited MKK7 expression in GFP-expressing HCT116 cells(11) by siRNA-mediated knockdown (Fig. 3a), and after mixing them with an equal number of the RFP-expressing HCT116 cells, injected the mixture of these cells into the spleen of immunocompromized NOG mice. Ten days after the splenic injection, formation of metastatic foci of both GFP-expressing and RFP-expressing cells was confirmed by dual fluorescence imaging (Fig. S2, left panel). Subsequently, the transfected HCT116/GFP and HCT116/RFP cells were recovered from the metastasized liver, and the ratio of the GFP cells versus the RFP-positive internal control cells was calculated by flow cytometry to determine the inhibitory effect of each oligonucleotide on liver metastasis.(11) Two different siRNAs that correspond to MKK7 were used (Fig. 3a), and in both cases the knockdown of MKK7 caused significant reduction (˜60%) of liver metastasis in comparison to the control siRNAs (Fig. 3b).


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)

Inhibition of MKK7 suppresses liver metastasis of colon cancer cells. (a, c) Western blot analyses of siRNA-transfected cells. HCT116/GFP cells (a) or DLD-1/GFP cells (c) were transfected with control or MKK7 siRNA. #1 and #2 designate a set of siRNAs from Invitrogen and Qiagen, respectively. The transfected cells were used for Western blot analyses with anti-MKK7 or anti-actin antibody. (b, d) Inhibition of liver metastasis by MKK7 siRNA. HCT116/GFP cells or DLD-1/GFP cells were transfected with the indicated siRNA, and the mixtures of the transfected HCT116/GFP cells and HCT116/red fluorescent protein (RFP) cells were injected into the spleen. (b) Ten days after the splenic injection of the mixture of the cells, metastasized cells were isolated from the liver and the number of GFP-positive and RFP-positive cells were counted by flow cytometry. The inhibitory effect of each siRNA was evaluated by calculating the GFP/RFP ratio. (d) Ten days after the splenic injection of a mixture of DLD-1/GFP and DLD-1/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.
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fig03: Inhibition of MKK7 suppresses liver metastasis of colon cancer cells. (a, c) Western blot analyses of siRNA-transfected cells. HCT116/GFP cells (a) or DLD-1/GFP cells (c) were transfected with control or MKK7 siRNA. #1 and #2 designate a set of siRNAs from Invitrogen and Qiagen, respectively. The transfected cells were used for Western blot analyses with anti-MKK7 or anti-actin antibody. (b, d) Inhibition of liver metastasis by MKK7 siRNA. HCT116/GFP cells or DLD-1/GFP cells were transfected with the indicated siRNA, and the mixtures of the transfected HCT116/GFP cells and HCT116/red fluorescent protein (RFP) cells were injected into the spleen. (b) Ten days after the splenic injection of the mixture of the cells, metastasized cells were isolated from the liver and the number of GFP-positive and RFP-positive cells were counted by flow cytometry. The inhibitory effect of each siRNA was evaluated by calculating the GFP/RFP ratio. (d) Ten days after the splenic injection of a mixture of DLD-1/GFP and DLD-1/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.
Mentions: Identification of MKK7 as a novel target gene of miR-493 prompted us to examine whether MKK7 is involved in liver metastasis of colon cancer cells. We inhibited MKK7 expression in GFP-expressing HCT116 cells(11) by siRNA-mediated knockdown (Fig. 3a), and after mixing them with an equal number of the RFP-expressing HCT116 cells, injected the mixture of these cells into the spleen of immunocompromized NOG mice. Ten days after the splenic injection, formation of metastatic foci of both GFP-expressing and RFP-expressing cells was confirmed by dual fluorescence imaging (Fig. S2, left panel). Subsequently, the transfected HCT116/GFP and HCT116/RFP cells were recovered from the metastasized liver, and the ratio of the GFP cells versus the RFP-positive internal control cells was calculated by flow cytometry to determine the inhibitory effect of each oligonucleotide on liver metastasis.(11) Two different siRNAs that correspond to MKK7 were used (Fig. 3a), and in both cases the knockdown of MKK7 caused significant reduction (˜60%) of liver metastasis in comparison to the control siRNAs (Fig. 3b).

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