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MiR-218 inhibits invasion and metastasis of gastric cancer by targeting the Robo1 receptor.

Tie J, Pan Y, Zhao L, Wu K, Liu J, Sun S, Guo X, Wang B, Gang Y, Zhang Y, Li Q, Qiao T, Zhao Q, Nie Y, Fan D - PLoS Genet. (2010)

Bottom Line: Here, we describe the regulation and function of miR-218 in gastric cancer (GC) metastasis. miR-218 expression is decreased along with the expression of one of its host genes, Slit3 in metastatic GC.The restoration of miR-218 suppresses Robo1 expression and inhibits tumor cell invasion and metastasis in vitro and in vivo.Taken together, our results describe a Slit-miR-218-Robo1 regulatory circuit whose disruption may contribute to GC metastasis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, Shaanxi, China.

ABSTRACT
MicroRNAs play key roles in tumor metastasis. Here, we describe the regulation and function of miR-218 in gastric cancer (GC) metastasis. miR-218 expression is decreased along with the expression of one of its host genes, Slit3 in metastatic GC. However, Robo1, one of several Slit receptors, is negatively regulated by miR-218, thus establishing a negative feedback loop. Decreased miR-218 levels eliminate Robo1 repression, which activates the Slit-Robo1 pathway through the interaction between Robo1 and Slit2, thus triggering tumor metastasis. The restoration of miR-218 suppresses Robo1 expression and inhibits tumor cell invasion and metastasis in vitro and in vivo. Taken together, our results describe a Slit-miR-218-Robo1 regulatory circuit whose disruption may contribute to GC metastasis. Targeting miR-218 may provide a strategy for blocking tumor metastasis.

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miR-218 targeted Robo1 by binding to its 3′-UTR.(A) The Robo1 3′-UTR was a potential target of miR-218. (B and C) miR-218 and Robo1 levels were analyzed by qRT–PCR and western blot, respectively. Robo1 levels decreased when miR-218 was upregulated in response to the miR-218-expression vector in MKN28-M cells, whereas the reverse was observed for Robo1 expression when miR-218 was knocked down in MKN28-NM cells. (D) MKN28-M cells were co-transfected with miR-218 and a luciferase reporter (Luc-Robo1) containing a fragment of the Robo1 3′-UTR harboring either the miR-218 binding site or a mutant (Luc-Robo1-mu) in which the first six nucleotides of the miR-218 binding site were deleted. A luciferase reporter construct engineered with a non-related fragment of cDNA was used as a negative control (Luc-control). The assays showed that luciferase activity in the Luc-Robo1 group was significantly decreased compared to the luciferase activity of the mutant and negative control groups. (E) MKN28-M-miR-218 cells, which stably over-expressed miR-218, were transiently transfected with a Robo1 expression construct or a Robo1 mutant construct lacking the miR-218 binding site. MKN28-M cells were transfected with Robo1 siRNA or a negative control siRNA. Western blot analysis for Robo1 showed that co-transfection of miR-218 and the Robo1 mutant construct produced higher levels of Robo1 protein than co-transfection of miR-218 and the Robo1 construct. Robo1 siRNA effectively reduced the amount Robo1 protein observed. (F) The cell invasion assay indicated that Robo1 mutant constructs could reverse the effect of miR-218-mediated suppression of cell invasion. Knockdown of Robo1 by siRNA in MKN28-M cells inhibited cell invasion. * P<0.05.
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pgen-1000879-g005: miR-218 targeted Robo1 by binding to its 3′-UTR.(A) The Robo1 3′-UTR was a potential target of miR-218. (B and C) miR-218 and Robo1 levels were analyzed by qRT–PCR and western blot, respectively. Robo1 levels decreased when miR-218 was upregulated in response to the miR-218-expression vector in MKN28-M cells, whereas the reverse was observed for Robo1 expression when miR-218 was knocked down in MKN28-NM cells. (D) MKN28-M cells were co-transfected with miR-218 and a luciferase reporter (Luc-Robo1) containing a fragment of the Robo1 3′-UTR harboring either the miR-218 binding site or a mutant (Luc-Robo1-mu) in which the first six nucleotides of the miR-218 binding site were deleted. A luciferase reporter construct engineered with a non-related fragment of cDNA was used as a negative control (Luc-control). The assays showed that luciferase activity in the Luc-Robo1 group was significantly decreased compared to the luciferase activity of the mutant and negative control groups. (E) MKN28-M-miR-218 cells, which stably over-expressed miR-218, were transiently transfected with a Robo1 expression construct or a Robo1 mutant construct lacking the miR-218 binding site. MKN28-M cells were transfected with Robo1 siRNA or a negative control siRNA. Western blot analysis for Robo1 showed that co-transfection of miR-218 and the Robo1 mutant construct produced higher levels of Robo1 protein than co-transfection of miR-218 and the Robo1 construct. Robo1 siRNA effectively reduced the amount Robo1 protein observed. (F) The cell invasion assay indicated that Robo1 mutant constructs could reverse the effect of miR-218-mediated suppression of cell invasion. Knockdown of Robo1 by siRNA in MKN28-M cells inhibited cell invasion. * P<0.05.

Mentions: To assess how a low level of miR-218 expression contributes to the invasion and metastasis of GC, we searched for the potential regulatory targets of miR-218 using prediction tools, including miRanda, PicTar, and TargetScan. Although hundreds of different targets were predicted, those genes involved in migration or invasion may be the relevant targets with respect to the biological functions of miR-218. We then performed a functional classification of the predicted targets using the DAVID program (http://david.abcc.ncifcrf.gov/). Of these genes, Robo1 is regarded as a proto-oncogene and harbors migration-promoting activity [30]–[35]. Mertsch et al. demonstrated that Robo1 facilitates glioma cell migration mediated by Slit2 [36]. Schmid et al. found that breast tumor cell migration is induced by the Slit2-Robo1 interaction in vitro[37]. These findings suggest that Robo1 may be a target for miR-218. To further test our hypothesis, we analyzed the expression of miR-218 and Robo1 in GES and in non-invasive (MKN28-NM and SGC7901-NM) and invasive (MKN28-M and SGC7901-M) GC cells. The results showed a negative correlation between the levels of miR-218 and Robo1 mRNA in these cells (Figure S3A). Furthermore, we observed that Robo1 mRNA (Figure S3B) and protein (Figure 5B2) levels were decreased when miR-218 was expressed by pGenesil-1-miR-218 in MKN28-M cells (Figure 5B1). The reverse was observed for Robo1 expression when miR-218 was knocked down in MKN28-NM cells (Figure 5C1 and Figure 5C2). The inverse relationship between miR-218 and Robo1 expression was further confirmed by immunohistochemistry (Text S1) in 40 cases of gastric cancer, in matched adjacent normal tissues that were also used in clinicopathological studies, and in 29 matched metastases. The results show that Robo1 was upregulated in GC, especially in metastatic GC (Figure S4), in which miR-218 has a relatively low expression.


MiR-218 inhibits invasion and metastasis of gastric cancer by targeting the Robo1 receptor.

Tie J, Pan Y, Zhao L, Wu K, Liu J, Sun S, Guo X, Wang B, Gang Y, Zhang Y, Li Q, Qiao T, Zhao Q, Nie Y, Fan D - PLoS Genet. (2010)

miR-218 targeted Robo1 by binding to its 3′-UTR.(A) The Robo1 3′-UTR was a potential target of miR-218. (B and C) miR-218 and Robo1 levels were analyzed by qRT–PCR and western blot, respectively. Robo1 levels decreased when miR-218 was upregulated in response to the miR-218-expression vector in MKN28-M cells, whereas the reverse was observed for Robo1 expression when miR-218 was knocked down in MKN28-NM cells. (D) MKN28-M cells were co-transfected with miR-218 and a luciferase reporter (Luc-Robo1) containing a fragment of the Robo1 3′-UTR harboring either the miR-218 binding site or a mutant (Luc-Robo1-mu) in which the first six nucleotides of the miR-218 binding site were deleted. A luciferase reporter construct engineered with a non-related fragment of cDNA was used as a negative control (Luc-control). The assays showed that luciferase activity in the Luc-Robo1 group was significantly decreased compared to the luciferase activity of the mutant and negative control groups. (E) MKN28-M-miR-218 cells, which stably over-expressed miR-218, were transiently transfected with a Robo1 expression construct or a Robo1 mutant construct lacking the miR-218 binding site. MKN28-M cells were transfected with Robo1 siRNA or a negative control siRNA. Western blot analysis for Robo1 showed that co-transfection of miR-218 and the Robo1 mutant construct produced higher levels of Robo1 protein than co-transfection of miR-218 and the Robo1 construct. Robo1 siRNA effectively reduced the amount Robo1 protein observed. (F) The cell invasion assay indicated that Robo1 mutant constructs could reverse the effect of miR-218-mediated suppression of cell invasion. Knockdown of Robo1 by siRNA in MKN28-M cells inhibited cell invasion. * P<0.05.
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pgen-1000879-g005: miR-218 targeted Robo1 by binding to its 3′-UTR.(A) The Robo1 3′-UTR was a potential target of miR-218. (B and C) miR-218 and Robo1 levels were analyzed by qRT–PCR and western blot, respectively. Robo1 levels decreased when miR-218 was upregulated in response to the miR-218-expression vector in MKN28-M cells, whereas the reverse was observed for Robo1 expression when miR-218 was knocked down in MKN28-NM cells. (D) MKN28-M cells were co-transfected with miR-218 and a luciferase reporter (Luc-Robo1) containing a fragment of the Robo1 3′-UTR harboring either the miR-218 binding site or a mutant (Luc-Robo1-mu) in which the first six nucleotides of the miR-218 binding site were deleted. A luciferase reporter construct engineered with a non-related fragment of cDNA was used as a negative control (Luc-control). The assays showed that luciferase activity in the Luc-Robo1 group was significantly decreased compared to the luciferase activity of the mutant and negative control groups. (E) MKN28-M-miR-218 cells, which stably over-expressed miR-218, were transiently transfected with a Robo1 expression construct or a Robo1 mutant construct lacking the miR-218 binding site. MKN28-M cells were transfected with Robo1 siRNA or a negative control siRNA. Western blot analysis for Robo1 showed that co-transfection of miR-218 and the Robo1 mutant construct produced higher levels of Robo1 protein than co-transfection of miR-218 and the Robo1 construct. Robo1 siRNA effectively reduced the amount Robo1 protein observed. (F) The cell invasion assay indicated that Robo1 mutant constructs could reverse the effect of miR-218-mediated suppression of cell invasion. Knockdown of Robo1 by siRNA in MKN28-M cells inhibited cell invasion. * P<0.05.
Mentions: To assess how a low level of miR-218 expression contributes to the invasion and metastasis of GC, we searched for the potential regulatory targets of miR-218 using prediction tools, including miRanda, PicTar, and TargetScan. Although hundreds of different targets were predicted, those genes involved in migration or invasion may be the relevant targets with respect to the biological functions of miR-218. We then performed a functional classification of the predicted targets using the DAVID program (http://david.abcc.ncifcrf.gov/). Of these genes, Robo1 is regarded as a proto-oncogene and harbors migration-promoting activity [30]–[35]. Mertsch et al. demonstrated that Robo1 facilitates glioma cell migration mediated by Slit2 [36]. Schmid et al. found that breast tumor cell migration is induced by the Slit2-Robo1 interaction in vitro[37]. These findings suggest that Robo1 may be a target for miR-218. To further test our hypothesis, we analyzed the expression of miR-218 and Robo1 in GES and in non-invasive (MKN28-NM and SGC7901-NM) and invasive (MKN28-M and SGC7901-M) GC cells. The results showed a negative correlation between the levels of miR-218 and Robo1 mRNA in these cells (Figure S3A). Furthermore, we observed that Robo1 mRNA (Figure S3B) and protein (Figure 5B2) levels were decreased when miR-218 was expressed by pGenesil-1-miR-218 in MKN28-M cells (Figure 5B1). The reverse was observed for Robo1 expression when miR-218 was knocked down in MKN28-NM cells (Figure 5C1 and Figure 5C2). The inverse relationship between miR-218 and Robo1 expression was further confirmed by immunohistochemistry (Text S1) in 40 cases of gastric cancer, in matched adjacent normal tissues that were also used in clinicopathological studies, and in 29 matched metastases. The results show that Robo1 was upregulated in GC, especially in metastatic GC (Figure S4), in which miR-218 has a relatively low expression.

Bottom Line: Here, we describe the regulation and function of miR-218 in gastric cancer (GC) metastasis. miR-218 expression is decreased along with the expression of one of its host genes, Slit3 in metastatic GC.The restoration of miR-218 suppresses Robo1 expression and inhibits tumor cell invasion and metastasis in vitro and in vivo.Taken together, our results describe a Slit-miR-218-Robo1 regulatory circuit whose disruption may contribute to GC metastasis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, Shaanxi, China.

ABSTRACT
MicroRNAs play key roles in tumor metastasis. Here, we describe the regulation and function of miR-218 in gastric cancer (GC) metastasis. miR-218 expression is decreased along with the expression of one of its host genes, Slit3 in metastatic GC. However, Robo1, one of several Slit receptors, is negatively regulated by miR-218, thus establishing a negative feedback loop. Decreased miR-218 levels eliminate Robo1 repression, which activates the Slit-Robo1 pathway through the interaction between Robo1 and Slit2, thus triggering tumor metastasis. The restoration of miR-218 suppresses Robo1 expression and inhibits tumor cell invasion and metastasis in vitro and in vivo. Taken together, our results describe a Slit-miR-218-Robo1 regulatory circuit whose disruption may contribute to GC metastasis. Targeting miR-218 may provide a strategy for blocking tumor metastasis.

Show MeSH
Related in: MedlinePlus