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miR-26a suppresses tumor growth and metastasis by targeting FGF9 in gastric cancer.

Deng M, Tang HL, Lu XH, Liu MY, Lu XM, Gu YX, Liu JF, He ZM - PLoS ONE (2013)

Bottom Line: In this study, we found that miR-26a was strongly downregulated in gastric cancer (GC) tissues and cell lines, and its expression levels were associated with lymph node metastasis and clinical stage, as well as overall survival and replase-free survival of GC.We also found that ectopic expression of miR-26a inhibited GC cell proliferation and GC metastasis in vitro and in vivo.In addition, miR-26a expression inversely correlated with FGF9 protein levels in GC.

View Article: PubMed Central - PubMed

Affiliation: Cancer Hospital and Cancer Research Institute, Guangzhou Medical University, Guangzhou, China ; Cancer Research Institute, University of South China, Hengyang, China.

ABSTRACT
The role of miR-26a in cancer cells seemed controversial in previous studies. Until now, the role of miR-26a in gastric cancer remains undefined. In this study, we found that miR-26a was strongly downregulated in gastric cancer (GC) tissues and cell lines, and its expression levels were associated with lymph node metastasis and clinical stage, as well as overall survival and replase-free survival of GC. We also found that ectopic expression of miR-26a inhibited GC cell proliferation and GC metastasis in vitro and in vivo. We further identified a novel mechanism of miR-26a to suppress GC growth and metastasis. FGF9 was proved to be a direct target of miR-26a, using luciferase assay and western blot. FGF9 overexpression in miR-26a-expressing cells could rescue invasion and growth defects of miR-26a. In addition, miR-26a expression inversely correlated with FGF9 protein levels in GC. Taken together, our data suggest that miR-26a functions as a tumor suppressor in GC development and progression, and holds promise as a prognostic biomarker and potential therapeutic target for GC.

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miR-26a directly targets FGF9.(A) The 3′-UTR element of FGF9 messenger RNA is partially complementary to miR-26a. miR-26a or scramble control and luciferase reporter containing either a wildtype or a mutant 3′-UTR were co-transfected into HEK-293T cells. And a Renilla luciferase expressing construct exerts as internal control. (B) Western blot analysis of FGF9 expression in SGC-7901 and AGS cells infected with miR-26a, and GES-1 transfected with miR-26a inhibitors (Anti-miR-26a). (C, D, E) FGF9 abrogates the suppressive roles of miR-26a in GC cell invasion and growth. SGC-7901 cells stably expressing miR-26a or scramble were transfect with or without FGF9 plasmids. Invasion assays(C), Apoptosis analysis (D), and Cell proliferation analysis (E) were performed with the above cells as described in Materials and Methods. Data are presented as mean±s.e.m from at least three independent experiments. (F) Spearman’s correlation scatter plot of the levels of miR-26a (determined by in situ hybridization) and FGF9 protein (determined by immunohistochemistry) in 126 GC specimens. Representative images of FGF9 expression by immunohistochemistry are shown (right panel). Original magnification: ×200.
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pone-0072662-g004: miR-26a directly targets FGF9.(A) The 3′-UTR element of FGF9 messenger RNA is partially complementary to miR-26a. miR-26a or scramble control and luciferase reporter containing either a wildtype or a mutant 3′-UTR were co-transfected into HEK-293T cells. And a Renilla luciferase expressing construct exerts as internal control. (B) Western blot analysis of FGF9 expression in SGC-7901 and AGS cells infected with miR-26a, and GES-1 transfected with miR-26a inhibitors (Anti-miR-26a). (C, D, E) FGF9 abrogates the suppressive roles of miR-26a in GC cell invasion and growth. SGC-7901 cells stably expressing miR-26a or scramble were transfect with or without FGF9 plasmids. Invasion assays(C), Apoptosis analysis (D), and Cell proliferation analysis (E) were performed with the above cells as described in Materials and Methods. Data are presented as mean±s.e.m from at least three independent experiments. (F) Spearman’s correlation scatter plot of the levels of miR-26a (determined by in situ hybridization) and FGF9 protein (determined by immunohistochemistry) in 126 GC specimens. Representative images of FGF9 expression by immunohistochemistry are shown (right panel). Original magnification: ×200.

Mentions: To understand how miR-26a suppresses GC growth and metastasis, we used three algorithms (Targetscan, Pictar and Miranda) to help identify miR-26a targets in human gastric cancers. Of these target genes that were predicted by all three algorithms (Table S1), FGF9 attracted our attention immediately as it has been implicated in tumorigenesis or metastasis [26]–[28]. We cloned the full-length FGF9 3′-UTR into a luciferase reporter vector. Luciferase assay revealed that miR-26a directly bound to FGF9 3′-UTR, and by which it remarkably reduced luciferase activities (Figure 4A). However, mutation of the putative miR-26a sites in the 3′-UTR of FGF9 abrogated luciferase responsiveness to miR-26a (Figure 4A). To directly assess the effect of miR-26a on FGF9 expression, we performed western blot analysis. As seen in Figure 4B, lentiviral induced ectopic miR-26a dramatically suppressed the FGF9 protein levels in SCG-7901 and AGS cells. Furthermore, knockdown of miR-26a, through transfection of anti-miR-26a, in GES-1 cells increased FGF9 protein levels (Figure 4B; Figure S1). Taken together, these results indicate that FGF9 is a direct downstream target for miR-26a in GC cells. The above results prompted us to examine whether miR-26a suppresses GC growth and metastasis through repressing FGF9 expression. For this purpose, FGF9 was re-expressed in miR-26a-transfected SGC-7901 cells. In miR-26a-expressing cells, re-expression of FGF9 rescued the invasion and growth defects of miR-26a (Figure 4C, D, E). Finally, we tested if miR-26a expression correlated with FGF9 protein levels in GC. There was an inverse correlation between the FGF9 protein levels, indicated by immunohistochemistry staining, and miR-26a expression assessed by in situ hybridization in 126 GC tissues on TMAs as used above (Figure 4F; Figure1C). Our findings demonstrate that miR-26a has properties consistent with tumor suppressor function. The ability to modulate FGF9 levels might explain, at least in part, why miR-26a can inhibit GC growth and metastasis.


miR-26a suppresses tumor growth and metastasis by targeting FGF9 in gastric cancer.

Deng M, Tang HL, Lu XH, Liu MY, Lu XM, Gu YX, Liu JF, He ZM - PLoS ONE (2013)

miR-26a directly targets FGF9.(A) The 3′-UTR element of FGF9 messenger RNA is partially complementary to miR-26a. miR-26a or scramble control and luciferase reporter containing either a wildtype or a mutant 3′-UTR were co-transfected into HEK-293T cells. And a Renilla luciferase expressing construct exerts as internal control. (B) Western blot analysis of FGF9 expression in SGC-7901 and AGS cells infected with miR-26a, and GES-1 transfected with miR-26a inhibitors (Anti-miR-26a). (C, D, E) FGF9 abrogates the suppressive roles of miR-26a in GC cell invasion and growth. SGC-7901 cells stably expressing miR-26a or scramble were transfect with or without FGF9 plasmids. Invasion assays(C), Apoptosis analysis (D), and Cell proliferation analysis (E) were performed with the above cells as described in Materials and Methods. Data are presented as mean±s.e.m from at least three independent experiments. (F) Spearman’s correlation scatter plot of the levels of miR-26a (determined by in situ hybridization) and FGF9 protein (determined by immunohistochemistry) in 126 GC specimens. Representative images of FGF9 expression by immunohistochemistry are shown (right panel). Original magnification: ×200.
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getmorefigures.php?uid=PMC3756000&req=5

pone-0072662-g004: miR-26a directly targets FGF9.(A) The 3′-UTR element of FGF9 messenger RNA is partially complementary to miR-26a. miR-26a or scramble control and luciferase reporter containing either a wildtype or a mutant 3′-UTR were co-transfected into HEK-293T cells. And a Renilla luciferase expressing construct exerts as internal control. (B) Western blot analysis of FGF9 expression in SGC-7901 and AGS cells infected with miR-26a, and GES-1 transfected with miR-26a inhibitors (Anti-miR-26a). (C, D, E) FGF9 abrogates the suppressive roles of miR-26a in GC cell invasion and growth. SGC-7901 cells stably expressing miR-26a or scramble were transfect with or without FGF9 plasmids. Invasion assays(C), Apoptosis analysis (D), and Cell proliferation analysis (E) were performed with the above cells as described in Materials and Methods. Data are presented as mean±s.e.m from at least three independent experiments. (F) Spearman’s correlation scatter plot of the levels of miR-26a (determined by in situ hybridization) and FGF9 protein (determined by immunohistochemistry) in 126 GC specimens. Representative images of FGF9 expression by immunohistochemistry are shown (right panel). Original magnification: ×200.
Mentions: To understand how miR-26a suppresses GC growth and metastasis, we used three algorithms (Targetscan, Pictar and Miranda) to help identify miR-26a targets in human gastric cancers. Of these target genes that were predicted by all three algorithms (Table S1), FGF9 attracted our attention immediately as it has been implicated in tumorigenesis or metastasis [26]–[28]. We cloned the full-length FGF9 3′-UTR into a luciferase reporter vector. Luciferase assay revealed that miR-26a directly bound to FGF9 3′-UTR, and by which it remarkably reduced luciferase activities (Figure 4A). However, mutation of the putative miR-26a sites in the 3′-UTR of FGF9 abrogated luciferase responsiveness to miR-26a (Figure 4A). To directly assess the effect of miR-26a on FGF9 expression, we performed western blot analysis. As seen in Figure 4B, lentiviral induced ectopic miR-26a dramatically suppressed the FGF9 protein levels in SCG-7901 and AGS cells. Furthermore, knockdown of miR-26a, through transfection of anti-miR-26a, in GES-1 cells increased FGF9 protein levels (Figure 4B; Figure S1). Taken together, these results indicate that FGF9 is a direct downstream target for miR-26a in GC cells. The above results prompted us to examine whether miR-26a suppresses GC growth and metastasis through repressing FGF9 expression. For this purpose, FGF9 was re-expressed in miR-26a-transfected SGC-7901 cells. In miR-26a-expressing cells, re-expression of FGF9 rescued the invasion and growth defects of miR-26a (Figure 4C, D, E). Finally, we tested if miR-26a expression correlated with FGF9 protein levels in GC. There was an inverse correlation between the FGF9 protein levels, indicated by immunohistochemistry staining, and miR-26a expression assessed by in situ hybridization in 126 GC tissues on TMAs as used above (Figure 4F; Figure1C). Our findings demonstrate that miR-26a has properties consistent with tumor suppressor function. The ability to modulate FGF9 levels might explain, at least in part, why miR-26a can inhibit GC growth and metastasis.

Bottom Line: In this study, we found that miR-26a was strongly downregulated in gastric cancer (GC) tissues and cell lines, and its expression levels were associated with lymph node metastasis and clinical stage, as well as overall survival and replase-free survival of GC.We also found that ectopic expression of miR-26a inhibited GC cell proliferation and GC metastasis in vitro and in vivo.In addition, miR-26a expression inversely correlated with FGF9 protein levels in GC.

View Article: PubMed Central - PubMed

Affiliation: Cancer Hospital and Cancer Research Institute, Guangzhou Medical University, Guangzhou, China ; Cancer Research Institute, University of South China, Hengyang, China.

ABSTRACT
The role of miR-26a in cancer cells seemed controversial in previous studies. Until now, the role of miR-26a in gastric cancer remains undefined. In this study, we found that miR-26a was strongly downregulated in gastric cancer (GC) tissues and cell lines, and its expression levels were associated with lymph node metastasis and clinical stage, as well as overall survival and replase-free survival of GC. We also found that ectopic expression of miR-26a inhibited GC cell proliferation and GC metastasis in vitro and in vivo. We further identified a novel mechanism of miR-26a to suppress GC growth and metastasis. FGF9 was proved to be a direct target of miR-26a, using luciferase assay and western blot. FGF9 overexpression in miR-26a-expressing cells could rescue invasion and growth defects of miR-26a. In addition, miR-26a expression inversely correlated with FGF9 protein levels in GC. Taken together, our data suggest that miR-26a functions as a tumor suppressor in GC development and progression, and holds promise as a prognostic biomarker and potential therapeutic target for GC.

Show MeSH
Related in: MedlinePlus