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MicroRNA-493 suppresses tumor growth, invasion and metastasis of lung cancer by regulating E2F1.

Gu Y, Cheng Y, Song Y, Zhang Z, Deng M, Wang C, Zheng G, He Z - PLoS ONE (2014)

Bottom Line: Our data indicated that the expression of miR-493 was markedly reduced in pulmonary carcinoma.The ectopic expression of miR-493 impaired cell growth and invasion in vitro and in vivo.Mechanically, miR-493 commonly directly targeted E2F1, which resulted in a robust reduction of the expression of mRNA and protein.

View Article: PubMed Central - PubMed

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

ABSTRACT
miRNAs have been proposed to be key regulators of progression and metastasis in cancer. However, an understanding of their roles and molecular mechanisms is needed to provide deeper insights for better therapeutic opportunities. In this study we investigated the role and mechanism of miR-493 in the development and progression of nonsmall-cell lung cancer (NSCLC). Our data indicated that the expression of miR-493 was markedly reduced in pulmonary carcinoma. The ectopic expression of miR-493 impaired cell growth and invasion in vitro and in vivo. Mechanically, miR-493 commonly directly targeted E2F1, which resulted in a robust reduction of the expression of mRNA and protein. This effect, in turn, decreased the growth, invasion and metastasis of lung cancer cells. Our findings highlight the importance of miR-493 dysfunction in promoting tumor progression, and implicate miR-493 as a potential therapeutic target in lung cancer.

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E2F1 is a direct target of miR-493.A, Sequence alignment of microRNAs of the miR-493 and the E2F1 3′-UTR. The E2F1 3′-UTR contains one predicted miR-493-binding site. The seed regions of miR-493 and the seed-recognizing sites in the E2F1 3′-UTR are indicated in red. B and C, Luciferase assay of 95D cells (left-hand side) and H1975 cells (right-hand side), which were co-transfected with miR-493 and a luciferase reporter containing full length E2F1 3′-UTR (Luc-wt) or a mutant (Luc-mut) in which the nucleotides of the miR-493-binding site were mutated. An empty luciferase reporter construct was used as a negative control (Luc-ctrl). The luciferase activities were measured 48 hours post transfection. miR-493 markedly suppressed the luciferase activity in Luc-wt reporter constructs. The data are the means ±s.e.m. for separate transfections (n = 4). *P<0.05 versus scramble. D and E, miR-493 stable transfection reduces the E2F1 protein and mRNA levels. F and G, The level of miR-493 inversely correlated with E2F1 expression. F, The E2F1 expression levels were measured by immunohistochemistry in tumor tissues. These paraffin-embedded tissues specimens were the same source of 65fresh lung cancer tissue aforementioned in figure 1. A representative image of the expression levels of E2F1 in human lung tumor tissues (200×) is shown. G, data are means± s.e.m. * means P<0.05.
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pone-0102602-g004: E2F1 is a direct target of miR-493.A, Sequence alignment of microRNAs of the miR-493 and the E2F1 3′-UTR. The E2F1 3′-UTR contains one predicted miR-493-binding site. The seed regions of miR-493 and the seed-recognizing sites in the E2F1 3′-UTR are indicated in red. B and C, Luciferase assay of 95D cells (left-hand side) and H1975 cells (right-hand side), which were co-transfected with miR-493 and a luciferase reporter containing full length E2F1 3′-UTR (Luc-wt) or a mutant (Luc-mut) in which the nucleotides of the miR-493-binding site were mutated. An empty luciferase reporter construct was used as a negative control (Luc-ctrl). The luciferase activities were measured 48 hours post transfection. miR-493 markedly suppressed the luciferase activity in Luc-wt reporter constructs. The data are the means ±s.e.m. for separate transfections (n = 4). *P<0.05 versus scramble. D and E, miR-493 stable transfection reduces the E2F1 protein and mRNA levels. F and G, The level of miR-493 inversely correlated with E2F1 expression. F, The E2F1 expression levels were measured by immunohistochemistry in tumor tissues. These paraffin-embedded tissues specimens were the same source of 65fresh lung cancer tissue aforementioned in figure 1. A representative image of the expression levels of E2F1 in human lung tumor tissues (200×) is shown. G, data are means± s.e.m. * means P<0.05.

Mentions: In addition to cell growth inhibition, the effect of miR-493 on tumor invasion and metastasis was also addressed in this study. A wound-healing assay showed that miR-493 could dramatically suppress tumor cell mobility in 95D cells compared with the empty vector-transfected cells (figure 3A). Furthermore, an invasion assay was also performed. The result demonstrated that miR-493 could suppress the invasive ability of lung cancer cells (figure 3B). An experimental animal model was utilized to further investigate the suppressive effect of miR-493 on tumor metastasis, 95D/miR-493 cells were injected into the tail veins of nude mice (five mice per group). Empty vector-transfected (95D/control) cells were used as controls. The mice were killed and the lungs were excised, and the metastatic lesions of the lung were then detected using a Bruker Small Animal Imaging System (Germany) after 28 days. The fields of metastatic lesions formation in the lung were significantly reduced compared to the control group (figure 3C). Although visible metastatic nodules were not observed on the surface of the lung, metastatic lesions were detected in the lung by haematoxylin and eosin staining (figure 4D). These results indicated that miR-493 could effectively suppress tumor metastasis in vitro and in vivo.


MicroRNA-493 suppresses tumor growth, invasion and metastasis of lung cancer by regulating E2F1.

Gu Y, Cheng Y, Song Y, Zhang Z, Deng M, Wang C, Zheng G, He Z - PLoS ONE (2014)

E2F1 is a direct target of miR-493.A, Sequence alignment of microRNAs of the miR-493 and the E2F1 3′-UTR. The E2F1 3′-UTR contains one predicted miR-493-binding site. The seed regions of miR-493 and the seed-recognizing sites in the E2F1 3′-UTR are indicated in red. B and C, Luciferase assay of 95D cells (left-hand side) and H1975 cells (right-hand side), which were co-transfected with miR-493 and a luciferase reporter containing full length E2F1 3′-UTR (Luc-wt) or a mutant (Luc-mut) in which the nucleotides of the miR-493-binding site were mutated. An empty luciferase reporter construct was used as a negative control (Luc-ctrl). The luciferase activities were measured 48 hours post transfection. miR-493 markedly suppressed the luciferase activity in Luc-wt reporter constructs. The data are the means ±s.e.m. for separate transfections (n = 4). *P<0.05 versus scramble. D and E, miR-493 stable transfection reduces the E2F1 protein and mRNA levels. F and G, The level of miR-493 inversely correlated with E2F1 expression. F, The E2F1 expression levels were measured by immunohistochemistry in tumor tissues. These paraffin-embedded tissues specimens were the same source of 65fresh lung cancer tissue aforementioned in figure 1. A representative image of the expression levels of E2F1 in human lung tumor tissues (200×) is shown. G, data are means± s.e.m. * means P<0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4126682&req=5

pone-0102602-g004: E2F1 is a direct target of miR-493.A, Sequence alignment of microRNAs of the miR-493 and the E2F1 3′-UTR. The E2F1 3′-UTR contains one predicted miR-493-binding site. The seed regions of miR-493 and the seed-recognizing sites in the E2F1 3′-UTR are indicated in red. B and C, Luciferase assay of 95D cells (left-hand side) and H1975 cells (right-hand side), which were co-transfected with miR-493 and a luciferase reporter containing full length E2F1 3′-UTR (Luc-wt) or a mutant (Luc-mut) in which the nucleotides of the miR-493-binding site were mutated. An empty luciferase reporter construct was used as a negative control (Luc-ctrl). The luciferase activities were measured 48 hours post transfection. miR-493 markedly suppressed the luciferase activity in Luc-wt reporter constructs. The data are the means ±s.e.m. for separate transfections (n = 4). *P<0.05 versus scramble. D and E, miR-493 stable transfection reduces the E2F1 protein and mRNA levels. F and G, The level of miR-493 inversely correlated with E2F1 expression. F, The E2F1 expression levels were measured by immunohistochemistry in tumor tissues. These paraffin-embedded tissues specimens were the same source of 65fresh lung cancer tissue aforementioned in figure 1. A representative image of the expression levels of E2F1 in human lung tumor tissues (200×) is shown. G, data are means± s.e.m. * means P<0.05.
Mentions: In addition to cell growth inhibition, the effect of miR-493 on tumor invasion and metastasis was also addressed in this study. A wound-healing assay showed that miR-493 could dramatically suppress tumor cell mobility in 95D cells compared with the empty vector-transfected cells (figure 3A). Furthermore, an invasion assay was also performed. The result demonstrated that miR-493 could suppress the invasive ability of lung cancer cells (figure 3B). An experimental animal model was utilized to further investigate the suppressive effect of miR-493 on tumor metastasis, 95D/miR-493 cells were injected into the tail veins of nude mice (five mice per group). Empty vector-transfected (95D/control) cells were used as controls. The mice were killed and the lungs were excised, and the metastatic lesions of the lung were then detected using a Bruker Small Animal Imaging System (Germany) after 28 days. The fields of metastatic lesions formation in the lung were significantly reduced compared to the control group (figure 3C). Although visible metastatic nodules were not observed on the surface of the lung, metastatic lesions were detected in the lung by haematoxylin and eosin staining (figure 4D). These results indicated that miR-493 could effectively suppress tumor metastasis in vitro and in vivo.

Bottom Line: Our data indicated that the expression of miR-493 was markedly reduced in pulmonary carcinoma.The ectopic expression of miR-493 impaired cell growth and invasion in vitro and in vivo.Mechanically, miR-493 commonly directly targeted E2F1, which resulted in a robust reduction of the expression of mRNA and protein.

View Article: PubMed Central - PubMed

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

ABSTRACT
miRNAs have been proposed to be key regulators of progression and metastasis in cancer. However, an understanding of their roles and molecular mechanisms is needed to provide deeper insights for better therapeutic opportunities. In this study we investigated the role and mechanism of miR-493 in the development and progression of nonsmall-cell lung cancer (NSCLC). Our data indicated that the expression of miR-493 was markedly reduced in pulmonary carcinoma. The ectopic expression of miR-493 impaired cell growth and invasion in vitro and in vivo. Mechanically, miR-493 commonly directly targeted E2F1, which resulted in a robust reduction of the expression of mRNA and protein. This effect, in turn, decreased the growth, invasion and metastasis of lung cancer cells. Our findings highlight the importance of miR-493 dysfunction in promoting tumor progression, and implicate miR-493 as a potential therapeutic target in lung cancer.

Show MeSH
Related in: MedlinePlus