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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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Ectopic expression of miR-493 in lung cancer cells inhibits proliferation.A, stable ectopic overexpression of miR-493 was generated in cell subsets 95D/miR-493 and H1975/miR-493. qRT-PCR was used to verify the transfection. ** means P<0.001 (t-test). B, growth curves demonstrated that the lung cells were capable of proliferation. Cells were seeded in 12-well plates at the desired cell concentrations and maintained in medium containing 10% FBS. The absorption value of cells was measured at the indicated time points in triplicate and their growth rates were recorded. * means P<0.05 (t-test). C, The cells cycle's distribution of lung cancer cells was analyzed with a FACScan flow cytometer. The proliferation index (PI) was used to describe the potentiality of cell proliferation. PI = G2+S fraction)/(G1+G2+S)×100%. Left, the representative image of cell cycle percentage changing. Right, the statistical analysis of cell cycle percentage, data are presented as mean± SEM of 3 independent experiments * means P<0.05 (t-test). D, colony formation assays were used to determine the effect of miR-493 on long-term cell survival. Left, the area covered on each plate by the colonies was measured using an imaging system and represented as the percentage of the total area of the plate. Right, the effect of miR-493 overexpression on colony formation of lung cancer cells: each column represents a mean value of triplicate experiments in each group. Data are mean ± SEM. * means P<0.05 (t-test). E, miR-493 inhibited pulmonary tumor growth in mouse xenograft models (12 animals). Left, representative tumors isolated from mice 25 days after the subcutaneous injection of 95D cells that were stably transfected with the miR-493-expressing or control vector. Right, Data are mean ± SEM. * p<0.05, (t test).
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pone-0102602-g002: Ectopic expression of miR-493 in lung cancer cells inhibits proliferation.A, stable ectopic overexpression of miR-493 was generated in cell subsets 95D/miR-493 and H1975/miR-493. qRT-PCR was used to verify the transfection. ** means P<0.001 (t-test). B, growth curves demonstrated that the lung cells were capable of proliferation. Cells were seeded in 12-well plates at the desired cell concentrations and maintained in medium containing 10% FBS. The absorption value of cells was measured at the indicated time points in triplicate and their growth rates were recorded. * means P<0.05 (t-test). C, The cells cycle's distribution of lung cancer cells was analyzed with a FACScan flow cytometer. The proliferation index (PI) was used to describe the potentiality of cell proliferation. PI = G2+S fraction)/(G1+G2+S)×100%. Left, the representative image of cell cycle percentage changing. Right, the statistical analysis of cell cycle percentage, data are presented as mean± SEM of 3 independent experiments * means P<0.05 (t-test). D, colony formation assays were used to determine the effect of miR-493 on long-term cell survival. Left, the area covered on each plate by the colonies was measured using an imaging system and represented as the percentage of the total area of the plate. Right, the effect of miR-493 overexpression on colony formation of lung cancer cells: each column represents a mean value of triplicate experiments in each group. Data are mean ± SEM. * means P<0.05 (t-test). E, miR-493 inhibited pulmonary tumor growth in mouse xenograft models (12 animals). Left, representative tumors isolated from mice 25 days after the subcutaneous injection of 95D cells that were stably transfected with the miR-493-expressing or control vector. Right, Data are mean ± SEM. * p<0.05, (t test).

Mentions: To assess the biological effects of overexpressing miR-493 in lung cancer cells, stable ectopic overexpression cell subsets 95D/miR-493 and H1975/miR-493 and their paired control cells were constructed. A qRT-PCR analysis showed that the transfection were successful (figure 2A). We determined that the overexpression of miR-493 in 95D and H1975 cells markedly impaired cell proliferation compared with the controls using a cell growth rate assay (figure 2B), the cell cycle distribution (figure 2C) and a colony formation assay (figure 2D). In addition, we tested whether miR-493 could play a role in tumor growth using nude mice xenograft models (six mice per group). 95D cells transfected either with miR-493 or a scrambled control were transplanted into nude mice and developed into solid tumors in 25 days. However, the tumor growth was significantly reduced when miR-493 was stably expressed in 95D cells (figure 2 E). These results implied that miR-493 can strongly suppress tumor cell growth.


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)

Ectopic expression of miR-493 in lung cancer cells inhibits proliferation.A, stable ectopic overexpression of miR-493 was generated in cell subsets 95D/miR-493 and H1975/miR-493. qRT-PCR was used to verify the transfection. ** means P<0.001 (t-test). B, growth curves demonstrated that the lung cells were capable of proliferation. Cells were seeded in 12-well plates at the desired cell concentrations and maintained in medium containing 10% FBS. The absorption value of cells was measured at the indicated time points in triplicate and their growth rates were recorded. * means P<0.05 (t-test). C, The cells cycle's distribution of lung cancer cells was analyzed with a FACScan flow cytometer. The proliferation index (PI) was used to describe the potentiality of cell proliferation. PI = G2+S fraction)/(G1+G2+S)×100%. Left, the representative image of cell cycle percentage changing. Right, the statistical analysis of cell cycle percentage, data are presented as mean± SEM of 3 independent experiments * means P<0.05 (t-test). D, colony formation assays were used to determine the effect of miR-493 on long-term cell survival. Left, the area covered on each plate by the colonies was measured using an imaging system and represented as the percentage of the total area of the plate. Right, the effect of miR-493 overexpression on colony formation of lung cancer cells: each column represents a mean value of triplicate experiments in each group. Data are mean ± SEM. * means P<0.05 (t-test). E, miR-493 inhibited pulmonary tumor growth in mouse xenograft models (12 animals). Left, representative tumors isolated from mice 25 days after the subcutaneous injection of 95D cells that were stably transfected with the miR-493-expressing or control vector. Right, Data are mean ± SEM. * p<0.05, (t test).
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pone-0102602-g002: Ectopic expression of miR-493 in lung cancer cells inhibits proliferation.A, stable ectopic overexpression of miR-493 was generated in cell subsets 95D/miR-493 and H1975/miR-493. qRT-PCR was used to verify the transfection. ** means P<0.001 (t-test). B, growth curves demonstrated that the lung cells were capable of proliferation. Cells were seeded in 12-well plates at the desired cell concentrations and maintained in medium containing 10% FBS. The absorption value of cells was measured at the indicated time points in triplicate and their growth rates were recorded. * means P<0.05 (t-test). C, The cells cycle's distribution of lung cancer cells was analyzed with a FACScan flow cytometer. The proliferation index (PI) was used to describe the potentiality of cell proliferation. PI = G2+S fraction)/(G1+G2+S)×100%. Left, the representative image of cell cycle percentage changing. Right, the statistical analysis of cell cycle percentage, data are presented as mean± SEM of 3 independent experiments * means P<0.05 (t-test). D, colony formation assays were used to determine the effect of miR-493 on long-term cell survival. Left, the area covered on each plate by the colonies was measured using an imaging system and represented as the percentage of the total area of the plate. Right, the effect of miR-493 overexpression on colony formation of lung cancer cells: each column represents a mean value of triplicate experiments in each group. Data are mean ± SEM. * means P<0.05 (t-test). E, miR-493 inhibited pulmonary tumor growth in mouse xenograft models (12 animals). Left, representative tumors isolated from mice 25 days after the subcutaneous injection of 95D cells that were stably transfected with the miR-493-expressing or control vector. Right, Data are mean ± SEM. * p<0.05, (t test).
Mentions: To assess the biological effects of overexpressing miR-493 in lung cancer cells, stable ectopic overexpression cell subsets 95D/miR-493 and H1975/miR-493 and their paired control cells were constructed. A qRT-PCR analysis showed that the transfection were successful (figure 2A). We determined that the overexpression of miR-493 in 95D and H1975 cells markedly impaired cell proliferation compared with the controls using a cell growth rate assay (figure 2B), the cell cycle distribution (figure 2C) and a colony formation assay (figure 2D). In addition, we tested whether miR-493 could play a role in tumor growth using nude mice xenograft models (six mice per group). 95D cells transfected either with miR-493 or a scrambled control were transplanted into nude mice and developed into solid tumors in 25 days. However, the tumor growth was significantly reduced when miR-493 was stably expressed in 95D cells (figure 2 E). These results implied that miR-493 can strongly suppress tumor cell growth.

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