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Down-regulation of eIF4GII by miR-520c-3p represses diffuse large B cell lymphoma development.

Mazan-Mamczarz K, Zhao XF, Dai B, Steinhardt JJ, Peroutka RJ, Berk KL, Landon AL, Sadowska M, Zhang Y, Lehrmann E, Becker KG, Shaknovich R, Liu Z, Gartenhaus RB - PLoS Genet. (2014)

Bottom Line: Deregulation of the translational machinery is emerging as a critical contributor to cancer development.The contribution of microRNAs in translational gene control has been established however; the role of microRNAs in disrupting the cap-dependent translation regulation complex has not been previously described.Our results provide evidence that the tumor suppressor effect of miR-520c-3p is mediated through repression of translation while inducing senescence and that eIF4GII is a key effector of this anti-tumor activity.

View Article: PubMed Central - PubMed

Affiliation: Marlene & Stewart Greenebaum Cancer Center, Department of Medicine, University of Maryland, Baltimore, Maryland, United States of America.

ABSTRACT
Deregulation of the translational machinery is emerging as a critical contributor to cancer development. The contribution of microRNAs in translational gene control has been established however; the role of microRNAs in disrupting the cap-dependent translation regulation complex has not been previously described. Here, we established that elevated miR-520c-3p represses global translation, cell proliferation and initiates premature senescence in HeLa and DLBCL cells. Moreover, we demonstrate that miR-520c-3p directly targets translation initiation factor, eIF4GII mRNA and negatively regulates eIF4GII protein synthesis. miR-520c-3p overexpression diminishes cells colony formation and reduces tumor growth in a human xenograft mouse model. Consequently, downregulation of eIF4GII by siRNA decreases translation, cell proliferation and ability to form colonies, as well as induces cellular senescence. In vitro and in vivo findings were further validated in patient samples; DLBCL primary cells demonstrated low miR-520c-3p levels with reciprocally up-regulated eIF4GII protein expression. Our results provide evidence that the tumor suppressor effect of miR-520c-3p is mediated through repression of translation while inducing senescence and that eIF4GII is a key effector of this anti-tumor activity.

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Analysis of microarray data in HeLa cells.(A) Functional categories of total and polysome associated mRNAs in Pre-miR-520c-3p compared to Pre-miR-Ctrl transfected cells. Heat map represents GO annotations with the most altered values in sucrose gradient fractions. Top 100 categories are illustrated in Figure S4. T indicates total RNA; lanes 1 through 11 represent RNA from sucrose fractions of increasing molecular weight. (B and C) The levels of total mRNAs of validated genes were measured by RT-qPCR in cells transfected with Pre-miR-520c-3p compared to Pre-miR-Ctrl. Graphs represent the means and SEM from three repeats of three independent assays. (D) Protein levels of miR-520c-3p target genes were measured by Western blotting. GAPDH was used as a loading control. (E) Cells were fractionated through sucrose gradients and the relative distribution of selected miR-520c-3p target mRNA (and housekeeping GAPDH mRNA) was studied by RT-qPCR analysis of RNA in each of 11 gradient fractions. Data are representative of three independent experiments.
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pgen-1004105-g003: Analysis of microarray data in HeLa cells.(A) Functional categories of total and polysome associated mRNAs in Pre-miR-520c-3p compared to Pre-miR-Ctrl transfected cells. Heat map represents GO annotations with the most altered values in sucrose gradient fractions. Top 100 categories are illustrated in Figure S4. T indicates total RNA; lanes 1 through 11 represent RNA from sucrose fractions of increasing molecular weight. (B and C) The levels of total mRNAs of validated genes were measured by RT-qPCR in cells transfected with Pre-miR-520c-3p compared to Pre-miR-Ctrl. Graphs represent the means and SEM from three repeats of three independent assays. (D) Protein levels of miR-520c-3p target genes were measured by Western blotting. GAPDH was used as a loading control. (E) Cells were fractionated through sucrose gradients and the relative distribution of selected miR-520c-3p target mRNA (and housekeeping GAPDH mRNA) was studied by RT-qPCR analysis of RNA in each of 11 gradient fractions. Data are representative of three independent experiments.

Mentions: To better understand the molecular mechanisms whereby miR-520c-3p elicits global translational repression and to identify miR-520c-3p targets and pathways involved in this regulation, we performed microarray analysis of total mRNA and mRNA isolated from polysomal fractions (translational profiling). Significantly altered genes were further analyzed for representation of Gene Ontology (GO) terms to identify key biologically functional categories that were significantly changed in miR-520c-3p overexpressed compared to control transfected cells. Noticeably, GO analysis revealed mainly translation and translation related categories with greatest representations of genes which had decreased protein synthesis (lowest values in the most actively translating fractions [fractions 10–11], and highest values in the non-translating and low-translating fractions of the gradient [fractions 1–7]) in miR-520c-3p overexpressed versus control cells (Figure 3A, Figure S4). Additionally, genes affected by miR-520c-3p overexpression in the most translationally active polysomal fraction 10 were put in the context of the known molecular interactions by using Ingenuity Pathways Analysis (IPA). IPA revealed that the top five networks, with the highest number of involved genes, were molecular functions linked to RNA post-transcriptional modification, cellular development, cellular growth and proliferation, cell cycle, and cancer (Table S1). The effect of miR-520c-3p overexpression was furthermore verified for several mRNAs (Figure 3B) chosen from the most significantly altered in microarray data genes (Table S2), which were computationally predicted as direct targets of miR-520c-3p (by miRanda and Microcosm database) and, functionally represented above ranked pathways found by GO and IPA analysis. By comparing miR-520c-3p upregulated to control transfected cells by RT-qPCR analysis, we observed no influence of miR-520c-3p on total mRNA levels of most of the validated miR-520c-3p target transcripts (Figure 3C). Instead, we found that increased miR-520c-3p levels lowered protein expression of studied target genes (Figure 3D).


Down-regulation of eIF4GII by miR-520c-3p represses diffuse large B cell lymphoma development.

Mazan-Mamczarz K, Zhao XF, Dai B, Steinhardt JJ, Peroutka RJ, Berk KL, Landon AL, Sadowska M, Zhang Y, Lehrmann E, Becker KG, Shaknovich R, Liu Z, Gartenhaus RB - PLoS Genet. (2014)

Analysis of microarray data in HeLa cells.(A) Functional categories of total and polysome associated mRNAs in Pre-miR-520c-3p compared to Pre-miR-Ctrl transfected cells. Heat map represents GO annotations with the most altered values in sucrose gradient fractions. Top 100 categories are illustrated in Figure S4. T indicates total RNA; lanes 1 through 11 represent RNA from sucrose fractions of increasing molecular weight. (B and C) The levels of total mRNAs of validated genes were measured by RT-qPCR in cells transfected with Pre-miR-520c-3p compared to Pre-miR-Ctrl. Graphs represent the means and SEM from three repeats of three independent assays. (D) Protein levels of miR-520c-3p target genes were measured by Western blotting. GAPDH was used as a loading control. (E) Cells were fractionated through sucrose gradients and the relative distribution of selected miR-520c-3p target mRNA (and housekeeping GAPDH mRNA) was studied by RT-qPCR analysis of RNA in each of 11 gradient fractions. Data are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3907297&req=5

pgen-1004105-g003: Analysis of microarray data in HeLa cells.(A) Functional categories of total and polysome associated mRNAs in Pre-miR-520c-3p compared to Pre-miR-Ctrl transfected cells. Heat map represents GO annotations with the most altered values in sucrose gradient fractions. Top 100 categories are illustrated in Figure S4. T indicates total RNA; lanes 1 through 11 represent RNA from sucrose fractions of increasing molecular weight. (B and C) The levels of total mRNAs of validated genes were measured by RT-qPCR in cells transfected with Pre-miR-520c-3p compared to Pre-miR-Ctrl. Graphs represent the means and SEM from three repeats of three independent assays. (D) Protein levels of miR-520c-3p target genes were measured by Western blotting. GAPDH was used as a loading control. (E) Cells were fractionated through sucrose gradients and the relative distribution of selected miR-520c-3p target mRNA (and housekeeping GAPDH mRNA) was studied by RT-qPCR analysis of RNA in each of 11 gradient fractions. Data are representative of three independent experiments.
Mentions: To better understand the molecular mechanisms whereby miR-520c-3p elicits global translational repression and to identify miR-520c-3p targets and pathways involved in this regulation, we performed microarray analysis of total mRNA and mRNA isolated from polysomal fractions (translational profiling). Significantly altered genes were further analyzed for representation of Gene Ontology (GO) terms to identify key biologically functional categories that were significantly changed in miR-520c-3p overexpressed compared to control transfected cells. Noticeably, GO analysis revealed mainly translation and translation related categories with greatest representations of genes which had decreased protein synthesis (lowest values in the most actively translating fractions [fractions 10–11], and highest values in the non-translating and low-translating fractions of the gradient [fractions 1–7]) in miR-520c-3p overexpressed versus control cells (Figure 3A, Figure S4). Additionally, genes affected by miR-520c-3p overexpression in the most translationally active polysomal fraction 10 were put in the context of the known molecular interactions by using Ingenuity Pathways Analysis (IPA). IPA revealed that the top five networks, with the highest number of involved genes, were molecular functions linked to RNA post-transcriptional modification, cellular development, cellular growth and proliferation, cell cycle, and cancer (Table S1). The effect of miR-520c-3p overexpression was furthermore verified for several mRNAs (Figure 3B) chosen from the most significantly altered in microarray data genes (Table S2), which were computationally predicted as direct targets of miR-520c-3p (by miRanda and Microcosm database) and, functionally represented above ranked pathways found by GO and IPA analysis. By comparing miR-520c-3p upregulated to control transfected cells by RT-qPCR analysis, we observed no influence of miR-520c-3p on total mRNA levels of most of the validated miR-520c-3p target transcripts (Figure 3C). Instead, we found that increased miR-520c-3p levels lowered protein expression of studied target genes (Figure 3D).

Bottom Line: Deregulation of the translational machinery is emerging as a critical contributor to cancer development.The contribution of microRNAs in translational gene control has been established however; the role of microRNAs in disrupting the cap-dependent translation regulation complex has not been previously described.Our results provide evidence that the tumor suppressor effect of miR-520c-3p is mediated through repression of translation while inducing senescence and that eIF4GII is a key effector of this anti-tumor activity.

View Article: PubMed Central - PubMed

Affiliation: Marlene & Stewart Greenebaum Cancer Center, Department of Medicine, University of Maryland, Baltimore, Maryland, United States of America.

ABSTRACT
Deregulation of the translational machinery is emerging as a critical contributor to cancer development. The contribution of microRNAs in translational gene control has been established however; the role of microRNAs in disrupting the cap-dependent translation regulation complex has not been previously described. Here, we established that elevated miR-520c-3p represses global translation, cell proliferation and initiates premature senescence in HeLa and DLBCL cells. Moreover, we demonstrate that miR-520c-3p directly targets translation initiation factor, eIF4GII mRNA and negatively regulates eIF4GII protein synthesis. miR-520c-3p overexpression diminishes cells colony formation and reduces tumor growth in a human xenograft mouse model. Consequently, downregulation of eIF4GII by siRNA decreases translation, cell proliferation and ability to form colonies, as well as induces cellular senescence. In vitro and in vivo findings were further validated in patient samples; DLBCL primary cells demonstrated low miR-520c-3p levels with reciprocally up-regulated eIF4GII protein expression. Our results provide evidence that the tumor suppressor effect of miR-520c-3p is mediated through repression of translation while inducing senescence and that eIF4GII is a key effector of this anti-tumor activity.

Show MeSH
Related in: MedlinePlus