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The role of p19 and p21 H-Ras proteins and mutants in miRNA expression in cancer and a Costello syndrome cell model.

García-Cruz R, Camats M, Calin GA, Liu CG, Volinia S, Taccioli C, Croce CM, Bach-Elias M - BMC Med. Genet. (2015)

Bottom Line: Our aim is to better understand the role of p19 and p21 H-Ras proteins in the cancer and Costello Syndrome development, concerning the miRNAs expression.These results allow us to conclude that the H-Ras G12S mutation plays an important role in miRNA expression and open up a new line of study to understand the consequences of this mutation on Costello syndrome.Furthermore, they suggest that oncogenes may have a sufficiently important impact on miRNA expression to promote the development of numerous cancers.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Biomédicas de Barcelona- CSIC, C/ Egipcíacas15, 08001, Barcelona, Spain. roseli_760901@hotmail.com.

ABSTRACT

Background: P19 H-Ras, a second product derived from the H-Ras gene by alternative splicing, induces a G1/S phase delay, thereby maintaining cells in a reversible quiescence state. When P21 H-Ras is mutated in tumour cells, the alternative protein P19 H-Ras is also mutated. The H-Ras mutation Q61L is frequently detected in different tumours, which acts as constitutive activator of Ras functions and is considered to be a strong activating mutant. Additionally, a rare congenital disorder named Costello Syndrome, is described as a H-Ras disorder in children, mainly due to mutation G12S in p19 and p21 H-Ras proteins, which is present in 90 % of the Costello Syndrome patients. Our aim is to better understand the role of p19 and p21 H-Ras proteins in the cancer and Costello Syndrome development, concerning the miRNAs expression.

Methods: Total miRNAs expression regulated by H-Ras proteins were first analyzed in human miRNA microarrays assays. Previously selected miRNAs, were further analyzed in developed cell lines containing H-Ras protein mutants, that included the G12S Costello Syndrome mutant, with PCR Real-Time Taq Man miRNA Assays primers.

Results: This study describes how p19 affects the RNA world and shows that: i) miR-342, miR-206, miR-330, miR-138 and miR-99b are upregulated by p19 but not by p19W164A mutant; ii) anti-miR-206 can restore the G2 phase in the presence of p19; iii) p19 and p21Q61L regulate their own alternative splicing; iv) miR-206 and miR-138 are differentially regulated by p19 and p21 H-Ras and v) P19G12S Costello mutants show a clear upregulation of miR-374, miR-126, miR-342, miR-330, miR-335 and let-7.

Conclusions: These results allow us to conclude that the H-Ras G12S mutation plays an important role in miRNA expression and open up a new line of study to understand the consequences of this mutation on Costello syndrome. Furthermore, they suggest that oncogenes may have a sufficiently important impact on miRNA expression to promote the development of numerous cancers.

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Related in: MedlinePlus

P19 H-Ras does not activate cell growth. DKO H-Ras(−/−)/N-Ras(−/−) fibroblasts that stably express pEGFP (negative control), pEGFP-p19 and pEGFP-p21 were studied by direct cell-proliferation assay. Cells were harvested and plated in 96 wells (10,000 cells/well) on six microplates in sextuplicate and incubated at 37 °C, 5 % CO2 with DMEM/10 % FCS. After the desired time, the microplates were washed and frozen. Cells were quantified with the green fluorescent dye CyQuant (Invitrogen) according to the manufacturer’s instructions. Fluorescence measurements were performed using a microplate reader with excitation at 485 nm and detection at 530 nnm
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Fig4: P19 H-Ras does not activate cell growth. DKO H-Ras(−/−)/N-Ras(−/−) fibroblasts that stably express pEGFP (negative control), pEGFP-p19 and pEGFP-p21 were studied by direct cell-proliferation assay. Cells were harvested and plated in 96 wells (10,000 cells/well) on six microplates in sextuplicate and incubated at 37 °C, 5 % CO2 with DMEM/10 % FCS. After the desired time, the microplates were washed and frozen. Cells were quantified with the green fluorescent dye CyQuant (Invitrogen) according to the manufacturer’s instructions. Fluorescence measurements were performed using a microplate reader with excitation at 485 nm and detection at 530 nnm

Mentions: In order to further understand how the alternative splicing of H-Ras alters miRNAs, we differentially expressed pEGFP-p19 or pEGFP-p21 in H-Ras(−/−) KO MEFs and analyzed their effect on miR-206 and miR-138. Figure 3a shows that p19 has a large effect on miR-206 expression whereas pGFP-p21 increases it only slightly. pEGFP-p19 also has a large effect on miR-138 in both KO MEFs and HeLa cells (Fig. 3b, c, respectively), thus indicating that the alternative splicing of H-Ras towards p19 or p21 affects the expression of several miRNAs. Figure 4 shows that pEGFP-p21 increases cell growth in H-Ras(−/−)/N-Ras(−/−) DKO MEFs whereas pEGFP-p19 does not. This finding is in accordance with our previous results, where we showed that p19 causes a G1/S delay in HeLa cells.Fig. 3


The role of p19 and p21 H-Ras proteins and mutants in miRNA expression in cancer and a Costello syndrome cell model.

García-Cruz R, Camats M, Calin GA, Liu CG, Volinia S, Taccioli C, Croce CM, Bach-Elias M - BMC Med. Genet. (2015)

P19 H-Ras does not activate cell growth. DKO H-Ras(−/−)/N-Ras(−/−) fibroblasts that stably express pEGFP (negative control), pEGFP-p19 and pEGFP-p21 were studied by direct cell-proliferation assay. Cells were harvested and plated in 96 wells (10,000 cells/well) on six microplates in sextuplicate and incubated at 37 °C, 5 % CO2 with DMEM/10 % FCS. After the desired time, the microplates were washed and frozen. Cells were quantified with the green fluorescent dye CyQuant (Invitrogen) according to the manufacturer’s instructions. Fluorescence measurements were performed using a microplate reader with excitation at 485 nm and detection at 530 nnm
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4631104&req=5

Fig4: P19 H-Ras does not activate cell growth. DKO H-Ras(−/−)/N-Ras(−/−) fibroblasts that stably express pEGFP (negative control), pEGFP-p19 and pEGFP-p21 were studied by direct cell-proliferation assay. Cells were harvested and plated in 96 wells (10,000 cells/well) on six microplates in sextuplicate and incubated at 37 °C, 5 % CO2 with DMEM/10 % FCS. After the desired time, the microplates were washed and frozen. Cells were quantified with the green fluorescent dye CyQuant (Invitrogen) according to the manufacturer’s instructions. Fluorescence measurements were performed using a microplate reader with excitation at 485 nm and detection at 530 nnm
Mentions: In order to further understand how the alternative splicing of H-Ras alters miRNAs, we differentially expressed pEGFP-p19 or pEGFP-p21 in H-Ras(−/−) KO MEFs and analyzed their effect on miR-206 and miR-138. Figure 3a shows that p19 has a large effect on miR-206 expression whereas pGFP-p21 increases it only slightly. pEGFP-p19 also has a large effect on miR-138 in both KO MEFs and HeLa cells (Fig. 3b, c, respectively), thus indicating that the alternative splicing of H-Ras towards p19 or p21 affects the expression of several miRNAs. Figure 4 shows that pEGFP-p21 increases cell growth in H-Ras(−/−)/N-Ras(−/−) DKO MEFs whereas pEGFP-p19 does not. This finding is in accordance with our previous results, where we showed that p19 causes a G1/S delay in HeLa cells.Fig. 3

Bottom Line: Our aim is to better understand the role of p19 and p21 H-Ras proteins in the cancer and Costello Syndrome development, concerning the miRNAs expression.These results allow us to conclude that the H-Ras G12S mutation plays an important role in miRNA expression and open up a new line of study to understand the consequences of this mutation on Costello syndrome.Furthermore, they suggest that oncogenes may have a sufficiently important impact on miRNA expression to promote the development of numerous cancers.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Biomédicas de Barcelona- CSIC, C/ Egipcíacas15, 08001, Barcelona, Spain. roseli_760901@hotmail.com.

ABSTRACT

Background: P19 H-Ras, a second product derived from the H-Ras gene by alternative splicing, induces a G1/S phase delay, thereby maintaining cells in a reversible quiescence state. When P21 H-Ras is mutated in tumour cells, the alternative protein P19 H-Ras is also mutated. The H-Ras mutation Q61L is frequently detected in different tumours, which acts as constitutive activator of Ras functions and is considered to be a strong activating mutant. Additionally, a rare congenital disorder named Costello Syndrome, is described as a H-Ras disorder in children, mainly due to mutation G12S in p19 and p21 H-Ras proteins, which is present in 90 % of the Costello Syndrome patients. Our aim is to better understand the role of p19 and p21 H-Ras proteins in the cancer and Costello Syndrome development, concerning the miRNAs expression.

Methods: Total miRNAs expression regulated by H-Ras proteins were first analyzed in human miRNA microarrays assays. Previously selected miRNAs, were further analyzed in developed cell lines containing H-Ras protein mutants, that included the G12S Costello Syndrome mutant, with PCR Real-Time Taq Man miRNA Assays primers.

Results: This study describes how p19 affects the RNA world and shows that: i) miR-342, miR-206, miR-330, miR-138 and miR-99b are upregulated by p19 but not by p19W164A mutant; ii) anti-miR-206 can restore the G2 phase in the presence of p19; iii) p19 and p21Q61L regulate their own alternative splicing; iv) miR-206 and miR-138 are differentially regulated by p19 and p21 H-Ras and v) P19G12S Costello mutants show a clear upregulation of miR-374, miR-126, miR-342, miR-330, miR-335 and let-7.

Conclusions: These results allow us to conclude that the H-Ras G12S mutation plays an important role in miRNA expression and open up a new line of study to understand the consequences of this mutation on Costello syndrome. Furthermore, they suggest that oncogenes may have a sufficiently important impact on miRNA expression to promote the development of numerous cancers.

Show MeSH
Related in: MedlinePlus