Molecular dissection of Cdc6 and the miR-148 family : two stories with common themes

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(1)Molecular dissection of Cdc6 and the miR-148 family : two stories with common themes Duursma, A.M.. Citation Duursma, A. M. (2008, May 14). Molecular dissection of Cdc6 and the miR-148 family : two stories with common themes. Retrieved from https://hdl.handle.net/1887/12848 Version:. Corrected Publisher’s Version. License:. Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden. Downloaded from:. https://hdl.handle.net/1887/12848. Note: To cite this publication please use the final published version (if applicable)..

(2) Chapter 6. miR-152 plays a role in S-phase and G2/M-phase cell cycle progression of diploid fibroblasts B>5' IIFGA5$C57<=A)I890F=9@=B?,9IJ9B;5A=. .

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(4) miR-152 plays a role in S-phase and G2/M-phase cell cycle progression of diploid fibroblasts B>5' IIFGA5$C57<=A)I890F=9@=B?,9IJ9B;5A= .<9(9H<9F@5B8G5B79F#BGH=HIH9*@9GA5B@55B

(5) 2AGH9F85A.<9(9H<9F@5B8G Eukaryotic cells have evolved several mechanisms to ensure proper proliferation. Upon detection of aberrant replication or DNA damage, cells activate an intra-S phase or a G2 phase checkpoint, which arrest cells to allow for repair and to prevent entry into mitosis. MicroRNAs (miRNAs) are small RNA molecules of approximately 22 nucleotides that bind to target mRNAs and inhibit translation. miRNAs have been implicated in many cellular processes, but only a few miRNAs have been shown to be involved in cell cycle regulation. In this study we aimed to study the role of miR-152 in diploid BJ ET fibroblasts by using antagomirs, which inhibit miRNA function. Interestingly, we observed that inhibition of miR-152 resulted in an increase of the relative percentage of S-phase and G2/M–phase cells. More detailed analysis revealed that these cells arrested in G2-phase. Together these results suggest that endogenous expression of miR-152 is required for proper cell cycle progression.. Introduction I?5FMCH=7 79@@G <5J9 9JC@J98 G9J9F5@ A97<5B=GAGHC9BGIF9DFCD9F(F9D@=75H=CB 5B8HCDF9J9BH;9BCA=7=BGH56=@=HM5B875B79F 7FI7=5@ FC@9 =G D@5M98 6M 7<97?DC=BHG H<5H 89@5M79@@7M7@9DFC;F9GG=CB=BH<9DF9G9B79 C: 85A5;98 ( (M69F; 9H 5@

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(48) A=, D@5MG5FC@9=B- D<5G95B8! ' D<5G9. A. B Relative Dnmt1 3'UTR Luciferase activity. Relative Luciferase activity. 1,20 1,00 0,80 0,60 0,40 0,20 0,00 pGL3. Dnmt1 wt. 2,5. 2. 1,5. 1. 0,5. 0. Dnmt1 mut. Scramble. AntagomiR-152. Figure 2. AntagomiR-152 inhibits miR-152 activity towards a Luciferase-Dnmt1-3’UTR. A A=, F9DF9GG9G D!& &I79LDF9GG=CBK=H<H<9K=@8 HMD9BAH U/.,BAH KH6IHBCH5AIH5BHK=H<5AIH5H98A=, H5F;9HG=H9 BAH AIH LDF9GG=CBK5G75@7I@5H98F9@5H=J9HC57C HF5BG:97H=CBK=H<57CBHFC@A=,( B BH5;CA=, F9DF9GG9G A=, A98=5H98F9;I@5H=CBC:BAH 9@@GHF5BG:97H98K=H<A=, 5B8BAH K9F9HF95H98K=H<BH5;CA=, CF 5-7F5A6@987CBHFC@ FFCF65FGF9DF9G9BHGH5B85F889J=5H=CB. HC=B<=6=HA=, 57H=J=HM #BH9F9GH=B;@M K9 BCH98 5 897F95G9 =B 79@@ DFC@=:9F5H=CBIDCB58A=B=GH9F=B;5BH5;CA=, HC $ . 79@@G .C EI5BH=:M H<=G 9::97H K9 G99898 79@@G =B 9EI5@ 5ACIBHG 5B8 HF95H98 H<9A K=H< 9=H<9F 5BH5;CA=, CF 5 G7F5A6@9 7CBHFC@ 5B8 7CIBH98 79@@G CB 85MG

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(51) <5DH9F. Total cell number / 1000. A 300 250 200. Scramble AntagomiR-152. 150 100 50 0 0. 1. 2. 3. 4. 5. days. B Scramble. AntagomiR-152. 500. 500. G1 S G2/M. 300. 400. 85% 5% 10%. Counts. Counts. 400. 200 100. G1 S G2/M. 300. 48% 15% 37%. 200 100. 0. 0 0. 200. 400. 600. 0. 200. PI. 400. 600. PI. C AntagomiR-152 104. 103. 103. 102. M. 101. 41%. G2 13%. 100. MPM-2. MPM-2. Scramble 104. 102. M. 101. 17%. G2 33%. 100 0. 200. 400. PI. 600. 0. 200. 400. 600. PI. Figure 3. Inhibition of endogenous miR-152 in BJ ET diploid fibroblasts results in a G2 arrest. A #B<=6=H=CB C:A=, 6MBH5;CA=, F9GI@HG=BF98I798DFC@=:9F5H=CB $.79@@GK9F9G998985B87CIBH98CB85M

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(138)

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