Functional study of MicroRNA 125b in vertebrate development 2

4.3. Spatio-temporal expression of miR-125b during zebrafish embryogenesis To examine if miR-125b expression is inversely correlated to p53 expression spatiotemporally during development, we analyzed miR-125b expression at different stages of zebrafish embryogenesis. Expression of miR-125b was first detected at 19 hours postfertilization (hpf) by whole mount in situ hybridization (Fig. 13a). miR-125b was present in the whole embryo with enrichment in the brain, the eyes and the somites at different stages (Fig. 13a-b). The expression pattern in the brain is consistent with previously published data (Wienholds et al., 2005). However, no enriched expression was detected in the spinal cord. Instead, we found a pronounced miR-125b expression in the somites between 22 and 30 hpf (Fig. 13a). By 22 hpf, miR-125b expression was enriched in the eyes, the somites, the telencephalon (tel) and the midbrain with stronger expression in the tegmentum (tg) and hindbrain (Fig. 13a-b). Between 26 and 30 hpf, miR-125b was strongly expressed in the hypothalamus (hyp), tegmentum, the midbrain-hindbrain boundary (mhb) and the hindbrain (Fig. 13b). miR-125b expression continues to increase in the brain such that the optic tectum (ot) became the only region with weak miR-125b expression by 48 hpf (Fig. 13a-b). Analysis of miR-125b expression in whole embryo lysate by quantitative RT-PCR showed that the expression initiated at 18 hpf and increased exponentially from 18 to 48 hpf (Fig. 13c). Interestingly, p53 and p21 expression were inversely correlated with miR-125b upregulation over time (Fig. 13c). We also compared the spatiotemporal expression pattern of miR-125b (our in situ hybridization analysis) with the expression pattern of p53 mRNA (Yamaguchi et al., 2008). p53 and miR-125b were observed to be coexpressed in the brain and the eyes at about 24 hpf. In the brain, miR-125b expression increases steadily from 24-48 hpf, while p53 expression decreases gradually during that same period. In the somites, miR-125b expression is 80 enriched from 22 to 30 hpf, while p53 expression is not observed. Western blots also showed that p53 protein can be detected at 18 hpf and decreases to undetectable levels by 48 hpf (data not shown). The inverse correlation between miR-125b and p53 expression/activity supports our hypothesis that p53 is downregulated by miR-125b during zebrafish embryogenesis. 81 Figure 13 - Spatio-temporal expression of miR-125b during zebrafish embryogenesis (a) Wholemount in situ hybridization of miR-125b in zebrafish embryos at 19 hpf, 22 hpf, 26 hpf, 30 hpf and 48 hpf. Side view of the whole body excluding the tail is shown. (b) Side view of zebrafish brain, in situ hybridization of with miR-125b at 22 hpf, 26 hpf, 30 hpf and 48 hpf. In (a) and (b), each image shows the expression pattern of miR-125b in a representative embryo. The same pattern was observed in all 20 embryos examined at each developmental stage. Abbreviation: ey, eye; hb, hindbrain; hyp, hypothalamus, mhb, midbrain-hindbrain boundary; ot, optic tectum; tel, telencephalon; tg, tegmentum. (c) The expression pattern of miR-125b, p53 and p21 during zebrafish development: transcript levels were quantified by real-time PCR, normalized to internal controls (18S or ȕ-actin) and presented as log2 fold change ± s.e.m. (n • 4) relative to the expression at 18 hpf. 82 4.4. miR-125b represses endogenous p53 and p53-induced apoptosis in human neuroblastoma cells To investigate the regulation of endogenous human p53 by miR-125b, we used the neuroblastoma cell line SH-SY5Y, which is known to express wild-type p53 (Vogan et al., 1993). The endogenous level of miR-125b in undifferentiated SH-SY5Y is relatively low and transfection of miR-125b duplex brought miR-125b level up by ~27 fold (Fig. 14a). Ectopic expression of miR-125b reduced the level of p53 protein in SH-SY5Y cells by ~40% (P < 0.01) (Fig. 15a-b). The level of p53 mRNA was also reduced by 125b-DP transfection although the fold change was smaller than that of p53 protein (Fig. 15c). The expression of p21 and bax, the two main targets of p53, also dropped significantly after 125b-DP transfection (Fig. 15c). Induction of p53 often leads to apoptosis (Almog and Rotter, 1997). However, in neuroblastoma cells, p53 protein is mainly localized to the cytoplasm, so the endogenous activity of nuclear p53 is usually insufficient to modulate apoptosis (Moll et al., 1996). Thus, we predicted that ectopic expression of miR-125b in SH-SY5Y cells will only suppress apoptosis when the p53 pathway is fully activated by an exposure to the drug 1-(5-isoquinolinyl sulfonyl)-2-methyl piperazine (H-7). Exposure to H-7 leads to an increased import of p53 into the nucleus where p53 becomes active and induces apoptosis (Ronca et al., 1997). Indeed, ectopic expression of miR-125b significantly suppressed H-7-induced apoptosis, but did not affect apoptosis in the untreated SH-SY5Y cells, as quantified by the staining of activecaspase-3 (Fig. 15d). 83 ** 125b-DP NC-DP3 -1 Mock Log2 fold change in miR-125b level ** -2 -4 ** 125b-AS NC-AS1 125b-DP NC-DP2 -6 Mock b Log2 fold change in miR-125b level a Figure 14 - Validation of miR-125b overexpression and knockdown in SH-SY5Y cells and in human lung fibroblast cells (a) The level of miR-125b in SH-SY5Y cells two days after a transfection with negative control duplex (NC-DP3) or miR-125b duplex (125b-DP). (b) The level of miR-125b in human lung fibroblast cells two days after a transfection with NC-DP2, 125b-DP, negative control antisense (NC-AS1) or miR-125b antisense (125b-AS). For both (a) and (b), the level of miR-125b was quantified by real-time PCR, normalized to the expression of U6 RNA and presented as log2 fold change ± s.e.m. (n • 4) relative to miR125b level in the mock-transfected cells. Two-tail T-test results are indicated by ** P < 0.01, relative to the mock-transfected control. 84 Figure 15 - miR-125b represses the endogenous p53 expression and suppresses p53induced apoptosis in human neuroblastoma SH-SY5Y cells. (a) The endogenous p53 protein level in SH-SY5Y cells two days after a transfection with mock (water), negative control duplex (NC-DP3), miR-125b duplex (125b-DP) or p53 siRNA. (b) The p53 protein level was quantified from the Western blot bands in (a), normalized to the GAPDH level and presented as fold change ± s.e.m. (n • 3) relative to the p53 level of mocktransfected cells. (c) The mRNA levels of p53, p21 and bax in SH-SY5Y cells two days after a transfection with NC-DP1 or 125b-DP. The expression was quantified by real-time PCR, normalized to the expression of ȕ-actin and presented as fold change ± s.e.m. (n • 4) relative to that in the cells transfected with NC-DP1. (d) The percentage of SH-SY5Y cells positive for active caspase-3 was quantified by the Cellomics® high-content screening system two days after a transfection with NC-DP1 or with 125b-DP. 10 μM H-7 treatment was applied 24 hours before fixing. The values represent average ± s.e.m. (n • 3). For each replicate, 20 images (including at least 10,000 cells) were analyzed. In all panels, two-tail T-test results are indicated by * P < 0.05 and ** P < 0.01, relative to the mock-transfected or NC-DP-transfected controls. 85 4.5. miR-125b represses endogenous p53 and apoptosis in primary human lung fibroblasts To further demonstrate the repression of human p53 by miR-125b in a physiological context, we examined this regulation in primary human lung fibroblasts which were cultured from normal fetal lungs to homogeneity. The level of miR-125b expression in human lung fibroblasts is relatively high. We were able to knockdown the endogenous miR-125b by ~24 fold with 125b-AS or overexpress miR-125b by ~26 fold with 125b-DP (Fig. 14b). Consistently, overexpression of miR-125b repressed p53 protein levels while knockdown of miR-125b elevated p53 levels significantly (Fig. 16a-b). The expression of p21 mRNA, a main target of p53, in human lung fibroblasts was also modulated by miR-125b in the same fashion as p53 protein (Fig. 16c). Here the effect of miR-125b on p21 mRNA level was solely dependent on p53 expression since knockdown of p53 by a siRNA was able to rescue the increase in p21 expression caused by the 125b-AS (Fig. 16c). In addition, 125b-DP represses p21 expression in a dose-dependent manner, with significant suppression still observable at a concentration as low as 10 nM of 125b-DP (Fig. 17a). The level of p53 mRNA in human lung fibroblasts was, however, not affected by the changes in miR-125b expression (Fig. 16c). This suggests that miR-125b inhibits the translation of p53 but does not modulate the stability of p53 mRNA in these cells. In addition, miR-125b knockdown led to a substantial increase in apoptotic cells, as quantified by activecaspase-3 staining, while miR-125b overexpression had the opposed effect (Fig. 16d). These data demonstrate that miR-125b expression is both necessary and sufficient for maintaining the physiological levels and the activity of p53 in human lung fibroblasts. 86 Figure 16 - miR-125b represses the endogenous p53 expression and suppresses apoptosis in human lung fibroblast cells. (a) The endogenous p53 level in human lung fibroblast cells two days after a transfection with mock (water), negative control duplex (NC-DP2) or miR-125b duplex (125b-DP); and one day after a transfection with mock, negative control antisense (NC-AS1) or miR-125b antisense (125b-AS). (b) The p53 protein level was quantified from the Western blot bands in (a), normalized to the GAPDH level and presented as fold change ± s.e.m. (n • 3) relative to the p53 level of mock transfected cells (dotted line). (c) The levels of p53 mRNA and p21 mRNA in human lung fibroblast cells two days after a transfection with mock, NC-DP2, 125b-DP, NC-AS1,125b-AS or cotransfection of 125b-AS and p53 siRNA. The expression was quantified by real-time PCR, normalized to the expression of ȕ-actin and presented as fold change ± s.e.m. (n • 4) relative to that in the mock-transfected cells (dotted line). (d) The percentage of human lung fibroblast cells positive for active caspase-3, two days after a transfection with mock, NC-DP2, 125b-DP, NC-AS1 or 125b-AS was quantified by the Cellomics® high-content screening system. The values represent average ± s.e.m. (n • 3). For each replicate, 20 images (including at least 10,000 cells) were analyzed. In all panels, two-tail T-test results are indicated by * P < 0.05 and ** P < 0.01, relative to the mock-transfected controls. 87 Fold change in p21 mRNA level a 1.2 * 0.8 ** ** 0.6 ** 0.4 0.2 0 10 20 30 40 50 60 70 80 90 100 0.4 ** de os i Etoposide DMSO Tubulin 0.8 Et op p53 1.2 M SO c D b Fold change in mir-125b expression Concentration of 125b-DP (nM) Figure 17 - Cellular responses to different doses of miR-125b and to etoposide treatments (a) Real-time PCR analysis of p21 mRNA levels in human lung fibroblasts transfected with miR-125b duplex (125b-DP) at different concentrations. The level of p21 mRNA was normalized to the expression of ȕ-actin and presented as fold change ± s.e.m. (n • 4) relative to the p21 level in mock transfected cells. Two-tail T-test results are indicated by * P < 0.05 and ** P < 0.01. (b) Western blot analysis of p53 protein in SH-SY5Y cells treated with 10 μM etoposide or with DMSO vehicle control for 24 hours. Tubulin was used as a loading control. (c) Real-time PCR analysis of miR-125b expression level in SH-SY5Y cells treated as in (b). The level of miR-125b was normalized to the expression of U6 RNA and presented as fold change ± s.e.m. (n • 4) relative to miR-125b level in DMSO treated cells. Two-tail T-test results are indicated by ** P < 0.01. 88 4.6. Loss of miR-125b increases p53 and p53-dependent apoptosis in zebrafish As a result of miR-125b knockdown by injection of m125bMO or lp125bMO1/2/3 into one-cell stage embryos, the endogenous level of p53 protein was elevated in zebrafish embryos at 24 hpf (Fig. 18a-b). p21 was also upregulated in both types of morphants (Fig. 18c). When the morphants were co-injected with a morpholino blocking translation of p53, p21 expression was restored to wild-type levels, indicating that the upregulation of p21 by miR-125b required p53 (Fig. 18c). At 24 hpf, an increase in TUNEL–positive apoptotic cells was observed in the midbrain and hindbrain domains of both m125bMO- and lp125bMO1/2/3- injected embryos (Fig. 18d). Enhanced apoptosis was observed in the m125bMO-morphants only from 18 hpf, consistent with the stage when miR-125b expression was first detected (Fig. 19). Apoptosis reached a peak at 24 hpf, when the brain defects were the most severe (Fig. 19). Apoptosis decreased gradually by 30 hpf (Fig. 19) but the hatched larvae were still defective with distorted heads and abnormal behaviors. We then asked whether the cell death phenotype in miR-125b morphants was caused by the elevation in p53 protein. To ablate p53 function, we used the zebrafish p53M214K mutant that is defective in p53 activity but still undergoes normal embryogenesis (Berghmans et al., 2005). Remarkably, knockdown of miR-125b, whether by m125bMO or by lp125bMO1/2/3, had no observable effects on brain apoptosis (Fig. 18d). 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Zhou,B., Wang,S., Mayr,C., Bartel,D.P., and Lodish,H.F. (2007). miR-150, a microRNA expressed in mature B and T cells, blocks early B cell development when expressed prematurely. Proc. Natl. Acad. Sci. U. S. A 104, 7080-7085. 126 BIOGRAPHY EDUCATION ƒ Bachelor of Science (Honours), National University of Singapore, 2005, majored in Life Sciences (Concentration: Molecular and Cell Biology) PUBLICATIONS 1. Minh Le TN, Cathleen Teh, Shyh Chang Ng, Huangming Xie, Beiyan Zhou, Vladimir Korzh, Harvey Lodish* and Bing Lim*; microRNA-125b is a novel negative regulator of p53; Genes & Development (2009), 27: 862-76 2. Minh Le TN, Huangming Xie, Beiyan Zhou, Poh Hui Chia, Moonkyoung Um, Gerald Udolph, Henry Yang, Bing Lim* and Harvey Lodish*; microRNA-125b promotes neuronal differentiation in human cells by repressing multiple targets; Molecular and Cellular Biology, In press. 3. Minh Le TN, MA Reza, Sanjay Swarup and Manjunatha Kini*; Gene duplication of coagulation factor V and origin of venom prothrombin activator in Pseudonaja textilis snake; Thrombosis and Haemostasis (2005) 93: 420 – 429. 4. MA Reza, Minh Le TN, Sanjay Swarup and Manjunatha Kini*; Molecular Evolution Caught In Action: Gene Duplication and Evolution of Molecular Isoforms of Prothrombin Activators In Pseudonaja textilis (Brown Snake); Journal of Thrombosis and Haemostasis (2006) 4: 1346-53. * Coresponding authors AWARDS 1. L’OREAL-UNESCO for Women in Science National Fellowship (2009-2010) 2. Singapore-MIT Alliance fellowship (2005-2009) 127 3. American Society for Cell Biology Worthington predoctoral award for top-five students in cell biology (2008) 4. Travel fellowship for the third Minisymposium on Small RNA (2008) 5. Reach the World Grant from the International Society on Thrombosis and Haemostasis (2005) 6. Second Poster Prize at the third International Conference on Structural Biology and Functional Genomics, Singapore (2004) 7. Second Poster Prize at the 8th Biological Sciences Graduate Congress, Singapore (2003) 8. Lee Foundation Study Grant (2004) 9. National University of Singapore Alumni Bursary Award (2005) 10. Third Prize at the National Biology Contest, Vietnam (2000) CONFERENCE PAPERS AND ORAL PRESENTATIONS 1. Le TNM, Teh C, Ng SC, Xie H, Zhou B, Korzh V, Lodish HF, Lim B; microRNA125b is a novel negative regulator of p53 (45-minute oral presentation); Graduate Student Seminar Series at Genome Institute of Singapore (Singapore, Mar 2009) 2. Le TNM, Xie H, Zhou B, Chia PH, Udolph G, Yang H, Lim B, Lodish HF; A systematic study of microRNA function in neurogenesis (15-minute oral presentation); The 10th Singapore-MIT Alliance Annual Symposium (Singapore, Jan 2009) 3. Le TNM, Xie H, Zhou B, Chia PH, Udolph G, Yang H, Lim B, Lodish HF; microRNA-125b promotes neuronal differentiation in human cells by repressing multiple targets (poster); The 48th annual meeting of American Society for Cell Biology (San Francisco, Dec 2008) 128 4. Le TNM, Xie H, Teh C, Zhou B, Chia PH, Udolph G, Korzh V, Yang H, Lim B, Lodish HF; The role of microRNAs in neurogenesis (45-minute oral presentation); Graduate Student Seminar Series at Genome Institute of Singapore (Singapore, Aug 2008) 5. Le TNM, Xie H, Teh C, Zhou B, Chia PH, Udolph G, Korzh V, Yang H, Lim B, Lodish HF; Revealing the conserved functions of microRNAs in neurogenesis (20minute oral presentation); The third Minisymposium on Small RNA (Vienna, May 2008) 6. Le TNM, Xie H, Teh C, Zhou B, Chia PH, Udolph G, Korzh V, Yang H, Lim B, Lodish HF; The role of microRNAs in neurogenesis (30-minute oral presentation); a public scientific seminar as part of Singapore-MIT Alliance road show at National University of Vietnam (Hanoi, Jan 2008) 7. Le TNM, Xie H, Teh C, Zhou B, Chia PH, Udolph G, Korzh V, Yang H, Lim B, Lodish HF; The role of microRNAs in neurogenesis (20-minute oral presentation); Computation and Systems Biology session at the 5th International Symposium on Nanomanufacturing (Singapore, Jan 2008) 8. Le TNM, Xie H, Zhou B, Lim B, Lodish HF; The role of microRNAs in neuronal differentiation (poster); the 5th Annual Meeting of the International Society for Stem Cell Research (Cairns, June 2007) 9. Le TNM, Xie H, Zhou B, Lim B, Lodish HF; Post-transcriptional mechanism modulating differentiation of SH-SY5Y cells (15-minute oral presentation); Singapore-MIT Alliance Annual Symposium (Singapore, Jan 2007) 10. Le TNM, Xie H, Zhou B, Lim B, Lodish HF; Post-transcriptional mechanism modulating differentiation of SH-SY5Y cells (45-minute oral presentation); 129 Graduate Student Seminar Series at Genome Institute of Singapore (Singapore, Nov 2006) 11. Le TNM, Xie H, Zhou B, Lim B, Lodish HF; Post-transcriptional mechanism modulating differentiation of SH-SY5Y cells (20-minute oral presentation); Computation and Systems Biology Minisymposium at MIT (Cambridge, May 2006) 12. Le TNM, Reza MA, Swarup S, Kini RM; Two parallel prothrombin activator systems in Pseudonaja textilis snake (poster); The International Society on Thrombosis and Haemostasis XXth Congress (Sydney, Aug 2005) 13. Le TNM, Reza MA, Swarup S, Kini RM; Two parallel prothrombin activator systems in Pseudonaja textilis snake (poster); The Third International Conference on Structural Biology and Functional Genomics (Singapore, Dec 2004) 14. Le TNM, Reza MA, Swarup S, Kini RM; Structural comparison between the nonenzymatic subunit of Pseutarin C and liver factor V in Pseudonaja textilis snake (poster); The Tenth National Undergraduate Research Opportunities Programme Congress (Singapore, Oct 2004) 15. Le TNM, Reza MA, Swarup S, Kini RM; Cloning and characterization of blood coagulation factor V cDNA from eastern brown snake (poster); The Eight Biological Sciences Graduate Congress (Singapore, Dec 2003) 16. Le TNM, Reza MA, Swarup S, Kini RM; Structural comparison between the nonenzymatic subunit of Pseutarin C and liver factor V in Pseudonaja textilis snake (poster); The Fourth Sino-Singapore Conference on Biotechnology (Singapore, Nov 2003) 130 TEACHING EXPERIENCES 1. Teaching assistant at the General Biology course at National University of Singapore (Mar 2009): guided undergraduate students at lab sessions 2. Teaching assistant at the Computation and Systems Biology boothcamp (Jul 2009): presented a 3-hour lecture on RNA interference to first year PhD students and assisted Prof. Harvey Lodish during his lectures OTHER EXPERIENCES 1. Editor-in-chief of The Mudskipper – Biological Science Society, National University of Singapore (2002-2004): coordinated the editorial board of 18 people (including editing, design, marketing, publicity and distribution teams) to produce a print and online magazine for Life Science students 2. Chairperson and speaker at the first career workshop for Vietnamese students in Life Sciences (Singapore, Aug 2008) 3. Assistant secretary at the second International Conference on Structural Biology and Functional Genomics (Singapore, Dec 2002) 4. Coordinator of the book “Molecular Cell Biology” (Lodish et al.) translation project: bringing together the publishers, translators, authors and sponsors to translate the book into Vietnamese and publish it in Vietnam (2007-present) 5. Coordinator of the Computation and Systems Biology journal club at SingaporeMIT Alliance (2005-2006) 6. Team leader of class 55A2 at the student talent contest, Hanoi College of Pharmacy, received the first team prize (Hanoi, 2000) 131 Appendix – Classification of miR-125b targets by functions Genes repressed by 2-day miR-125b overexpression and predicted by TargetScan were classified by their functions using Ingenuity Pathway analysis. Category Cellular Growth and Proliferation Cell Death Cell Morphology Cellular Assembly and Organization Lipid Metabolism Nucleic Acid Metabolism Small Molecule Biochemistry Molecular Transport Cell Cycle Cellular Movement Molecules GAB2, TP53INP1, EPOR, MAPK3, TNFRSF10B, APAF1, FANCC, DLC1, BAK1, PTPRO, CXCL12, RAG1, AKT3, RET, EIF5A2 EME1, SARM1, GAB2, MAPK3, BCL2L12, SGPL1, PPP1R9B, PTPRO, AKT3, BCL2L2, PHF17, SORT1, RET, E2F2, ARC, PRKRA, TP53INP1, MAPKAP1, GORASP1, CDC25C, EPOR, TNFRSF10B, APAF1, THY1, MDC1, FANCC, DLC1, KCNH2, BAK1, BMF, CXCL12, BAP1, RAG1, RPS6KA1 TNFRSF10B, APAF1, KCNH2, BAK1, GGA2, CXCL12, PIP5KL1, CDC42SE1, RPS6KA1, RET, PPP1CA, E2F2, PVRL2 SUV39H1, TNFRSF10B, APAF1, BAK1, RAB22A, ZFYVE1, NIN, PPP1R9B, PTPRO, CXCL12, PIP5KL1, STX3, RET, CDC42EP4 ZFYVE1, SGPL1, ACACB, ST6GALNAC6, CXCL12, ESRRA, H6PD, DGAT1, MLYCD, ST8SIA3 ACACB, TDG, CXCL12, NT5M, APAF1, H6PD, MLYCD, RGS12 ST6GALNAC6, MAPK3, SUV39H1, NFKBIE, ESRRA, RGS12, ZFYVE1, CHM, SGPL1, PTPRO, AKT3, SLC25A15, PPP1CA, RET, PRKRA, CDC25C, ACACB, TDG, APAF1, H6PD, MKNK2, EPM2A (includes EG:7957), CXCL12, NT5M, SIK1, DGAT1, MLYCD, RPS6KA1, ST8SIA3 ACACB, NFKBIE, ESRRA, H6PD, SLC8A2, RGS12, BAK1, EPM2A (includes EG:7957), CXCL12, AKT3, SLC25A15, DGAT1, MLYCD, PPP1CA EME1, GAB2, SH3BP4, PHC2, CCNC, MAPK3, POLE, PPP1R9B, PTPRO, RET, PPP1CA, E2F2, GORASP1, CDC25C, TP53INP1, ACACB, EPOR, APAF1, MDC1, FANCC, BAK1, CXCL12, BAP1, SIK1, HINFP GAB2, CXCL12, DLC1, RET [...]... processing of all pre-mir - 125 b isoforms Injection of each lp 125 bMO individually resulted in an incomplete knockdown of miR - 125 b (Fig 10b) and led to a mild neural cell death In particular, the level of mature miR - 125 b was reduced more by lp 125 bMO1 and lp 125 bMO2 than by lp 125 bMO3, probably due to the lower expression of pre-mir - 125 b- 3 in the embryos as well as a cross binding of lp 125 bMO1 and lp 125 bMO2 to... misMO in p53M214K mutant misMO + p53MO misMO * * 100%, n = 105 * m 125 bMO in p53M214K mutant 92% , n = 111 * 95%, n = 85 m 125 bMO + p53MO m 125 bMO * * 98%, n = 1 12 100%, n = 114 * lp 125 bMO1 /2/ 3 in p53M214K mutant 100%, n = 113 * lp 125 bMO1 /2/ 3 + p53MO lp 125 bMO1 /2/ 3 Figure 20 - Rescue of miR - 125 b morphants by the loss of p53: The phenotype of miR - 125 b morphants were reversed by co-injection of miR - 125 b morpholinos... Figure 21 - Synthetic miR - 125 b rescues apoptosis in miR - 125 b morphants (a) TUNEL assay for detecting apoptotic cells in the 24 -hpf brains: embryos were injected with a standard negative control morpholino, miR - 125 b duplex ( 125 b- DP), m 125 bMO or lp 125 bMO1 /2/ 3 Two different concentrations of 125 b- DP ( 12. 5 fmole and 37.5 fmole per injection) were used to rescue the embryos injected with lp 125 bMO1 /2/ 3 Each... knockdown of miR - 125 b by either m 125 bMO or lp 125 bMOs resulted in the same phenotype: upregulation of p53 protein and increase in neural cell death at 24 hpf (Fig 10c and 18a-d) In fact, the severity of the phenotype was dependent on the dose and the efficacy of the morpholinos For m 125 bMO or for the combination of lp 125 bMO1 /2/ 3, injection of 0 .25 pmole morpholino had no effect; 0.5 pmole morpholino induced... change in expression c 2 5 4 3 2 1 0 Mock 1 .2 125 b- DP 1 0.8 0.6 ** ** ** 0.4 0 .2 0 bax p53 125 b- DP 1.4 p21 b Fold change in expression 6 Mock Log2 fold change in miR - 125 b level a SH-SY5Y NC-DP1 SH-SY5Y 125 b- DP 3T3 Mock 1.5 3T3 125 b- DP 1 * ** 0.5 * * ** ** ** ** ** ** ** PPP2CA PLK3 PLAGL1 BAK1 PRKRA TP53INP1 PPP1CA 0 Figure 23 – miR - 125 b function in the mouse Swiss-3T3 cells (a) The level of miR - 125 b in. .. with co-injection of synthetic miR - 125 b duplex into lp 125 bMO1 /2/ 3 morphants Indeed, the number of apoptotic cells in the morphants was reduced significantly by miR - 125 b duplex in a dose-dependent manner (Fig 21 a) The efficiency of rescue was quantified by the number of embryos with visible dead cells in the live brain at 24 hpf Both m 125 bMO and lp 125 bMO1 /2/ 3 injection caused more than 90% of embryos... anticipated, the level of p53 protein in the treated embryos was reduced significantly by miR - 125 b duplex (Fig 22 a) Staining of apoptotic cells in the embryonic brain further demonstrated that the severe apoptosis induced by gamma-irradiation or camptothecin was rescued significantly by the injection of miR - 125 b duplex (Fig 22 c) In fact, the rescue effects of miR - 125 b duplex during the DNA damage response... of miR - 125 b during the early development of zebrafish 90 Figure 18 - Loss of miR - 125 b elevates p53 and triggers p53-dependent apoptosis in zebrafish embryos (a) Elevation of p53 protein caused by loss of miR - 125 b in zebrafish embryos: embryos were injected with misMO, m 125 bMO or lp 125 bMO1 /2/ 3 Western blot was performed at 24 hpf (b) The p53 protein level was quantified from the Western blot bands in. .. 22 a) Interestingly, both treatments resulted in a significant drop in miR - 125 b expression (Fig 22 b), suggesting that the downregulation of miR - 125 b allows a smooth upregulation of p53 in this stress response pathway To test whether an ectopic expression of miR - 125 b can reduce the extent of the DNA damage stress response, we exposed miR - 125 b- duplex-injected embryos to gammairradiation or camptothecin... whereas co-injection of the miR - 125 b duplex reduced this number to only 6% (Table 6) This rescue of apoptosis corresponds to the reduction in p53 protein by miR - 125 b duplex (Fig 21 bc) Importantly, since this duplex does not bind to the lp 125 bMOs, rescue of the lp 125 bMO1 /2/ 3 morphants demonstrates that synthetic miR - 125 b duplex produced mature miR - 125 b that replenished the endogenous miR - 125 b to repress . p 53MO m 125 bMO + p 53MO l p 125 bMO1 /2/ 3 + p 53MO * * misMO in p 53 M214K mutant * * m 125 bMO in p 53 M214K mutant l p 125 bMO1 /2/ 3 in p 53 M214K mutant * m 125 bMO misMO l p 125 bMO1 /2/ 3 . reduction in p53 protein by miR - 125 b duplex (Fig. 21 b- c). Importantly, since this duplex does not bind to the lp 125 bMOs, rescue of the lp 125 bMO1 /2/ 3 morphants demonstrates that synthetic miR - 125 b. assay for detecting apoptotic cells in the 24 -hpf brains: embryos were injected with a standard negative control morpholino, miR - 125 b duplex ( 125 b- DP), m 125 bMO or lp 125 bMO1 /2/ 3. Two different