This action might not be possible to undo. Are you sure you want to continue?
MiR-125b in Leukemia
Abstract Introduction Materials and Methods Results and Discussion Differentiation blockage and apoptosis inhibition of 32DCl3 and HL60 cell lines by miR-‐125b overexpression Apoptosis suppression by miR-‐125b in HL60 and 32DCl3 Binding sites cloning into psicheck2 vector Generation of binding site mutants Validation of putative targets involved in cell proliferation, apoptosis and cell signaling DUSP6 and ETS1 MAP4K2 PPP2R1B Validation of putative targets involved in myeloid differentiation SP1 CBFB General discussion and conclusion Acknowledgement References Appendix 3 4 7 15 16 18 19 20 23 27 29 32 35 37 38 39
MiR-125b in Leukemia
ABSTRACT MiR-‐125b was shown to be overexpressed in leukemic patients carrying the chromosomal translocation t(2;11)(p21;q23), which resulted in blocked differentiation and apoptosis of myeloid progenitors in vitro. This was hypothesized to be the cause of leukemia in these patients. The mechanism underlying this phenomenon remains to be elucidated. The aim of this study is to determine the direct targets of miR-‐125b which lead to differentiation and apoptosis blockage. In my first internship, six putative targets were selected based on mRNA-‐sequencing, RQ-‐PCR and bioinformatic approaches, including DUSP6, ETS1, MAP4K2, PPP2R1B, SP1 and CBFB. To validate these gene candidates as real targets of miR-‐125b, the binding sites were inserted into 3’UTR of luciferase reporter gene and then co-‐transfected with miR-‐125b into 293T cells. Mutations of these binding sites were created and used as controls. In a further step, miR-‐125b was overexpressed in three different cells lines, namely NB4, HL60 (human cell lines) and 32DCl3 (mouse cell line) which could be induced to differentiate or to die in program. Protein levels of the putative targets were examined before and after induction according to physiological functions of the genes. The results revealed that CBFB is most likely a direct target of miR-‐125b. Since CBFB forms complexes with Runx protein family, which is essential for maturation of hematopoietic cells, the down-‐regulation of it by miR-‐125b could explain the differentiation blockage observed in vitro.
MiR-125b in Leukemia
INTRODUCTION MicroRNAs modulate a variety of cellular pathways, including development, differentiation, cell proliferation and apoptosis (Bartel 2004; Bartel 2009) and dysregulation of miRNA expression underlies specific oncogenic events in human cancer (Calin and Croce 2006; Esquela-‐Kerscher and Slack 2006; Garzon, Fabbri et al. 2006; Calin, Liu et al. 2007) In a previous study, Bousquet M. et al. showed that a chromosomal translocation t(2;11)(p21;q23) in 19 patients suffering from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) caused a six to ninety fold up-‐regulation of miR-‐125b compared to healthy individuals and leukemic patients lacking the translocation t(2;11)(p21;q23) (Bousquet, Quelen et al. 2008). The gene for miR-‐125b is located on chromosome 11 (miR-‐125b-‐1 locus), but it is unclear how the t(2;11)(p21;q23) translocation leads to miR-‐125b overexpression. When miR-‐ 125b was transiently transfected into the human promyelocytic leukemia cell lines NB4 and HL60 it blocked the differentiation of these cell lines upon chemical treatment. It is noted that NB4 cells become mature granulocytes and HL60 undergoes monocytic maturation after treatment with ATRA and DMSO, respectively. They were also observed to have endogenous miR-‐125b up-‐ regulated at the very end of cell maturation. Transient transfection of miR-‐125b resulted in maturation arrest, shown by a reduction in the markers of terminal differentiated myeloid cells (CD11b, CD14 and CD15) and by morphological observation(Bousquet, Quelen et al. 2008). Recently, miR-‐125b was shown to inhibit apoptosis in NB4 cells upon induction of apoptosis with camptothecin (unpublished data, Bousquet M. et al.). These properties may account for the differentiation and apoptosis blockage in leukemic cells in vivo by miR-‐125b(Bousquet et al. 2008). Moreover, miR-‐125b was found to have oncogenic functions in prostate cancer where it was found to suppress bak1 and induce androgen-‐independent growth of the cancer cells (Shi, Xue et al. 2007). It was also described as a negative regulator of pro-‐apoptotic gene p53 and BMPR1B associated with breast cancer pathogenesis (Le, Teh et al. 2009; Saetrom, Biesinger et al. 2009). To search for targets of miR-‐125b involved in leukemia (AML and MDS), in the first internship I used mRNA sequencing (next generation sequencing) and RQ-‐PCR, together with integrative bioinformatics approach to screen for putative target candidates. To this end, I used the promyelocytic cell line, NB4, transfected with miR-‐125b mimic. Six genes, namely DUSP6, ETS1, MAP4K2, PPP2R1B, SP1 and CBFB were found to be down-‐regulated by miR-‐125b. Among them, DUSP6, ETS1, PPP2R1B have been demonstrated to play roles in cell growth control and apoptosis and were reported to be down-‐regulated in several types of cancer (Furukawa, Sunamura et al. 2003; Tamaki, Goi et al. 2004; Esplin, Ramos et al. 2006; Okudela, Yazawa et al. 2009). SP1 and CBFB are both key players in hematopoiesis, particularly in myeloid maturation (Clarke and Gordon 1998; Hauses, Tonjes et al. 1998; Kundu and Liu 2003). SP1, a universal transcription
that targeting has no biological significance and hence is not worth being taken into account in attempt of deciphering the phenotypes caused by miR-‐125b overexpression. the detected changes in mRNA level do not always and necessarily lead to changes in protein level. For this purpose. The verification of the function of miR-‐125 by this assay indicates the potential of being a direct target of the gene but does not guarantee its function in vivo. 1998). that performs the ultimate function of a gene and second.D. western blot was finally used to quantitate and compare the effect of miR-‐125b on the protein level of the putative targets. which were found in the primary study. NB4 cell line was purchased from DSMZ. To achieve this goal. 32DCl3 and HL60ER stably infected with vector XZ or XZmiR-‐125b were gifts from Marina Bousquet. This internship was aimed at validating the six putative target genes of miR-‐125b. Interestingly. they could explain the myeloid differentiation inhibition and apoptosis suppression observed in NB4 cell line. 32DCl3ER and HL60ER were generous gifts from Dr. Nguyen MiR-125b in Leukemia factor. Therefore. It is important to validate a real target of a miRNA at the protein level for two reasons: first. CBFB does not function by direct interaction with DNA but forms complexes with other DNA-‐binding transcription factors. it is the protein. Runx proteins. besides the NB4 cell line. in which the target sequences were inserted into the 3’UTR of luciferase reporter gene which was then co-‐transfected with miR-‐125b. however. respectively. Tonjes et al. not mRNA. Wei Tong and Dr. regulates many genes. The results revealed that CBFB was the most likely to be direct targets. penicillin and streptomycin. MATERIALS AND METHODS Cell lines and pre-‐microRNAs. NB4 cells were cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum. HL60 (human cell line) and 32DCl3 (mouse cell line).T. our data indicated significant decreases in mRNA levels of these two SP1 down-‐ stream targets. Brad Fletcher. L-‐glutamine. 293T cell line was purchased from ATCC. such as HCK and CD11b which are essential for myeloid differentiation. were infected with retroviral vectors expressing miR-‐125b. Cell culture and transfection. in which overexpression of miR-‐125b was obtained by transient transfection with mimics. In fact. the target binding sites of miR-‐125 in the mRNAs of these genes were tested in an artificial system.T. two other promyelocytic cell lines. It was reported that a defect in CBFB could largely prevent definitive hematopoiesis and lead to lethality in embryogenesis. Transient transfection of 5 . MiR negative control #1 and miR-‐125b mimics were purchased from Dharmacon. there is a possibility that a decrease in mRNA level as a consequence of destabilization by miR-‐ 125b would not cause any change at the protein level. The expectation was that if these candidates are direct targets. through its binding to their promoter regions (Hauses.
the cells were analysed by FACS. 8 hours and 24 hours of induction.5g/L sodium bicarbonate. 10% IL3 (WEHI media) and 1% penicillin and streptomycin RPMI (GIBCO). day 2 and day 4 after IL3 removal. In this study. 32DCl3ER were grown in 10% fetal bovine serum. 4 hours.T. Nguyen MiR-125b in Leukemia miR negative control #1 and miR-‐125b (22. Constructing 3’UTR gene fragments flanking binding sites of miR-‐125b 6 . 2 and 4 days of induction. The rest of the induced culture was kept until day 5 for FACS and morphological analysis. these two cell lines carry vectors (XZ) containing miR-‐125b gene and GFP as a reporter for selection.D. After 24h. HL60ER were cultured in IMDM (GIBCO) supplemented with 1.5uL of each mimic at the concentration of 50uM) into NB4 cells (3 x 106) were performed by electroporation at 200V and 950uF. 5µl of Annexin V and 5µl of 7-‐AAD were added to 100µl of cells and incubated for 15min in the dark. The cells were harvested for protein quantification after 1. 10% fetal bovine serum. using Pulser (BioRad). the transfected cells were induced to differentiate into granulocytes by addition of ATRA at the final concentration of 10uM for 5 days. apoptosis was triggered by removing IL3 from the medium. The cells were harvested at four time points after 2 hours. Apoptosis assay was performed using the Annexin V-‐PE apoptosis detection kit (BD Biosciences) according to the manufacturer’s instructions. Camptothecin was added into the growing culture of HL60ER XZ and XZmiR-‐125b at the density of 3x105cell/mL with the final concentration of 10µM.T. cells were washed twice with PBS and resuspended at 1x106cell/mL in 1X Binding Buffer. HL60ER and 32DCl3ER are cell lines expressing murine ecotropic receptor. the cells were washed three times in IL3 lacking medium and then resuspended at 3x105 cell/mL in the same medium plus G-‐CSF at the final concentration of 100ng/ml. Induction of differentiation in 32DCl3ER To differentiate 32DCl3ER into myeloid cells. After adding 400µl of 1X binding buffer. The cells were harvested at day 1. which allows a stable gene transfer into the cells with murine retroviral vectors. Induction of apoptosis in HL60ER and 32DCl3ER To induce apoptosis in HL60ER. The cells were washed three times with the medium without IL3 and then resuspended in the same medium at 5x105 cell/mL. Briefly. For 32DER.
Using primers listed in the table below.T.1 R M4K2. To confirm the presence of the inserts.2 R PPP2R1B F PPP2R1B R Oligonucleotide sequence (5’-‐>3’) taaCTCGAGATTTGCAGCATGCTTGACTTTACCA ataGCGGCCGCAGCTCTAAACTTTACACCACGAACA atgcCTCGAGgaagcaaagatggacttcagtg gcatGCGGCCGCGCCCTGGTTCTACTCTTACCCA taaCTCGAGACTCTGTACTCTGCCCTAGATTGT ataGCGGCCGCGACTTAGCACTGTTTTCCACTCTG atgcCTCGAGTTCAGGTTTTCAGGTTGCCCAT gcatGCGGCCGCCTGCAGCTTCCAGTATTCACAC taaCTCGAGAATGACCGCTTGTGGATCTGCA ataGCGGCCGCCGTCACATAGCTCATTGTAGCC taaCTCGAGAACTGCATGAGGATACGCTGGA ataGCGGCCGCTCAGCAGCAGAAACTTCTGCAG atgcCTCGAGACTCTTCGGGAGACATGTTGAG gcatGCGGCCGCTGGCCAAGTTATGAGCCAAAGC 7 . Hluc+ is firely luciferase gene. which plays a role as an internal control of the assay. Psicheck-‐2 map HRluc encodes the Renilla luciferase. Table 1.D. coli DH5α was transformed with the constructs and plated onto semi-‐solid medium containing Ampicillin for selection of the plasmids containing the wanted inserts. Primers for cloning binding sites. Nguyen a MiR-125b in Leukemia Figure 1. fragments surrounding the binding sites of the corresponding genes were amplified by PCR.1 F M4K2. The multicloning sites.2 F SP1. followed by DNA sequencing. are within 3’UTR of hRluc. The recognition sites of XhoI and NotI are in red Primer name DUSP6 F DUSP6 R ETS1 F ETS1 R CBFB F CBFB R SP1. The PCR products were cloned into the reporter vector psicheck2 (Promega) using XhoI and NotI (at its 5’ end and 3’ end respectively).T.2 F M4K2. E. selected plasmids were digested with XhoI and NotI.2 R M4K2. including several restriction sites.
then 22 cycles of 95 oC for 30s. 53 oC for 1 min and 66 oC for 8 min.com/qcprimerdesign (Table 2 . The principle of site-‐directed mutagenesis is illustrated in Figure 2. coli DH5α. it was chilled down to 4 oC before adding DpnI which digested the non-‐mutated parental DNA templates. designed with the support from http://www. The mixture were run in thermal cycler (BioRad) at 95oC for 30s. 8 .T.0 µL of primers (10µM.5U/ µL.0 µL dNTPs mixture (10mM each) and PfuTurbo DNA polymerase at the concentration of 2. 1. forward and reverse). Selected plasmids were then sequenced to verify the mutations.stratagen. After. First mutagenesis PCR reaction was performed with 50 ng of plasmid. 1. Nguyen MiR-125b in Leukemia TP53INP1 f TP53INP1 R ZNF148 F ZNF148 R APAF1 F APAF1 R APC F APC R STAT3 F STAT3 R PPP1CA F PPP1CA R PPP2CA F PPP2CA R taaCTCGAGGTGTATCACATAATGCCTTGCCT ataGTTTAAACGCACTAATGGGTTAGTTACAGAC atgcCTCGAGTGGGTCTACATGCAAATGTGGT gcatGCGGCCGCTAGCACATTGGTACTCACTTCA atgcCTCGAGCATACCTTGTTGTACTGTTGGTA gcatGCGGCCGCTCATGAGGTCAAGAGATCGAGA atgcCTCGAGAGGCACTCTTGATGGTTAGGAA gcatGTTTAAACCCATCTCACCTCAAATACCAGA atgcCTCGAGTGGCCTTTTAGTGAGTAAGGCT gcatGCGGCCGCTTCCACAGAAACTCTGATCAGC taaCTCGAGCCAGATGATGGATTGATTGTACAG ataGCGGCCGCTGCGAGAATCCAGCTTTGACCT taaCTCGAGCAGTTTCTGGCATAGCGCTA ataGCGGCCGCAACACTGCAGTGCTCTTGTGCA Site-‐directed mutagenesis Constructs carrying binding sites were mutated at the 6-‐nucleotide sequence complementary to the seed sequence of miR-‐125b using primers with the desired mutation .T. The digestion was incubated overnight at 37 oC before being transformed into E.D.
T. Nguyen MiR-125b in Leukemia Figure 2.D. Overview of site-‐directed mutagenesis (source: Stratagene) 9 .T.
T. 1% L-‐Glutamin DMEM without Peni-‐ Streptomycin) were added.2 FM SP1. Oligonucleotide sequence (5’-‐>3’) gctaaacagtatattacctctgtataaaattctagtgtcacctcaaatgc gcatttgaggtgacactagaattttatacagaggtaatatactgtttagc cacaggctggcttctgttttattcatcgatttttttaaaaagtcaatcagaaa tttctgattgactttttaaaaaaatcgatgaataaaacagaagccagcctgtg Tggctcacagcctgtcacccacgtgactaccatcagttctgcc ggcagaactgatggtagtcacgtgggtgacaggctgtgagcca Cccacaccgtcttccttttccaactggcactc Gagtgccagttggaaaaggaagacggtgtggg Gggctccaccacctgcattccacgtggaagatccacagagacatc Gatgtctctgtggatcttccacgtggaatgcaggtggtggagccc Acaacgtgctgctgtcactccacgtgaaatccacgcacatctggg Cccagatgtgcgtggatttcacgtggagtgacagcagcacgttgt ctcatgtgaaaggggaatgtggaagccacgtgttaatgtagtggttgttttggtttt aaaaccaaaacaaccactacattaacacgtggcttccacattcccctttcacatgag 10 . Prior to the transferring. Dharmacon). Primers for generation of mutated binding sites. After 24h. when the cells reached a confluence of 60-‐70%. DEL: Deletion. mixture A was added to mixture B and incubated at room temperature for 20 min before they were transferred onto the plate containing cells. 1µL of 1µM mature microRNA (miR-‐125b or miRcontrol.1 Rm M4K2. M: Substitution Primer name DUSP6 F Del DUSP6 R del CBFB FM CBFB RM SP1.T.D. 293T cells were seeded into 96 well white plates at 1-‐2x104 cell/well. Nguyen MiR-125b in Leukemia Table 2.2 F dEL SP1. the old medium in the wells was aspirated by taping the plate onto a paper towel or by vacumm and 50µL of new medium (10% FBS.2 RM SP1. Non-‐inserted psicheck2 vector and miR-‐125b perfect match inserted vector were included into all of the experiments as the control of transfection and proper working of the mimics. Two mixtures were prepared for the transfection.2 Rm PPP2R1B FM1 PPP2R1B RM1 Luciferase assay One day before transfection. Mixture B included 25µL of OptiMEM. 10ng of constructs. The assay was performed in a triplicate for each construct and repeated at least two times independently.1 Fm M4K2.2 R dEL M4K2.2 Fm M4K2. they were used for co-‐ transfection. After 10 min. Mixture A contained 25µL of OptiMEM (GiBCO) and 1µL of Lipofectamin 2000 and was incubated at room temperature for 10 minutes.
the medium was discarded by inverting the plate. The mixture was incubated at 25oC for 10 minutes. which is an internal control of transfection efficiency. Nguyen MiR-125b in Leukemia Figure 3.T. Total RNA was isolated from miR-‐125b and miRcontrol transfected cells treated and untreated with ATRA. 0. was normalized with Firefly luciferase signal. 0. Reagent 2 (Renilla luciferase substrate: buffer as 1:100) was added after to measure Renilla luminescence. After 48h of co-‐transfection.2 ug random primers. 42 oC for 50 11 . The first strand cDNA synthesis was performed in a mixture of 20uL including 4ug RNA.T. Source: Promega.D. The mixture was shaken at RT for 10 min before measurement. shRNA is replaced by miRNA but the mechanism of action is similar. The cells were lysed by lysis buffer in the first reagent of the kit which also contained substrate for Firely luciferase reaction (internal control).5 mM dNTPs and 1µL Superscript II reverse transcriptase (Invitrogen). Renilla luciferase signal. Principle of Luciferase assay. Luminescence was measured using Dual-‐Glo Luciferase kit (Promega) and TECAN luminescence reader. which accounts for the effect of miR-‐125b on the 3’UTR of the Renilla gene. Note that in this case. Real time quantitative PCR.
D. Table 3. cDNA product solution was diluted 20 times with water and 5µL of the diluted solution was used for real-‐time PCR reaction with SYBR Master Mix (Applied Biosystems) and the following primers for genes of interest (designed by Diu Nguyen.T. Nguyen MiR-125b in Leukemia minutes and 70 oC for 15 minutes. purchased from Invitrogen). RQ-‐PCR primers Primer name Apaf1 hF Apaf1 hR Stat3 hF Stat3 hR Znf148 hF Znf148 hR Etv6 hF Etv6 hR Ppp1ca hF Ppp1ca hR Ppp1ca mF Ppp1ca mR Ppp2ca hF Ppp2ca hR Ppp2ca mF Ppp2ca mR TP53INP1 hF TP53INP1 hR Tp53inp1 mF Tp53inp1 mR MLN51 hF MLN51 hR Actine hF Actine hR 18s mF 18s mR Β-actin mF Β-actin mR Oligonucleotide sequence (5’-‐>3’) TTAGCTGCAGAAGCTGTTAGAG CAGTTTCATCAGAAGCCCAGAT AACATCTGCCTAGATCGGCTAG TTGCTGCAACTCCTCCAGTTTC TCCTCAAGGACTACAGTATGCA TGTAAGTCTACTGGCTCTCTGA TGAACCACATCATGGTCTCTGT GTCAGAAAGCAACTGATAGACG GTGCCAGCATCAACCGCATCT GCAGGCAGTTGAAGCAGTCAG CATCTGCAGAGCACATCAGGTT CATCATGGCACCAGCATTGTCA GGTGCCATGACCGGAATGTAG GAGGTGCTGGGTCAAACTGCA ACCAGCTGGTGATGGAGGGAT TTGCAGCTTGGTTACCACAACG CCACCCGTGGGACTGATGAAT GAGCAGCAAGAGCTGCAACATA AACTCAAGTGGTCCCAGAATGG GGGCGAAAACTCTTGGGTTGTT TAATCCCAGTTACCCTTATGCTCCA GTTATAGTAGGTCACTCCTCCATATACCTGT TCCCTGGAGAAGAGCTACGA AGGAAGGAAGGGTGGAAGAG GTAACCCGTTGAACCCCATT CCATCCAATCGGTAGTAGC GGCTGTATTCCCCTCCATCG CCAGTTGGTAACAATGCCATGT 12 .T. After that.
Infected 32DCl3 cells (1x105 ) at day 5 of GCSF induction were spun using the cytospin (SHANDON) at 500 rpm for 5 minutes on a glass slide and then dried for 20 minutes at room temperature. FACS analysis. The supernatant containing total protein of the cells was stored at -‐80oC after protein concentration being quantified. Morphological analysis.T. For western blotting. Cell lysis. The staining mixture was incubated at room temperature for 30 minutes in dark and then washed again in 2% FBS PBS to remove all of spare antibodies.T. The cell lysate was kept on ice for 30 min and centrifuged at 13. the slides were stained with Giemsa solution (Sigma-‐Aldrich) (1:20 solution in water) for 15 minutes and washed with water again. 0. Western Blots were probed with the following antibodies: GCK (MAP4K2) (Cell Signaling). 50 mM HEPES [pH 7. the stained cells were resuspended in 500 uL 2% FBS PBS and analysed using Fluorescence activated cell sorting FACS LSRII (BD Biosciences).0]. 50µg protein (in supernatant) was denatured by NuPAGE loading buffer (Invitrogen) at 70oC for 10 min. Nguyen MiR-125b in Leukemia Quantitative PCR was performed on 96 well plate using ABI 7600. SP1 (Santa Cruz) and CBFB (Abcam). The amplified product length ranges from 100 to 120 bp.1% Nonidet P-‐40.D. Cytospun cell slides were then stained with May-‐Grunwald solution (Sigma Aldrich) for 5 minutes and washed with water three times. Unstained and stained untransfected 32DCl3ER cells were used as a control.000 rpm for 10 min. Infected 32DCl3ER (5x105-‐1x106) cells after 5 days removal of IL3 and addition of GCSF were washed in 3 ml of 2% FBS PBS and then resuspended in 50 uL of 2% FBS PBS containing anti-‐mouse CD11b coupled with PE-‐Cy7 at ratio of 1:100. 5 mM EDTA) containing proteinase inhibitor cocktail (complete tablet-‐Roche). PPP2R1B (Abcam). Complete drying of the slides was followed before visualizing under microscope AxioCam MRc (Zeiss). protein quantification and Western Blot The cells were harvested and lysed by ELB buffer (250 mM NaCl. After that. 13 . After that.
thus the level of CD11b as they are conjugated. Note that as miR125b gene is incorporated into the genome of 32DCl3 cells as the result of retroviral vector infection the expression of miR-‐125b is constitutive and more physiological than in NB4 cells. the horizontal axis represents the intensity of chromophore PE (Comp-‐PE-‐A).T. A Figure 4.D. the result means there are more mature cells in 32DCl3ER XZ (red) than that in 32DCl3ER XZmiR-‐125b (green). which were transiently tranfected with microRNA mimics. transiently transfected into the cells. Nguyen MiR-125b in Leukemia RESULTS AND DISCUSSION Confirmation of differentiation blockage in 32DCl3 cells by miR-‐125b In the previous work. Histogram FACS analysis of CD11b (coupled with PE) expression on NB4 transfected with miR-‐control (red) and with miR-‐125b (green) (A) and changes in morphology of May-‐Grunwald and Giemsa stained cells at day 5 of induction of differentiation in 32DCl3ER XZmiR-‐125b (C) compared to 32DCl3ER XZ empty vector (B) 14 . et al.T. In this histogram. Confirmation of differentiation blockage of 32DCl3ER XZmiR-‐125b. In this study. demonstrated that the NB4 cell line’s ATRA-‐induced differentiation was suppressed by miR-‐125b. Bousquet M. Because CD11b is a marker of differentiated myeloid cells. The second peak of the red curve indicates a CD11b positive population in the 32DCl3ER XZ while there is no such peak in 32DCl3ER XZmiR-‐125b. Figure 4 shows FACS analysis on 32DCl3ER XZ and 32DCl3ER XZmiR-‐125b at day 5 after induction with G-‐CSF (as described in Materials and Methods). we confirmed that miR-‐125b had the same effect on the murine cell line 32DCl3.
A clearer picture of this can be seen in the case of 32DCl3ER cells which were stained with both Annexin V and 7AAD. respectively. NB4. which is necessary for DNA synthesis In IL3-‐dependent 32DCl3ER cells. This result supports our data of the previous work on the human cell line NB4 and hence strengthens the hypothesis that miR-‐125b overexpression blocks myeloid differentiation.T. HL60 and 32DCl3. we also addressed the question of whether miR-‐125b is a causative factor of leukemia or in other words. It is obvious that 32DCl3ER XZ (Figure 4B) is more differentiated than 32DCl3ER XZmiR-‐ 125b (Figure 4C). HL60ER and 32DCl3ER cells expressing miR-‐125b at stable and high levels were forced to undergo programmed cell death by adding Camptothecin and removing IL3 from their culture.T. To next examine whether miR-‐125b prevents apoptosis and promotes cell growth. miR-‐125b inhibits granulocytic maturation in the murine cell line 32DCl3. the removal of IL3 results in DNA fragmentation and hence cell death (Pommier. Taken together. indicating that the cells overexpressing miR-‐125b are more resistant to apoptosis than the control cells.D. In HL60ER. Nguyen MiR-125b in Leukemia B C Figure 4B and C show the results of cell staining (May-‐Grunwald and Giemsa) of infected cells at day 5 after induction. which distinguishes cell death by apoptosis (Annexin V positive) and necrosis (Annexin V 15 . Terminally differentiated myeloid cells are typically characterized by multilobulated and segmented shape of their nuclei and cytoplasmic granules (Sung. Gao et al. As can be seen in Figures 5A1-‐2 in two independent experiments the proportion of Annexin V negative cells in HL60ER XZmiR-‐125b is significantly greater than that in HL60XZ. which detect apoptosis and necrosis at an early stage. Apoptosis prevention by miR-‐125b in HL60 and 32DCl3 In our study. the cells were stained with only Annexin V or Annexin V and 7AAD (see Materials and Methods for details). In the case of miR-‐125b. the first evidence has been confirmed with three different cell lines. After 24h of induction for HL60ER and 4 days of induction for 32DCl3ER. A gene is considered to be an oncogene if when mutated or in increased expression it can cause (1) differentiation blockage (2) apoptosis suppression and/or (3) cell proliferation stimulation. Camptothecin promotes apoptosis by inhibiting Topoisomerase I. 1998). Pourquier et al. 2006). whether miR-‐125b is an oncogene.
81 % vs 27.15% and 60. Nguyen MiR-125b in Leukemia and 7AAD double positive). B1 Annexin V B2 A1 A2 B3 B4 16 .T.98% vs 30.D.T.36%). Figures 5B1-‐4 show that the percentage of double negative cells in 32DCl3ER XZmiR-‐125b (live cells) was more than twice as much as those in 32DCl3ER XZ (60.
B1-‐B4: FACS dot plots of two independent apoptosis assays on 32DCl3ER XZmiR-‐125b and 32DCl3ER XZ. The horizontal axes are Annexin V-‐PE intensity indicating apoptosis and the vertical ones are the number of cells counted. PPP2R1B. In the first internship.T.D. which allow testing whether a putative gene is a direct target of miR-‐125b. Nguyen MiR-125b in Leukemia Figure 5. respectively. M4K2-‐1/chk2 and M4K2-‐2/chk2 carry the first and the second binding site. Apoptosis suppression by miR-‐125b in HL60 (A1 and A2) and 32DCl3 (B1-‐B4) A1-‐A2: FACS histogram of two independent apoptosis assays on HL60ER XZ125b (Red) and HL60ER XZ (Green). SP1 and CBFB. The horizontal axes represent Annexin V-‐PE and the vertical axes. CBFB/chk2 and SP1. These genes are involved in cell proliferation. ETS1/chk2 and PPP2R1B/chk2 contain all three and two binding sites. ETS1. This resulted in the identification of six candidate genes. DUSP6/chk2. MAP4K2. 2010. namely DUSP6. followed by sequencing of the fragments. In the following sections. Binding sites cloning into the psicheck2 vector To be able to do luciferase assays. respectively in their 3’UTR. validation of these putative targets by luciferase assay and western blot will be presented and discussed. Kundu. apoptosis and myeloid differentiation (Okudela. B1 and B3 are duplicates of the cells infected with control retroviral vectors XZ. Fragments of the genes flanking the binding sites were cloned into 3’UTR of the Renilla gene in psicheck2 (chk2) vector (Promega) using the primers mentioned in Materials and Methods. B2 and B4 are duplicates of the cells infected with retroviral vector XZmiR-‐125b.T. Yang. they were stained with Annexin V coupled to PE. we first constructed vectors carrying a part of 3’UTR sequence of the putative targets containing binding sites for miR-‐125b. Hauses. 2003). 17 500 400 . Zhang. I used mRNA-‐seq and RT-‐PCR to find putative target genes of miR-‐125b.2/chk2 carry one miR-‐125b binding site in 3’UTR of the corresponding genes. 1998. Tamaki. Selected plasmids were confirmed to have the binding sites by digestion with two restriction enzymes whose restriction sequences are at the ends of each insertion. in the ORF of MAP4K2. Apoptosis was induced by adding Camptothecin (See Materials and Methods) and after 24h. 2004. 2009. 2003. 7AAD-‐PE-‐Cy-‐5-‐5 which indicates necrosis.
.ttccaactgg ||||||||||||||||||||||||||||||||||||| |||||||||| tttacccagaacagtaacaacccacaccgtcttccttcagggatttccaactgg SP1. Nguyen MiR-125b in Leukemia Figure 6. Generation of binding site mutants Deletion and/or substitution mutations of the binding sites were created through site-‐directed mutagenesis (see Materials and Methods) using primers bearing 2-‐4 mismatched nucleotides in the binding sites complementary to the seed sequence of miR-‐125b. PPP2R1B.. PPP2R1B XhoI/NotI.D...2 Mut tttacccagaacagtaacaacccacaccgtcttcctcttaagctttccaactg 18 . I was unsuccessful in making mutants of M4K2.T. Figure 7 shows the side-‐ by-‐side alignment between mutated and wild-‐type binding sites (Blat).2 Del tttacccagaacagtaacaacccacaccgtcttcctt. therefore the mutants were expected to abolish the effect of miR-‐125b in the cells.2 XhoI/NotI. In this study. Cloning miR-‐125b binding sites into the 3’UTR of luciferase gene in the psicheck2 vector..agtgtcac |||||||||||||||||||||||||||||||||||| |||||||| tgtgctaaacagtatattacctctgtataaaattcttcagggagtgtcac right: DUSP6 XhoI/NotI.. psicheck2 XhoI/NotI and DNA ladder 1Kb plus... M4K2-‐2 XhoI/NotI. SP1 and CBFB. DNA ladder 1Kb plus. DUSP6 Del tgtgctaaacagtatattacctctgtataaaattct.. SP1. M4K2-‐1 XhoI/NotI. due to difficulties around the binding sites.. Left to Lower bands in all the lanes in agarose gel (Figure 6). except empty psicheck2 vector (the band at lower than 100 bp in this lane probably is the fragment between two recognition sites of XhoI and NotI) indicate the inserted fragments with the expected sizes suggesting that all of the desired binding sites were cloned successfully. PPP2R1B Mut aagccacgtgttaatgtagtggttgttttggtttttatttgctcaattttgtc |||| | |||||||||||||||||||||||||||||||||||||||||||| aagctcagggttaatgtagtggttgttttggtttttatttgctcaattttgtc SP1. The purpose of generating mutated binding sites was to provide necessary and sufficient evidence that these sites are direct targets of miR-‐125b. CBFB XhoI/NotI .T. we attempted to create mutants of seven substitution or deletion mutants of five putative targets DUSP6. ETS1 XhoI/NotI.. M4K2. however.
The same was observed with ETS1 even though this gene was predicted to be a miR-‐125b target by Targetscan and has three conserved binding sites (Figures 8 and 9). Alignment of wild-‐type and mutated DNA sequences surrounding the putative miR-‐125b binding sites. DUSP6 (has one miR-‐125b binding site in its 3’UTR) and ETS1 (three binding sites in the 3’UTR) were shown to be down-‐regulated by miR-‐125b overexpression at mRNA levels in NB4 cell lines.4 0. apoptosis and cell signaling DUSP6 and ETS1 In the previous study.2010 ) However.. the insertion of the 3’UTR miR-‐125b binding site of DUSP6 in the Renilla gene in chk2 vector did not result in a decrease of Renilla luminescence.2 1 0. Therefore. Mut: substitution.T.6 0. ETS1 was demonstrated to regulate DUSP6 expression in ERK signaling pathway controlling cell growth (Zhang.2 0 Q-‐PCR mRNA-‐seq Q-‐PCR mRNA-‐seq miRctl miR125b A undiﬀerenqated diﬀerenqated 19 . it can be concluded that these two genes are not direct targets of miR-‐125b.T.8 0. Nguyen MiR-125b in Leukemia |||||||||||||||||||||||||||||||||||| | |||||||||| tttacccagaacagtaacaacccacaccgtcttccttcagggatttccaactg CBFB Mut aagtcacaggctggcttctgttttattcatcgatttttttaaaaagtcaa ||||||||||||||||||||||||||||| ||||||||||||||||||| aagtcacaggctggcttctgttttattcagggatttttttaaaaagtcaa Figure 7. Validation of putative targets involved in cell proliferation. In addition. Kobayashi et al.D. The decrease in DUSP6 mRNA level was also confirmed by RQ-‐PCR in HL60ER XZmir-‐125b compared to HL60ER XZ. Del: Deletion. The interpretation of this result is that miR-‐125b has no effect in the stability of DUSP6. The sequences complementary to seed sequence of mir-‐125b are highlighted in yellow. Relagve DUSP6 expression 1.
B.T. n = 2. Fold change in mRNA level (mRNA-‐seq and RQ-‐PCR data from my first internship) in transiently transfected NB4 cell line. Chk2 is empty psicheck2 vector and perfectM 125b is chk2 containing a sequence perfectly 20 . Data is represented with mean+/-‐SE.T. A. The bars represent means of relative expression measured from three independent experiments. DUSP6 is not a direct target of miR-‐125b.D. Luciferase assay of 3’UTR of DUSP6 carrying one binding site of miR-‐125b and mutated binding site. Nguyen MiR-125b in Leukemia B C Figure 8. The error bars indicate standard deviation of the means. Fold change in mRNA level (quantified by RQ-‐PCR) in stably miR-‐125b-‐expressing HL60ER. C.
6 0. ETS1 1.T. B 21 .2 0 Q-‐PCR mRNA-‐seq Q-‐PCR mRNA-‐seq miRctl miR125b A undiﬀerenqated diﬀerenqated Figure 9.T. Data represents means of relative luminescence units +/-‐ SE of two independent experiments (n=2). A.D. Nguyen MiR-125b in Leukemia complementary with miR-‐125b. ETS1 is not a direct target of miR-‐125b.8 0.4 Relagve ETS1 expression 1.2 1 0. The error bars indicate standard deviation of the means.4 0. Fold change in ETS1 mRNA level (mRNA-‐seq and RQ-‐PCR data from the first internship) in transiently transfected NB4 cell line. The bars represent means of relative expression measured from three independent experiments.
Oddly. In order to check whether or not MAP4K2 could be a direct target of miR-‐125b. It is hard to find a biological explanation for this result having in mind that both binding sites are conserved in human and mouse. HL60ER XZmiR-‐125b (human cell line) and 32DCl3ER XZmiR-‐125b (mouse cell line). It was observed to be decreased at mRNA level during differentiation of NB4 transfected with miR-‐125b mimics in our previous study. Our RQ-‐PCR results of this gene in stably miR-‐125b-‐overexpressed HL60ER at 0 and 2 hours after induction of apoptosis also show a decrease in the mRNA level. namely NB4 transfected with mimics. which plays an important role in signaling pathways and cell responses to stress. 2003). there was almost no significant and consistent change in MAP4K2 protein levels in transfected NB4 and HL60ER XZ125b compared to the controls.T. Data represents means of relative luminescence units +/-‐ SE of two independent experiments (n=2). This supports the hypothesis that MAP4K2. is modulated by miR-‐125b. Zhao et al. MAP4K2 MAP4K2 was reported to be up-‐regulated about three fold during normal NB4 cell maturation to granulocytes (Yang.D. carrying three miR-‐125b binding sites. there was a clear decrease of this protein in 32DCl3ER XZ125b compared to 32DCl3ER XZ cells (empty vector). Chk2 is psicheck2 without an insert and perfectM 125b is chk2 containing a sequence perfectly complementary to miR-‐125b. Nguyen MiR-125b in Leukemia B. we are unable to conclude whether or not MAP4K2 is a direct target of miR-‐125b. It can be seen from the Figure. 22 . Luciferase assay with the 3’UTR of ETS1. Because of a lack of consistent evidence at protein level. two binding sites in the ORF of the gene were inserted separately into psicheck2 and co-‐transfected with miR-‐125b mimics into 293T cells.T. Luciferase assays revealed that they both responded to miR-‐125b and that the first binding site (closer to 5’UTR) showed a great effect (luciferase activity decreased by more than 50% in comparison to the Chk control) than the second site (Figure 10C) We next examined the protein level changes due to miR-‐125b overexpression in different cell lines. The western blot results are shown in Figure 11.
T.D.8 0.2 1 0.2 0 Q-‐PCR mRNA-‐seq Q-‐PCR mRNA-‐seq miRctl miR125b A undiﬀerenqated diﬀerenqated B 23 .T.4 0.6 0. Nguyen Relagve MAP4K2 expression MiR-125b in Leukemia 1.
T. Data is represented with mean+/-‐SE. C. Nguyen MiR-125b in Leukemia C A. Luciferase assay of luciferase constructs containing two binding sites located in the ORF of MAP4K2 (M4K2-‐1 and M4K2-‐2). A. The error bars indicate standard deviation of the means. B. The bars represent means of relative expression measured from three independent experiments. Fold change in MAP4K2 mRNA level (quantified by RQ-‐PCR) in constantly miR-‐125b-‐expressing HL60ER before and during apoptosis induction. 24 . Figure 10.D.T. Fold change in MAP4K2 mRNA level (mRNA-‐seq and RQ-‐PCR data from the first internship) in transiently transfected NB4 cell line. n = 2. Chk2 is empty psicheck2 without an insert and perfectM 125b is chk2 containing a sequence perfectly complementary with the seed sequence of miR-‐125b. Data represents means of relative luminescence units +/-‐ SE of two independent experiments (n=2). Validation of MAP4K2 as a potential target of miR-‐125b.
MAP4K2 protein quantification A.T. The error bars represent the standard deviation of the means. 25 . HL60ER and 32DCl3ER. GAPDH was used as a loading control. Data was obtained using densitometry on blot films using PhotoShop CS4.D.5 0 NB4 -‐RA HL60 32D miR125b B Figure 11.0.5 2 ctl 1. B.T.5 1 0. Nguyen MiR-125b in Leukemia D2 of transfection D2 of transfection A 2. The bars are the means of density of the bands from two independent experiments. Relative fold change of MAP4K2 protein in miR-‐125b transfected/infected cells compared to controls. Western Blot of MAP4K2 in three cell lines NB4.
the degree of protein reduction did not match with the degree of mRNA level observed in NB4 and HL60ER. such as colorectal cancer. Relative PPP2R1B mRNA level in HL60ER XZ125b was also dramatically decreased by more than 50% and further dropped by approximately 70-‐80% (2h and 4h) compared to the controls after apoptosis was induced in the cells by Camptothecin. involved in cell cycle control and growth factor signaling. 2004. which was found to be down-‐regulated at mRNA level in NB4 cells after transfection with miR-‐125b. Esplin. indicating that PPP2R1B is potentially a direct target of miR-‐ 125b.T. This gene is a component of Serine/Threonine Protein phosphotase 2 (PP2A). Ramos et al. 2007). an important enzyme that down-‐ regulates the mitogen-‐activated protein kinase (MAPK) cascade. Apparently.T. the western blot in NB4 transfected with miR-‐125b mimics and HL60ER XZmir-‐125b during apoptosis (Figure 12D). Nguyen PPP2R1B MiR-125b in Leukemia The next target candidate to be validated at the protein level is PPP2R1B.D. Goi et al. Yeh. however. compared to the control. Hsieh et al. The effect of miR-‐125b was abolished when the highly conserved site (one of the two sites) was mutated (Figure 12C). lung cancer and breast cancer (Tamaki. that could not explain why a significant reduction in protein level was not detected in HL60ER XZ125b after induction of apoptosis when PPP2R1B mRNA dipped at 30-‐20%. It is necessary to repeat the same validation in another constantly miR-‐ 125b-‐expressed cell line. In the next step of validation. PPP2R1B gene encodes the beta isoform of the constant regulatory subunit A of PP2A and has been found to be mutated. Western Blot in HL60ER XZ125b at day 0 provided more evidence for PPP2R1B being a real target (Figure 12D). 26 . it could be that low transfection efficiency and unstable level of the mimics in the cells led to a small reduction (about 30%) of mRNA resulting in undetectable change of the protein. cervical cancer. inactivated or deleted in many types of cancers. for example 32DCl3. to be able to draw a satisfactory conclusion of whether PPP2R1B is a direct miR-‐125b target. However. Given that miR-‐125b overexpression in NB4 was achieved by transient transfection while in HL60ER it was obtained through infection. 2006. luciferase assay performed with a fragment found in the 3’UTR of the gene containing both miR-‐125b binding sites (7mer) showed a reduction of luciferase activity by about 55%.
D.2 1 0. Fold change in PPP2R1B mRNA level (mRNA-‐seq and RQ-‐PCR data from the first internship) in transiently transfected NB4 cell line. n = 2. Chk2 is empty psicheck2 without an insert and perfectM 125b is chk2 27 . Fold change in PPP2R1B mRNA level (quantified by RQ-‐PCR) in constantly miR-‐125b-‐expressing HL60ER before and during apoptosis induction.T.8 0.T.2 0 Q-‐PCR mRNA-‐seq Q-‐PCR mRNA-‐seq miRctl B undiﬀerenqated diﬀerenqated A C Figure 12. Data is represented with mean+/-‐SE .6 0. Validation of PPP2R1B as a potential target of miR-‐125b. B. Luciferase assay of 3’UTR of PPP2R1B with two binding sites and 3’UTR with one mutated binding site (PPP2R1B Mut1). C.4 0. Nguyen MiR-125b in Leukemia Relagve PPP2R1B expression 1. A.
namely CD11b and HCK.T. No reduction was observed in NB4 transfected with miR-‐125b mimics and HL60ER XZ125b after 4h of apoptosis induction. Nguyen MiR-125b in Leukemia containing a sequence perfectly complementary with 5’strand of miR-‐125b. there are several binding sites of miR-‐125b along the SP1 mRNA sequence.D. we observed the consequential down-‐regulation of down-‐stream SP1 targets. Note that these two genes are essential for cell differentiation. Second. Validation of putative targets involved in myeloid differentiation SP1 In my previous study. including 4 sites in the ORF (one site 8mer. First.T. one site 7mer and two sites 6mer) and 2 others in the 3’UTR (one site 7merA and one site 6mer). D2 of transfection D2 of transfection D. whose regulation of transcription is typically achieved through binding of SP1 to the promoter regions. Data represents means of relative luminescence units +/-‐ SE of two independent experiments (n=2). it was shown to be decreased at mRNA level by miR-‐125b overexpression in NB4 cells during RA-‐induced differentiation. SP1 was found to be a promising miR-‐125b target. 28 . PPP2R1B protein levels in HL60ER XZ125b compared to the control. Third.
luciferase expression was recovered significantly when the site was mutated or deleted (Figure 13B). Furthermore.2 0 Q-‐PCR mRNA-‐seq Q-‐PCR mRNA-‐seq miRctl miR125b undiﬀerenqated diﬀerenqated A 29 .6 0. 1. a fragment containing all binding sites in the ORF was cloned in the vector psicheck2. No change in the protein level of SP1 was observed in NB4 treated with RA while the reduction observed in 32DCl3ER XZ125b and HL60ER XZ125b was not significant.D. Luciferase assays on the ORF binding sites altogether and the second site in 3’UTR showed no effect (data not shown) whereas the first 3’UTR site.8 0.2 (7merA) responded strongly to miR-‐125b leading to a reduction of about 45% of luciferase activity (Figure 13B). the verification by Western Blotting did not support conclusively the results from mRNA quantification and luciferase assay (Figure 13C and D).2 Relagve SP1 expression 1 0. SP1. The two binding sites in 3’UTR were put into two separated clones. Nguyen MiR-125b in Leukemia To further examine the possibility that SP1 is a real target of miR125b and to indentify which of the 6 binding sites has the main response element of miR-‐125b. However.T.4 0. We therefore conclude that SP1 is not a direct target of miR-‐ 125b in promyelocytic cell lines.T.
4 1. Nguyen MiR-125b in Leukemia B C 1.D.6 0.2 0 NB4 +RA HL60 32D ctl miR125b D 30 .4 0.8 0.2 1 0.T.T.
CBFB Core binding factor beta is one of the two putative targets involved in myeloid differentiation. The protein levels were compared between control cells and miR-‐125b transfected/infected cells (NB4. The site in the ORF had no effect (data not shown) while the site in 3’UTR was clearly targeted by miR-‐125b. However. de Bruijn and Speck 2004). 31 . Consistent with the data from mRNA quantification. this does not match the mRNA data (Figure 15). Interestingly. Data represents means of relative luminescence units +/-‐ SE of two independent experiments (n=2). PicTar and MiRanda (Iorio. Chk2 is empty psicheck2 without an insert and perfectM 125b is chk2 containing a sequence perfectly complementary with 5’strand of miR-‐125b. 2000. there was an approximate 1.T. The explanation for this could not be that the gene becomes inactivated after RA addition because CBFB is demonstrated to be highly expressed in hematopoietic stem cells. CBFB protein quantity was reduced before induction in all three cell lines. Interestingly. 2005). TargetScan. 35% in HL60 at day 0 after infection and 25% in 32DCl3 at day 0 after infection (Figure 17A and B). which indicated no significant change after RA induction. The western blots strongly indicate that CBFB might indeed be a direct target of miR-‐125b. this enormous suppression was completely disabled when only two nucleotides in the MRE (mirna responsive element) were replaced (Figure 16A and B). Kundu. early staged progenitors of myeloid lineages and mature myeloid cells (Blake. Adya et al. The reduction was about 30% in NB4 at day 2 of transfection.D. Western Blot and Protein quantification by densitometry shows inconsistent and insignificant reduction in SP1 protein under overexpression of miR-‐125b. Nguyen MiR-125b in Leukemia Figure 13. C. Based on the mRNA-‐seq and RQ-‐PCR data. Ferracin et al.T. It was predicted by three independent miRNA target prediction softwares. the reduction was still observed when the cells (NB4 and 32DCl3) underwent differentiation (Figure 17A). and D. B. Fold change in SP1 mRNA level (mRNA-‐seq and RQ-‐PCR data from the first internship) in transiently transfected NB4 cell line. Chen et al. Luciferase assay with miR-‐125b binding site in 3’UTR of SP1 and its two mutations (a deletion and a substitution). A. The hypothesis therefore would be that miR-‐125b destabilizes CBFB mRNA (in both undifferentiated and differentiated cells).6 fold decrease in mRNA level of this gene in miR-‐125b transfected NB4 cells before induction of differentiation (Figure 15). We cloned two miR-‐125b binding sites of CBFB (one located in the ORF and another one in the 3’UTR) separately and co-‐transfected each clone with miR-‐125b into 293T cells. Validation of SP1 as a potential target of miR-‐125b. Kundu and Liu 2003. indicating that the MRE but not any other sequences within the 3’UTR cloned is sufficient to respond to the regulation of miR-‐125b. HL60 and 32DCl3) before and during differentiation. suppressing around 45% of luciferase activity (Figure 16A). 2002.
Nguyen MiR-125b in Leukemia subsequently leading to a decrease in protein levels.T.4 Relagve CBFB expression 1. mRNA levels of CBFB measured by mRNA-‐seq and Q-‐PCR before and after induction of differentiation (normalized to Actine and MLN51). We propose that CBFB is a direct target of miR-‐125b in promyelocytic cell lines. Q-‐PCR data: n=3. mRNA seq data: n=2.6 0.D. 1. the reduction in CBFB protein may explain the blockage of differentiation observed in NB4 and 32DCl3 cells.2 0 miRctl Q-‐PCR mRNA-‐seq Q-‐PCR mRNA-‐seq undiﬀerenqated diﬀerenqated Figure 15. A 32 .T. Data represents means +/-‐ standard error of the mean (SE) normalized to control.2 1 0.4 0. Because CBFB is essential for myeloid differentiation (as discussed in the first report).8 0.
Relative luciferase activity is presented as means +/-‐SE of n=2 independent experiments in relation to controls (n=1).D. Nguyen MiR-125b in Leukemia B Figure 16. Empty vector (chk2) and vector containing perfect match sequence with miR-‐125b (PerfectM 125b) are used as controls. (A) Dual-‐luciferase reporter assay of HEK293T cells co-‐transfected with Luc-‐CBFB3’UTR/chk2 or Luc-‐CBFB3’UTR-‐Mut and miR-‐125b or mircontrol mimics. D2 of transfection D2 of transfection D2 of transfection A 33 .T. (B) Alignment of wild-‐type CBFB3’UTR and mutated CBFB3’UTR with miR-‐125b. Replaced nucleotides are in red.T.
Nguyen MiR-125b in Leukemia Relagve CBFB expression to GAPDH 1.013 P= 0. 34 .11 P= 0.003 P= 0. SP1 and CBFB. HL60ER infected with XZ or XZmiR-‐125b at day 0 (triplicate). NB4 transfected with miR-‐125b at day 2 after differentiation induction with retinoid acid (RA). Data represents means of relative density of the bands on western blotting films +/-‐ SE of three independent independent experiments with NB4 (D2 of transfection) and HL60 (D0). Reduction in CBFB protein level under the effect of miR-‐125b was observed in three different cell lines .28 P= 0.11 ctl miR125b B Figure 17. Quelen et al. ETS1. 2008) and second.T. validating six putative targets of miR-‐125b which were found in the first internship: DUSP6.q23) (Bousquet. Statistical significance of the difference between the control and the experimental cells were performed using T-‐test with p-‐values on the top of the bars of each group.4 0.17 P= 0.D. NB4 at day 2 of transfection (triplicate). 32DCl3ER infected with XZ or XZmiR-‐125b at day 0.2 0 P= 0. first obtaining more in vitro evidence to support the hypothesis that miR-‐125b is the causative factor of leukemia (AML and MDS) which carry the chromosomal translocation t(2.2 1 0.T.8 0. day 1 and day 4 after induction of differentiation by GCSF.6 0. MAP4K2. and of two independent experiments with all other groups. (A) Data from Western Blot in three different cell lines. PPP2R1B. GENERAL DISCUSSION AND CONCLUSION We I achieved two goals of the project.(B) Protein quantification by densitometry (B).11)(p21.
The hurdle in accomplishing a physiological overexpression lies in the inability of the NB4 cell line to harbor a retroviral vector carrying miR-‐125b. the reduction of the messengers was not large enough to be significantly different from the control. (2009) it was shown to block apoptosis in this cell line as well. In this study. However. there is still a raising controversy of whether this is physiologically relevant. However. Note that the means of overexpressing miR-‐125b in these experiments was through the introduction of an immense amount of mature microRNA directly into the cells through electroporation. Yazawa et al.. of which mRNA data from NB4 indicated that about 40% of its total transcripts were degraded by miR-‐125b. Furthermore.T. the decrease was greater (approximate 45% at day 0) and became even more 35 .D. Kobayashi et al. The modification of the cell lines and the selection were done and will be described in detail by Bousquet M. DUSP6 has been demonstrated to be a member of the ERK signaling pathway which controls cell growth (Furukawa. when up-‐regulated in NB4 cells. Okudela. all miR-‐125b target sequences in the six gene candidates were tested in the luciferase reporter assay and protein quantification by immunoblotting. I propose that the decrease in mRNA levels of both DUSP6 and ETS1 could be the result of the regulation of some up-‐stream direct targets of miR-‐125b. MAP4K2. Sunamura et al. together with the evidence that mRNA levels of these two genes decreased by induction of the microRNA lead us to think that these genes were miR-‐125b direct targets (see Results and Discussion). However. 2010 (unpublished). Interestingly. by which it is possible to select cells expressing miR-‐125b. in unpublished data by Bousquet M. Later. All of this. HL60 and 32DCl3 into the ones bearing murine ecotopic receptors on their cellular membrane (namely HL60ER and 32DCl3ER). PPP2R1B and SP1 all passed the validation by the luciferase reporter assay. this may not be the case of PPP2R1B. Nguyen MiR-125b in Leukemia MiR-‐125b has been proven to possess typical features of an oncomir. 2009) and ETS1 involved in a variety of cellular processes including cell proliferation and apoptosis. therefore although the phenotypes were undoubted and impressive. Yazawa et al. 2003. which allow them to be infectable by murine retrovirus. 2009). a recent study revealed that ETS1 actually regulates the expression of DUSP6 (Zhang. It is possible that in the case of MAP4K2 and SP1. In HL60ER where miR-‐125b was expressed constantly. In the primary study. miR-‐125b prevented cells from developing into mature granulocytes. Our attempt to look for a system that stably expresses miR-‐125b in a more physiological degree led to the modification of two cell lines. Another advantage of this method is that the vectors also contain GFP. The next three putative targets. Okudela.T. . our results suggest that they are not real direct targets because we did not observe changes in the protein levels. they were predicted to be miR-‐125b targets with highly conserved binding sites. The data from FACS analysis and morphological observation on 32DCl3ER XZ125b and HL60ER XZ125b presented here strengthens our conclusion from the previous study that miR-‐125b inhibits maturation of myeloid progenitors and apoptosis. when it came to protein quantification in vivo the results were ambiguous.
D. Yagi et al. It is mostly the concentration of proteins and the interaction among them that are the true causative factors and therefore. It could be argued that there are other factors affecting translation. flow cytometry using chromophore-‐coupled antibodies. Kundu and Liu 2003) supports the notion that CBFB might be a direct target of miR-‐125b and along this way involved in the origin of leukemias characterized by a t(2. the correlation between our data and the physiological underlyings that have been demonstrated for CBFB (Sasaki. a small difference between the experimental and the control samples could not be detected. Nguyen MiR-125b in Leukemia dramatic when apoptosis was induced (about 65% after 2h and 75% after 4h of Camptothecin addition). 2001. 36 . Colangelo et al. Haga et al. Adya et al. a recent study has found an alternative way. 1996. Even though mRNA levels have been proven to be very useful in the diagnosis of diseases (e. Taken these points into account when considering CBFB as a real target of miR-‐125b. a 50% reduction of that protein would unlikely to have a biological effect.11)(p21. immune-‐ blotting has been the only choice used in the majority of the microrna targets research. There is another important point that needs to be discussed in microRNA target studies in general and this work in particular. Another explanation to all the results observed in these three putative targets is the technical limitations of western blot (nonspecificity of the antibodies or low sensitivity). Blake. the question is what magnitude of down-‐regulation in protein level can lead to a change in phenotype. To the best of our knowledge.T. but the quantification by western blot did not support this. In fact. In general. which seems to be more sensitive (Gururajan. if the detected down regulation in protein level is small but statistically significant. Such a tremendous reduction in mRNA was expected to result in a drop in the translation yield of PPP2R1B. On the other hand. for instance. it is necessary to generate the same phenotype of blockage of myeloid differentiation in the tested cell lines by siRNA in order to draw a firm conclusion. if only half of the normal level of some protein is enough for the cell to perform its role in a cell. protein quantification and identification still lags behind the advanced and high-‐throughput techniques used for mRNA level determination. Nevertheless. 2000. they are correlative not causative. a phenotype caused by overexpression of a microRNA could be the collective outcome of small changes in many of its targets. as the final step of determining true targets. Shigesada et al. the correlation between mRNA and protein abundance data was reported to be low or limited for all three genes (Greenbaum. 2003). This review also pointed out one reason which was that even with the significant development in the technologies used to measure protein quantity over the past few years. the cells are not in a high demand of the protein so only a small proportion of the synthesized mRNA are used to produce a basal level of PPP2R1B and therefore. Colangelo et al. Nevertheless.g.q23) translocation.2010). 2003).T. even a huge decrease of mRNA makes no difference. cancers). their quantities should be studied in much greater detail (Greenbaum. Huang. For example.
M. Haga. (2006). C. Whitehead Institute for helping with mRNA-‐seq data analysis. Jan Kooter for helpful lectures." Nat Rev Cancer 6(11): 857-‐ 66. Victor Yeh and Violeta Rayon for their proofreading this report and fruitful discussion. et al. (1998). "Comparing protein abundance and mRNA expression levels on a genomic scale. P." J Leukoc Biol 63(2): 153-‐68. (2000). 37 . Colangelo. (2009). Marina Bousquet for all the techniques teaching. Croce (2006). Quelen.. Esplin. (2001). "Myeloid-‐specific gene expression.. Shigesada." EMBO J 20(4): 723-‐33.. and F. G." Trends Mol Med 12(12): 580-‐7..q23) translocation. A. "MicroRNA 125b inhibition of B cell differentiation in germinal centers. G. daily supervising on the project. "Ultraconserved regions encoding ncRNAs are altered in human leukemias and carcinomas. R. E. D. Greenbaum." Cell 136(2): 215-‐33. and function. Garzon. S. "MicroRNAs: target recognition and regulatory functions." J Exp Med 205(11): 2499-‐506.. comments on the report and a great friendship. J.. "Myeloid cell differentiation arrest by miR-‐125b-‐1 in myelodysplastic syndrome and acute myeloid leukemia with the t(2. REFERENCES Bartel. "The transcription factor Sp1 regulates the myeloid-‐specific expression of the human hematopoietic cell kinase (HCK) gene through binding to two adjacent GC boxes within the HCK promoter-‐proximal region. D. mechanism. Furukawa." Blood 96(13): 4178-‐84. A.. (2007). Slack (2006). et al.. et al. et al. T. (2004). D. Bartel." Cancer Cell 12(3): 215-‐29. M.. (2006). R. I thank Sumeet Gupta and all the staff in Genomic Core facility. "The glycine 90 to aspartate alteration in the Abeta subunit of PP2A (PPP2R1B) associates with breast cancer and causes a deficit in protein function. Liu." Am J Pathol 162(6): 1807-‐15.D. Adya. C. I thank Dr. C.T. biogenesis." J Biol Chem 273(48): 31844-‐52. L. Blake. to Dr. G. "Oncomirs -‐ microRNAs with a role in cancer." Nat Rev Cancer 6(4): 259-‐69. Harvey Lodish for providing me with the stipend. "MicroRNA signatures in human cancers. M. Gordon (1998). Nguyen ACKNOWLEDGMENT MiR-125b in Leukemia I thank Nuffic (Netherlands organization for higher education) and Vrije Universiteit Amsterdam for the studentship. Sunamura. Ramos. Calin. Esquela-‐Kerscher. Calin. et al.11)(p21. and S. Gururajan." Cell 116(2): 281-‐97. P. general laboratory support and a great supervision. (2008). Fabbri. M. P. C. et al. M. et al. et al. My special thanks to Prof." Genes Chromosomes Cancer 45(2): 182-‐90. M. "Dimerization with PEBP2beta protects RUNX1/AML1 from ubiquitin-‐proteasome-‐mediated degradation. et al.." Genome Biol 4(9): 117. Bingbing Yuan and George Bell with their help on gene function and network analysis. advice and comments on the report.T. G. "Zebrafish homolog of the leukemia gene CBFB: its expression during embryogenesis and its relationship to scl and gata-‐1 in hematopoiesis. N. T. Bousquet. Tonjes. "MicroRNAs: genomics. "Potential tumor suppressive pathway involving DUSP6/MKP-‐3 in pancreatic cancer. K." Int Immunol 22(7): 583-‐92. R. D. Clarke. Hauses. I thank all the Lodish lab members for technical support. (2003). and C. Huang. et al. A. "MicroRNA expression and function in cancer. (2003).
"An androgen-‐regulated miRNA suppresses Bak1 expression and induces androgen-‐independent growth of prostate cancer cells. Saetrom. M. Z.. Pourquier. (2005). Okudela. "Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer. as shown in figure 18A-‐18C. Tamaki. Yazawa. APPENDIX Searching for other putative targets A number of other candidates chosen from both the list of mRNA-‐sequencing data and literature review were also tested by RQ-‐PCR and luciferase assays." Biochim Biophys Acta 1400(1-‐3): 83-‐105. Biesinger. Gao.. Y.T. Le.T. "MicroRNA-‐125b is a novel negative regulator of p53." J Mol Diagn 5(4): 212-‐21. et al. L. C.. V. (2009). T. H. 38 . M. (2004). (2009). Yeh. et al. "Mechanism of action of eukaryotic DNA topoisomerase I and drugs targeted to the enzyme. M. L. et al.D. (2006). Kundu. Ferracin. "Cbf beta is involved in maturation of all lineages of hematopoietic cells during embryogenesis except erythroid. and P. Y. H. J. "A risk variant in an miR-‐125b binding site in BMPR1B is associated with breast cancer pathogenesis. T. (2007). "Down-‐regulation of DUSP6 expression in lung cancer: its mechanism and potential role in carcinogenesis. Shi. Zhang DE. et al." Proc Natl Acad Sci U S A 104(50): 19983-‐ 8." Blood Cells Mol Dis 30(2): 164-‐9. Zhao. Chen HM." Am J Pathol 175(2): 867-‐81." Int J Gynecol Cancer 17(4): 868-‐71. "Mutation analysis of the tumor suppressor gene PPP2R1B in human cervical cancer.. Zhang. P. Tenen DG. et al. "Dual specificity phosphatase 6 (DUSP6) is an ETS-‐regulated negative feedback mediator of oncogenic ERK signaling in lung cancer cells. et al. K. P. (2009).. X. Teh. et al. Yang. K. T. Scheibe RJ." Proc Natl Acad Sci U S A 93(22): 12359-‐63. (1996)." Carcinogenesis 31(4): 577-‐86. B. S. "PPP2R1B gene alterations inhibit interaction of PP2A-‐Abeta and PP2A-‐C proteins in colorectal cancers. "Gene expression profiling during all-‐trans retinoic acid-‐induced cell differentiation of acute promyelocytic leukemia cells. Pommier. M... Sasaki. et al. (2003)..268(11):8230-‐9.” J Biol Chem.. 1993 Apr 15." Cancer Res 69(18): 7459-‐65. et al... et al. Pahl HL. Nguyen MiR-125b in Leukemia Iorio. see internship report 1." Oncol Rep 11(3): 655-‐9. (2007). "MicroRNA gene expression deregulation in human breast cancer. (1998). S. Y. Liu (2003)." Cancer Res 65(16): 7065-‐70. Goi. P. "Absence of fetal liver hematopoiesis in mice deficient in transcriptional coactivator core binding factor beta. (1993). “The Sp1 transcription factor binds the CD11b promoter specifically in myeloid cells in vivo and is essential for myeloid-‐specific promoter activity.. For mRNA sequencing data and biological pathways of these genes. L. et al." Genes Dev 23(7): 862-‐76." Nat Genet 38(11): 1323-‐8. M. et al. S. Sung. Kobayashi. Yagi. Y. L. Xue. Hsieh.
6 1. Nguyen MiR-125b in Leukemia 1. If the red x still appears. under miR-‐125b overexpression at different time points during apoptosis induction. under miR-‐125b overexpression.8 0. The data correspond to the mean from two independent experiments.8 1. The image cannot be displayed. which is considered as 1 or 100%.2 1 0. all involved in apoptosis.T. transfected or infected with miR-‐125b. measured by RQ-‐PCR in the infected HL60ER and 32DCl3ER. Figure 18B. Fold change of expression of three genes. and then open the ﬁle again. 39 . you may have to delete the image and then insert it again. Fold change of expression of four genes. STAT3 and ZNF148. PPP1CA.2 0 APAF1 ETV6 STAT3 ZNF148 HL60 miRctl HL60 miR125b NB4 miRctl NB4 miR125b Figure 18A.T. Your computer may not have enough memory to open the image. respectively.4 Relagve expression 1.4 0. The blue and green bars indicate the expression of corresponding genes in NB4 and HL60ER transfected or infected with miR-‐ control. respectively. Restart your computer. measured by RQ-‐PCR in transfected NB4 and infected HL60ER. or the image may have been corrupted.D.6 0. The red and violet bars represent the expression of corresponding genes in NB4 and HL60ER. ETV6. APAF1. PPP2CA and TP53INP1. The data correspond to the mean from at least two independent experiments.
T.T.D. Empty vector (chk2) and vector containing perfect match sequence with miR-‐125b (PerfectM 125b) are used as controls. thus. no result of luciferase assay is available for it here. 40 . The binding site of ETV6 was not successfully cloned into psicheck2. Nguyen MiR-125b in Leukemia Figure 18C. Luciferase activity is reduced significantly with the binding sites in most of the genes tested by RQ-‐PCR in figure 18A and B.
This action might not be possible to undo. Are you sure you want to continue?
We've moved you to where you read on your other device.
Get the full title to continue listening from where you left off, or restart the preview.