72
NC 181a
83
A
B
Figure 15. ETV6/RUNX1 expression in miRNA mimics transfected REH cells.
Expression of (A) ETV6/RUNX1 mRNA in REH cell (no transfection control), NC-transfected REH cells (miRNA transfection control), and 181a-transfected REH cells was determined by Taqman qRT-PCR, and (B) ETV6/RUNX1 fusion protein was analyzed by Western blot with anti-RUNX1 (left). Quantification was conducted by densitometric analysis (right). GAPDH and anti-β-actin were used as internal controls for mRNA and protein expression, respectively. Bars represent the mean ± SD of three independent experiments. * P ≤ 0.05 (ANOVA).
181a NC
β-actin
kDa
100
E/R
REH
84
A
B
C
85
Figure 16. miR-181a targets the 3ʹ-UTR of RUNX and ETV6/RUNX1.
(A) The putative miR-181a binding site in the RUNX1 3ʹ UTR, both wild type and mutated version. (B) The last 678 bp of the human RUNX1 3' UTR containing normal (WT) or mutated (mut) miR-181a targeting sequences were cloned and inserted to the downstream of a pGL3-SV40Fluc vector in which a Firefly reporter gene (FLuc) was driven by a SV40 promotor (SV40). A Bgl II restriction enzyme site was added into the mutated version of miR-181a targeting sequence for easy recognition by restriction enzyme mapping. (C) pGL3-SV40FLuc-181WT/mut and pRLuc-TK (a Renilla luciferase control reporter vector used as a calibration control for transfection efficiency) were transfected into 293FT cells with expression vectors for miR-181a (181a) or negative control shRNA (NC). Luciferase activity was adjusted by FLuc/RLuc.
Bars represent the mean ± SD of three independent experiments. ** P ≤ 0.01 (ANOVA).
86
A B
Figure 17. Stable expression of MIR181A1 in REH cells via lentiviral transduction.
REH cells were infected with lentiviral vector expressing the negative control shRNA (NC) or miR-181a (181A1). Infected cells were undergone a week of puromycin selection. Relative (A) miR-181a and (B) miR-181a-1 level were determined by Taqman microRNA assays. Bars show the mean ± SD from three independent experiments. *** P ≤ 0.001 (ANOVA).
87
A
B C
Figure 18. Assessment of cell growth of lentivirus transduced REH cell.
(A) Growth curve was determined as following: 10,000 cells per well were seeding in a 96-well plate and cultured for 48, 96, and 144 hours, and then assessed with the MTT assay. (B) Cells were seeded at a density of 1*105 cells/ml and cultured. 72hr later, cells were stained with trypan blue and counted.
(C) Cell count at 72hr was demonstrated as percentage of infection control (NC).
Bars show the mean ± SD from three independent experiments. 181A1 vs. NC * P
≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001; NC vs. REH ##P ≤ 0.01 (ANOVA).
88
A
B
Figure 19. Apoptosis analysis of lentivirus transduced REH cell.
(A) Lentiviral transduced REH cells were seeded at a density of 1*105 cells/ml and cultured. 72hr later, cells were collected and assessed the apoptotic cells by flow cytometric analysis of annexin V/PI staining of lentivirus-transduced cells.
(B) Representative histograms demonstrate the proportion of annexin V-positive cells. Bars show the mean ± SD from three independent experiments. ** P ≤ 0.01 (ANOVA).
89
A
B
Figure 20. Analysis of cell cycle and proliferation of lentivirus transduced REH cells.
Cells were seeded at a density of 1*105 cells/ml and cultured for 72hr, and then cell cycle and cell proliferation were assessed by Biparametric BrdU/DNA analysis. (A) During the last 30 minutes of culture, 1 mM BrdU was added to the cells, and then the cells were stained with anti-BrdU and 7-aminoactinomycin D (7-AAD) and detected by flow cytometry. (B) The percentage of cells in each of the cell-cycle phases (G0/G1, S, and G2+M) was quantified. Bars show the mean
± SD from three independent experiments. * P ≤ 0.05 (ANOVA).
BrdU-FITC
7-AAD
NC 181A1
S
G2+M G0/G1
90
A
B
Figure 21. Analysis of CD10 expression on lentivirus-infected REH cells.
(A) Percentage of lentivirus-infected REH cells stained for cell-surface marker CD10-PE/CD34-PerCP/CD19-APC as analyzed by flow cytometry. (B) The results were quantified and are presented as the average ± SD of three independent evaluations. (REH: 13.1%; NC: 14.9 ± 3.43%; 181A1: 37.9 ± 3.35),
** P ≤ 0.01 (ANOVA).
181A1 REH NC
CD19-APC
CD10-PE
91
A
B
Figure 22. Analysis of CD20 expression on lentivirus-infected REH cells.
(A) Percentage of lentivirus-infected REH cells stained for cell-surface marker CD20-FITC/CD45-PerCP as analyzed by flow cytometry. (B) The results were quantified and are presented as the average ± SD of three independent evaluations. (REH: 2.2%; NC: 1.29 ± 0.51%; 181A1: 4.61 ± 1.66), * P ≤ 0.05 (ANOVA).
181A1 REH NC
CD45-PerCP
CD20-FITC
92
A
B
Figure 23. Analysis of surface IgM expression on lentivirus-infected REH cells.
(A) Percentage of lentivirus-infected REH cells stained for cell-surface marker IgM-FITC/CD19-PE/CD45-PerCP as analyzed by flow cytometry. (B) The results were quantified and are presented as the average ± SD of three independent evaluations. (REH: 2.44%; NC: 1.55 ± 0.49%; 181A1: 4.34 ± 1.5),
* P ≤ 0.05 (ANOVA).
181A1 NC
REH
CD19-PE
sIgM-FITC
93
A
B
Figure 24. Analysis of К-chain expression on lentivirus-infected REH cells.
(A) Percentage of lentivirus-infected REH cells stained for cell-surface marker К-chain-FITC/CD19-PE/CD45-PerCP as analyzed by flow cytometry. (B) The results were quantified and are presented as the average ± SD of three independent evaluations. (REH: 0.59%; NC: 1.3 ± 0.3%; 181A1: 4.2 ± 0.58),
*** P ≤ 0.001 (ANOVA).
181A1 NC
REH
CD19-PE
К-chain-FITC
94
A
B
Figure 25. Analysis of λ-chain expression on lentivirus-infected REH cells.
(A) Percentage of lentivirus-infected REH cells stained for cell-surface marker λ-chain-FITC/CD19-PE/CD45-PerCP as analyzed by flow cytometry. (B) The results were quantified and are presented as the average ± SD of three independent evaluations. (REH: 0.74%; NC: 0.79 ± 0.31%; 181A1: 1.8 ± 0.5),
* P ≤ 0.05 (ANOVA).
181A1 REH NC
CD19-PE
λ-chain-FITC
95
A
B
Figure 26. Analysis of CD10 expression on apoptotic REH cells.
(A) Percentage of lentivirus-infected REH cells stained for cell-surface marker Annexin V-FITC/CD10-PE was analyzed by flow cytometry. (B) The results were quantified and are presented as the average ± SD of three independent evaluations. Annexin V＋CD10＋: REH 0.99 ± 0.15%; NC 1.05 ± 0.19%; 181A1:
1.67 ± 0.09; Annexin V＋CD10－: REH 3.43 ± 1.66%; NC 3.1 ± 0.86%; 181A1:
8.74 ± 1.62. ** P ≤ 0.01 (ANOVA).
181A1 REH NC
CD10-PE
Annexin V-FITC
96
A B
Figure 27. Ectopic expression of MIR181A1 in primary ALL cells via lentiviral transduction.
Patient cells were infected with lentiviral vector expressing the negative control shRNA (NC) or miR-181a (181A1). Infected cells were undergone a week of puromycin selection. Relative (A) miR-181a and (B) miR-181a-1 level were determined by Taqman microRNA assays. Bars show the mean ± SD from three independent experiments. *** P ≤ 0.001 (ANOVA).
97
Figure 28. Detection of ETV6/RUNX1 mRNA in cultured primary ALL cells.
Patient cells infected with lentiviral vector and undergone a week of puromycin selection were detected the ETV6/RUNX1 (upper) and endogenous GAPDH (lower) expression by qRT-PCR or RT-PCR. The Ct value was indicated below each sample.
98
Figure 29. Surface marker analysis of lentivirus-infected ETV6/RUNX1- positive primary ALL cells derived from patient #747.
After puromycin selection, lentivirus-infected patient cells were stained for cell-surface markers CD20-FITC/CD10-PE/CD34-PerCP/CD19-APC and analyzed by flow cytometry. Mean fluorescence intensity (MFI) of (A) CD10 and (B) CD20 expression in CD45+CD19+ cells were as indicated in the graphics. (C) Detail analysis of the percentage of CD10High and CD10Low in CD45+CD19+
99
A
B
Figure 30. Surface marker analysis of lentivirus-infected ETV6/RUNX1- positive primary ALL cells derived from patient #752.
After puromycin selection, lentivirus-infected patient cells were stained for cell-surface markers CD10-PE/CD34-PerCP/CD19-APC and analyzed by flow cytometry. Mean fluorescence intensity (MFI) of (A) CD10 expression in CD10High and CD10Low population of CD45+CD19+ cells were as indicated in the graphics. (B) Detail analysis of the percentage of CD10High and CD10Low in CD45+CD19+ lentiviral-transduced cells.
Non-infected NC 181A1
100
Figure 31. Surface marker analysis of lentivirus-infected ETV6/RUNX1- positive primary ALL cells derived from patient #745.
After puromycin selection, lentivirus-infected patient cells were stained for cell-surface markers CD20-FITC/CD10-PE/CD34-PerCP/CD19-APC and analyzed by flow cytometry. Mean fluorescence intensity (MFI) of (B) CD10 and (C) CD20 expression of CD45+CD19+ cells were as indicated in the graphics.
Non-infected NC181A1 Non-infected NC181A1
67.64
101
Figure 32. Surface marker analysis of lentivirus-infected ETV6/RUNX1- negative primary ALL cells derived from patient #754.
After puromycin selection, lentivirus-infected patient cells were stained for cell-surface markers CD20-FITC/CD10-PE/CD34-PerCP/CD19-APC and analyzed by flow cytometry. Mean fluorescence intensity (MFI) of (A) CD10 and (B) CD20 expression in CD45+CD19+ cells were as indicated in the graphics.
B-ALL-754.003
Non-infected NC181A1 Non-infected NC181A1
5.11
102
A
B
Figure 33. Viability of lentivirus-infected primary ALL cells derived from patient #745 (E/R-positive) and #752 (E/R-positive).
After puromycin selection, growth curve of lentivirus-infected primary ALL cells derived from (A) patient #745 and (B) patient #752 were determined as following: 10,000 cells per well were seeding in a 96-well plate and cultured for 48 and 96 hours, and then assessed with the MTT assay.
103
Figure 34. Schematic representation of the double negative loop comprising ETV6/RUNX1 and MIR181A1.
In leukemia cells with frequent chromosome rearrangement t(12;21)(p13;q22), ETV6/RUNX1 oncoprotein occupies the putative RUNX1-binding site upstream of MIR181A1 and restricts transcription by recruiting co-repressors such as HDAC3. This repression of MIR181A1 expression consequently upregulates the target of miR-181a, ETV6/RUNX1 ― the oncoprotein itself, and enhances ETV6/RUNX1’s oncogenic potential
104
Tables
105
106
Table 2. siRNA sequences siE/R-siRNA 1
Sense 5'-CCAUUGGGAGAAUAGCAGAAUGCAU-3'
Antisense 5'-AUGCAUUCUGCUAUUCUCCCAAUGG-3' siE/R-siRNA 5
Sense 5'-UGGGAGAAUAGCAGAAUGCAUACUU-3'
Antisense 5'-AAGUAUGCAUUCUGCUAUUCUCCCA-3' Scramble-siRNA S
Sense 5'-GAAGACGGUAAAUACGUUCGAUAAU-3'
Antisense 5'-AUUAUCGAACGUAUUUACCGUCUUC-3'
107
Table 3. Clinical features of the ALL patients included in miRNA expression profiling study
SR: standard risk; HR: high risk; VHR: very high risk
*Calculated by Fisher's Exact test
†Calculated by Student's t-test
108
Table 4. The statistic signature of 17 miRNAs
miRNA* Expression level,
* Selected by differential expression in patients with or without t(12;21)
109
110
Appendixes
111