Chapter 2. Materials and Methods
3.4 The cellular effects of MIR181A1 on B-ALL cells
3.4.1. Ectopic expression of MIR181A1 impedes REH cell growth
To elucidate the cellular function of MIR181A1 in B-ALL and how MIR181A1
participates in the preleukemic events induced by ETV6/RUNX1, REH cells were
transduced by a lentiviral vector carrying MIR181A1 (181A1-LV) to express miR-181a
and miR-181a-1 stably and constitutively (Figure 17). We found that ectopic
overexpression of MIR181A1 resulted in growth retardation of the cells, and
181A1-LV-transduced REH cells showed a nearly 40% decrease in both MTT activity
(Figure 18A) and cell density after 72 hours of seeding (Figure18B-C).
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3.4.2. Apoptotic cells increases in MIR181A1-lentivirus transduced REH cells
The growth retardation of 181A1-LV transduced cells might be caused by
increased cell death or decreased proliferation. To investigate the possible mechanism,
lentivirus-transduced cells were stained with annexin V and PI to detect the apoptotic
cells (Figure 19A). Compared with REH cells, the infection control (NC) did not show
difference in annexin V–positive (apoptotic) cell population, while 181A1-LV
transduction induced an nearly 8% increase in apoptosis (Figure 19B).
3.4.3. The percentage of G0/G1 phase population increases in MIR181A1-lentivirus transduced REH cells
We further assessed the proliferation activity by biparametric BrdU/DNA flow
cytometry. The total DNA was stained by 7-AAD dye and proliferative cells were
labeled by BrdU incorporation and detected by anti-BrdU-FITC. G0/1, S, or G2/M
phase was defined by 7-AAD staining intensities, and proliferative cells that were
actively synthesizing DNA were characterized by BrdU-positive (Figure 20A). We
found that MIR181A1 expression resulted in slightly decrease in the percentage of S and
G2/M phase cells without statistical significance, while the most obvious change was
the increase of the proportion of cells in G0/G1 phase (Figure 20B).
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3.4.4. Ectopic expression of MIR181A1 enhances REH cell differentiation
The oncogenic effect of ETV6/RUNX1 has been postulated to operate through
impairment of B-cell differentiation in a bone marrow transplantation model, and
consequently it results in the accumulation of pro-B-cells (38). We investigated whether
the greatly reduced MIR181A1 expression seen in ETV6/RUNX1-positive pro-B ALL
blasts plays a role in the ETV6/RUNX1-mediated blockade of B-cell differentiation.
The stages of B lymphocyte maturation are characterized by specific expression patterns
of immunoglobulins and other membrane proteins. To gain insight into the effect of
miR-181a overexpression on REH cell maturation, we stained cells for differentiation
markers and found an increase in CD10-negative (Figure 21), CD20-positive (Figure
22), surface IgM-positive (Figure 23), κ-chain-positive (Figure 24), and λ-chain-positive
(Figure 25) cell populations in 181A1-LV-transduced cells compared with NC. In
normal progression of B cell differentiation, decreased CD10 expression and increased
CD20, IgM, κ-chain, and λ-chain expression represent a gradual maturation of B
lymphoid cells from pre-B I cells to immature B cells (74), indicating MIR181A1
expression may induce REH cells toward partial differentiation.
3.4.5. MIR181A1 expression enhances apoptosis of differentiated cells
Because the decrease in CD10 expression was the most notable change of
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181A1-LV-transduced cells, we further stained cells for CD10 and annexin V (Figure
26A). As expected that there was no difference between REH cells and cells transduced
with NC. MIR181A1 transduction induced apoptosis in both CD10-positive and
CD10-negative population. Moreover, we found that most apoptotic 181A1-transduced
cells were CD10-negative (Figure 26B), implying a loss of survival ability in these
differentiated cell.
3.4.6. Ectopic expression of MIR181A1 induces partial differentiation of ETV6/RUNX1-positive primary ALL blasts
Loss of the marker CD10 and a gain of CD20 have been associated with differentiation
of normal B-cell precursors from HSCs to naive mature B lymphocytes in the bone
marrow (75). The infection of primary blasts isolated from the bone marrow of B-ALL
patients with a lentiviral vector expressing miR-181a increased the level of
miR-181a/miR-181a-1 by an average of 2.5-fold (1.5 to 3-fold)/ 3-fold (1.3 to 5.2-fold)
in three ETV6/RUNX1-positive samples compared with the controls (Figure 27). We
have also confirmed that the ETV6/RUNX1-positive primary ALL blasts survived after
lentivirus infection and puromycin selection were ETV6/RUNX1-positive (Figure 28).
We observed that lentiviral infection may alter certain properties of primary ALL blasts,
thus we mainly compared the NC and 181A1-LV transduced cells. This induction
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altered the lymphocytic differentiation as shown by the decrease in the high expression
of CD10 in cells in two of three ETV6/RUNX1-positive samples, though the intensity of
CD20 were not changed in all three samples (Figure 29-31). We also explore the effect
of MIR181A1 transduction in one ETV6/RUNX1-negative sample, but no significant
change in surface CD10 and CD20 expression was found (Figure 32). We were also
determine the growth rate of lentivirus-infected primary ALL blasts, however, the
patient cells seemed just survived in our culture medium rather than proliferation and
the growth activity was gradually lost after thawing out (Figure 33), which may cause
the low induction of miR-181a expression and reduce the influence of miR-181a.
Taken together, our data suggests that the level of miR-181a expression may be
important for the perturbation of the lymphocytic differentiation program in
ETV6/RUNX1-expressing ALL.
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Chapter 4. Discussion
Despite having an extremely short sequence, miRNAs have diverse functions via
targeting of multiple genes simultaneously. They have been implicated in virtually all
aspects of biology, including cell proliferation, cell differentiation, cell cycle, apoptosis,
developmental timing, metabolism, and hematopoiesis. They participate in endogenous
transcriptional networks that control early development, lineage decision, and
differentiation in many cell types, including hematopoietic cells (76).