Table 1.
Primers for qRT-PCR.
Fig 1.
MiR-326 targets the 3′-untranslated region (UTR) of Bcl-xL but not that of Bak.
(a) Putative miR-326 binding sequences in the 3′-UTR of the Bcl-xL and Bak genes and the mut sequences. (b) Transfection of miR-326 mimic compared to the negative control (miR-NC) significantly inhibited luciferase activity from a reporter vector containing the 3′-UTR of Bcl-xL (**P<0.01). However, there was no inhibition of a reporter vector with mutations in the miR-326-binding site (Bcl-xL-mut) (P>0.05). (c) Transfection of miR-326 did not significantly reduce the luciferase activity from reporter vectors containing the 3′-UTR of Bak or Bak-mut (P>0.05). Results represent the means ± SD of luciferase values in 3 independent experiments and are normalized to 1.0 for the miR-NC samples.
Fig 2.
MiR-326 regulates the expression of endogenous Bcl-xL.
(a, b) Transfection efficiency of miR-326 mimic and miR-326 inhibitor in leukocyte-depleted apheresis platelets (LDPs) were determined by qRT-PCR. LDPs were transfected using a transfection kit and mimic or inhibitor at a final concentration of 50, 100, or 200 nM and were collected after 24 h for analysis. A blank control sample and non-targeting negative RNA (miR-NC or inhibitor NC) were used as controls. *P<0.05, **P<0.01. (c) qRT-PCR of Bcl-xL mRNA was performed 24 h after transfection of LDPs as indicated. *P<0.05. (d) qRT-PCR of Bak mRNA was performed 24 h after transfection of LDPs as indicated. (e–g) Levels of Bcl-xL and Bak protein were determined by western blotting. The mean ± SD expression levels are shown. *P<0.05. Results are representative of 3 independent experiments.
Fig 3.
Bcl-xL siRNA effectively knocks down Bcl-xL mRNA and protein and induces platelet apoptosis.
Leukocyte-depleted apheresis platelets (LDPs) were transfected with siBcl-xL or negative control siRNA (siNC) at a final concentration of 50 nM. LDPs without transfection were used as blank controls (control). Bcl-xL expression and apoptosis were assessed 48 h after transfection. (a) Expression levels of the Bcl-xL mRNA were assessed by qRT-PCR. 5s rRNA was used as an endogenous control. *P<0.05. (b,c) Bcl-xL protein levels were assessed by western blotting. β-actin was used as a loading control. *P<0.05. (d, e) Depolarization of platelet ΔΨm was determined by JC-1 fluorochrome staining and flow cytometry. Histograms of platelets with JC-1 staining are shown for mock treated platelets (control) and platelets transfected with siNC or siBcl-xL. Depolarization is characterized as the decrease in the content of JC-1 aggregates, as reflected in the decrease of red (FL2) fluorescence. Dots inside R1 indicate that platelets are out of the main population and are considered to be undergoing ΔΨm depolarization. *P<0.05. (f, g) Annexin V-FITC staining (R2) demonstrated that the annexin V positive cells in LDPs significantly increased as compared to the mock-transfected control and siNC (**P<0.01 vs. control, ##P<0.01 vs. siNC). (h) The caspase-3 activity in LDPs was significantly increased as compared to control and negative siRNA (**P<0.01 vs. control, ##P<0.01 vs. siNC). Values are expressed as the mean ± SD; n = 3 in each group.
Fig 4.
MiR-326 promotes platelet apoptosis.
Leukocyte-depleted apheresis platelets (LDPs) were untransfected (control) or transfected with 50 nM of miR-326 mimic or a non-targeting negative control miRNA (miR-NC). LDPs were cultured under standard blood banking conditions and harvested at 24 or 72 h. Flow cytometry was used to analyze the apoptosis status of platelets after transfection. (a, b) Depolarization of ΔΨm was determined by JC-1 fluorochrome assay. *P<0.05. (c) Annexin V positive staining of platelets increased by nearly two-fold after transfection of miR-326 mimic. *P<0.05. (d) Overexpression of miR-326 mimic increased caspase-3 activity in LDP. *P<0.05.
Fig 5.
Expression of additional Bcl-2 family members after transfection.
Lysates from washed platelets (20 μg) were subjected to western blot analysis for expression of proteins. (a) Western blotting analysis is shown for untransfected platelets (control) and platelets transfected with miR-326 mimic or non-targeting negative control (miR-NC). Leukocyte-depleted apheresis platelets (LDPs) were cultured under standard blood banking conditions and harvested at the indicated times after transfection. Results are representative of 3 independent experiments. (b, c) The effect of miR-326 mimic transfection on Bcl-2 and Bak mRNA expression (P>0.05 vs. siNC).
Fig 6.
Platelets are not activated by miR-326 expression.
Leukocyte-depleted apheresis platelets (LDPs) were untransfected (control) or treated with 50 nM miR-326 mimic or non-targeting negative control (miR-NC). LDPs were cultured under standard blood banking conditions and harvested at the indicated times. Expression and activity was determined by flow cytometry. (a) Relative number of P-selectin (CD62p)-positive platelets. (b) Relative number of CD63-positive platelets. (c) Relative number of PAC-1-positive platelets. Data were quantified from 3 separate experiments and are shown as mean ± SD.