MALAT1 promotes platelet activity and thrombus formation through PI3k/Akt/GSK-3β signalling pathway

Figures

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  Figure 1. MALAT1 is present in the cytoplasm of MEG01 and downregulated in activated MEG01 cells. (A) RNA fluorescence in situ hybridisation (FISH) of the MALAT1 and 18S in MEG01 cells. Scale bar, 20 µm. There were three separate tests carried out, and each image represents a different one of those experiments. (B) MALAT1 distribution in the nucleus and cytoplasm of MEG01 cells by RT-PCR. Y-axis represents the mean of CT value. GAPDH is used as a cytoplasmic reference. (C) After treating MEG-01 cells with thrombin at a concentration of 1 U/mL for 6, 12, and 24 hours, relative MALAT1 expression was evaluated (**p<0.01) in these cells on collagen-coated coverslips. RT-PCR, reverse transcription PCR.

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  Figure 2. MALAT1 knockdown (KD) in CD34+ megakaryocytes represses adhesion and spreading. Each experiment was performed in triplicate. (A) Comparison of the capacity of MALAT1 KD and control CD34+megakaryocytes (green) to adhere to collagen-coated coverslips following stimulation with or without thrombin 1 U/mL (scale bar, 200 µm). (B) Statistical analysis of the adhesion area of CD34+ megakaryocytes in collagen-coated coverslips. Values are mean±SD (***p<0.001). (C) Comparison of CD34+ megakaryocyte spreading potential on collagen-coated coverslips between MALAT1 KD and control groups without and with 1 U/mL thrombin (scale bar, 20 m). (D) Spreading area was compared with that of platelets of the wild type (**p<0.01), and the mean±SEM was plotted. Around 30 platelets from each genotype were counted on average.

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  Figure 3. MALAT1 represses PLP adhesion, spreading and activity. (A) Adhesion of PLPs (green) from MALAT1 KD and control CD34+ megakaryocytes after treatment by thrombin 1 U/mL (scale bar, 200 μm). Three separate sets of data were collected for each experiment. (B) A statistical examination of the adhesion area of PLPs in coverslips that were coated with collagen. The values are shown as the mean±SD (*p<0.05). (C) A comparison of the capacity of PLPs to spread derived from MALAT1 kD CD34+ megakaryocytes and the scramble in the presence of thrombin at a concentration of 1 U/mL on coverslips coated with collagen (scale bar, 20 µm). (D) Histogram showing the relative area of PLPs for MALAT1 KD and scramble. Data are from three independent experiments. Error bars denote SD *p<0.05. (E) Flow cytometric analysis of CD61 and CD62P expressions on the PLPs isolated from MALAT1 kD megakaryocytes and scramble. (F) Histogram showing the proportions of CD61+CD62P+ cells for MALAT1 kD megakaryocytes and scramble group. Data are from three independent experiments. Error bars denote SD **p<0.01. KD, knockdown; PLPs, platelet-like particles.

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  Figure 4. MALAT1 regulates platelet spreading, aggregation, and activity. (A) Platelets with a deficiency in MALAT1 and platelets with a wild-type genotype (WT) were spread out over fibrinogen-coated coverslips with or without thrombin (0.1 U/mL). (B) Quantification analysis of the spreading area of MALAT1-deficient and WT platelets. Values are mean±SD (**p<0.001). (C) Flow cytometric analysis of integrin aIIβ3 and P-selectin expressions of MALAT1-deficient and WT platelets in the absence or presence of thrombin (0.1 U/mL), using JON/A PE-labelled JON/A antibody and FITC-labelled Wug.E9 antibody (n=6). (D) Aggregation of washed MALAT1-deficient and WT platelets using thrombin (0.1 U/mL). (E) Histogram showing the proportions of aIIβ3 and P-selectin cells for MALAT1 KD megakaryocytes and scramble group. Data are from three independent experiments. Error bars denote SD **p<0.01, ***p<0.001. (F) Panel D depicts the percentage of platelet maximum aggregation based on the findings of at least five separate studies, and the data are presented as the mean±SD (**p<0.01). FITC, fluorescein isothiocyanate; KD, knockdown.

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  Figure 5. Downregulated MALAT1 in platelets increases thrombus formation in vitro and in vivo. (A) Photomicrographs that show the progression of platelet adhesion from MALAT1 KD and scramble mice on collagen. Blood from MALAT1 KD and scrambling animals was collected in sodium citrate (3.8% w/v) and fluorescently tagged with calcein (1 µM) for 1 hour before being perfused through fibrillar collagen-coated bioflux plates at a shear rate of 40 dynes/cm2 for 5 min. (B) Dot plot comparing the area coverage of platelets derived from scramble mice and MALAT1 knockout mice. There were a total of three separate trials carried out. *p<0.05 (C) Images showing representative stages of thrombus formation at 0 min, 5 min, and 25 min after an injury caused by FeCl3 to mesenteric arterioles in MALAT1 KD mice and scrambling mice. A, arteriole; V, venule. (D) The occlusion periods for arteriole thrombosis produced by FeCl3 in scramble and MALAT1 KD mice are displayed as box plots (n=5). **p<0.01. (E) MALAT1 KD accelerates haemostatic plug formation in mice. horizontal lines represent the mean (eight individuals in each group). KD, knockdown.

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  Figure 6. MALAT1 deficiency in platelets activates PI3k/Akt signal pathway. (A) Western blot analysis of WT and MALAT1-deficient platelets in the absence or presence of LY294002 (50 nmol/kg) with antibodies that recognise phosphorylated GSK-3β, phosphorylated Akt Thr308, phosphorylated Akt Ser473, and total Akt. (B) Western blot results were quantified and presented as mean±SD (**p<0.01, ***p<0.001). (C) Analysis of phosphorylated PDK1 and PTEN by Western blotting in MALAT1-deficient and WT platelets in the absence or presence of LY294002 (50 nmol/kg), respectively. (D) The findings of the Western blot were analysed quantitatively and displayed as the mean±SD (*p<0.05, **p<0.01, ***p<0.001). (E) Flow cytometric examination of integrin aIIβ3 and P-selectin expressions with JON/A PE-labelled JON/A antibody and FITC-labelled Wug.E9 antibody (n=6). This was done in the absence or presence of the PI3K inhibitor LY294002. (F) A histogram depicting the percentages of cells expressing aIIβ3 and P-selectin with or without the PI3K inhibitor LY294002. Data are from three independent experiments. Error bars denote SD **p<0.01. WT, wild-type.

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  Figure 7. MALAT1 regulates platelet activity through PI3k/Akt/GSK-3β pathway. MALAT1 is a potent negative regulator of platelet activity. In the absence of this lncRNA, integrin outside-in signalling can be activated by PI3k/Akt/GSK-3β pathway.