Figure 1.
MiRNA microarray images of global and RISC-immunoprecipitated (RISC-IP) miRNA from human U-87 astrocytoma and primary human astrocyte cells.
The representative chip images are displayed in pseudo colors to expand the visual dynamic range. As signal intensity increases from 1 to 65,535, the corresponding color changes from blue to green, to yellow and to red.
Figure 2.
RISC-IP miRNA expression compared to global miRNA expression in primary human astrocytes.
(A) MiRNAs with a log2 fold change >2 (p<0.01). MiRNAs with a positive fold change were increased in the RISC compared to the global cellular milieu in primary astrocytes and miRNAs with a negative fold change are decreased in RISC compared to the global cellular milieu in primary astrocytes. (B) Hierarchical cluster heatmap showing all significantly expressed miRNAs (p<0.01) in four independent samples/cell types. Each row shows the relative expression level for a single miRNA and each column shows the expression level for a single sample. The red or green color indicates relative high or low expression, respectively. (C) Disease/disorders associated with the miRNAs differentially expressed in RISC compared to the global cellular milieu of primary astrocytes with a Fisher's exact test p-value threshold set at 0.01 (yellow threshold line) using Ingenuity Pathway Analysis software.
Figure 3.
RISC-IP miRNA expression compared to global miRNA expression in human U-87 astrocytoma cells.
(A) MiRNAs with a log2 fold change >2 (p<0.01). MiRNAs with a positive fold change are increased in RISC compared to the global cellular milieu in astrocytoma cells and miRNAs with a negative fold change are decreased in RISC compared to the global cellular milieu in astrocytoma cells. (B) Hierarchical cluster heatmap showing all significantly expressed miRNAs (p<0.01) in four independent samples/cell type. Each row shows the relative expression level for a single miRNA and each column shows the expression level for a single sample. The red or green color indicates relative high or low expression, respectively. (C) Disease/disorders associated with the miRNAs differentially expressed in RISC compared to the global cellular milieu of U-87 astrocytoma cells with a Fisher's exact test p-value threshold set at 0.01 (yellow threshold line) using IPA software. (D) MiRNAs in RISC compared to the global cellular fraction in U-87 astrocytoma cells. U-87 astrocytoma cell miRNA expression was normalized to primary astrocytes miRNA expression. MiRNAs displayed have a log2 fold change >3. Green and red arrows indicate decreased and increased miRNA levels respectively.
Figure 4.
MiRNA microarray validation with qRT-PCR analysis for (A) global miRNA and (B) RISC-specific miRNA.
Nine random miRNAs were selected from the miRNA microarray datasets and examined by qRT-PCR. Fold change from the miRNA microarray are given by log2 values (left y-axis, light grey bars). Fold change from the qRT-PCR was determined using the 2-ΔΔCt method and all miRNA expression values were normalized to the RNU6B endogenous control (right y-axis, dark grey bars). Error bars represent the standard deviation of the mean (SD). Note: only the general trend of up-regulation and down-regulation can be compared but the fold change (y-axis) cannot be directly compared between assays due to differences in calculation methods.
Figure 5.
RISC-IP miRNA expression in human U-87 astrocytoma cells compared to primary human astrocytes.
(A) MiRNAs with a log2 fold change >2 (p<0.01). MiRNAs with a positive fold change are increased in RISC-IP astrocytoma cells compared to RISC-IP astrocytes and miRNAs with a negative fold change are decreased in RISC-IP astrocytoma cells compared to RISC-IP astrocytes. (B) Hierarchical cluster heatmap showing all significantly expressed miRNAs (p<0.01) in four independent samples/cell type. Each row shows the relative expression level for a single miRNA and each column shows the expression level for a single sample. The red or green color indicates relative high or low expression, respectively. (C) Disease/disorders and (D) molecular and cellular functions associated with the miRNAs differentially expressed in RISC of U-87 astrocytoma cells compared to primary astrocytes with a Fisher's exact test p-value threshold set at 0.01 (yellow threshold line) using IPA software.
Table 1.
Significantly expressed (p<0.01) RISC-immunoprecipitated miRNAs in human U-87 astrocytoma cells compared to primary human astrocytes with log2>3.
Table 2.
Significantly expressed (p<0.01) global miRNAs in human U-87 astrocytoma cells compared to primary human astrocytes with log2>3.
Figure 6.
Global mRNA levels to RISC-IP mRNA levels in U-87 astrocytoma and primary astrocytes.
A hierarchical heatmap comparing global mRNA levels to RISC-IP mRNA levels in U-87 astrocytoma and primary astrocytes. MRNAs included in the heatmap had a fold change >1.4 and were significantly expressed (p<0.01).
Table 3.
RISC-immunoprecipitated mRNA compared to global cellular mRNA in U-87 astrocytoma cells and primary astrocytes with a fold change > ±1.8 (p<0.01).
Figure 7.
(A) MRNA microarray validation with qRT-PCR analysis in grouped RISC-IP U-87 astrocytoma and primary astrocytes samples. Grouped RISC-IP data were compared to the grouped global mRNA from U-87 astrocytoma and primary astrocytes samples. Eight mRNAs were selected from the grouped mRNA microarray dataset and examined by qRT-PCR. Fold change from the mRNA microarray are given by log2 values (left y-axis, light grey bars). Fold change from the qRT-PCR was determined using the 2-ΔΔCt method and all mRNA expression values were normalized to the beta-actin endogenous control (right y-axis, dark grey bars). Error bars represent the standard deviation of the mean (SD). Importantly, the fold change (y-axis) cannot be directly compared between assays due to differences in calculation methods, but the general trend of up-regulation and down-regulation can be compared. (B) MRNAs in RISC compared to the global cellular milieu in U-87 astrocytoma cells. MRNA expression in U-87 astrocytoma cells were normalized to primary astrocytes mRNA expression. All mRNAs had a fold change >2.5 and were significantly expressed (p<0.01). Green and red arrows indicate decreased and increased levels respectively.
Table 4.
RISC-immunoprecipitated mRNA in human U-87 astrocytoma cells compared to RISC-immunoprecipitated mRNA in primary human astrocytes with a fold change >±2.6 (p<0.01).
Figure 8.
Disease and disorder representation of the mRNA in the RISC of U-87 astrocytoma cells compared to primary astrocytes and the mRNA in the global cellular milieu of U-87 astrocytoma cells compared to primary astrocytes.
Bar charts display the relative number (-log(p-value)) of mRNAs with a fold change >2.5 and were considered significant (p<0.01). RISC-IP mRNA were indicated with dark blue bars and the global mRNA were indicated with light blue bars. The threshold (yellow lines) were set at p<0.01 and were calculated using Fischer's exact p-value test using IPA software.
Figure 9.
Molecular and cellular functional assessment of the mRNA in the RISC of U-87 astrocytoma cells compared to primary astrocytes and the mRNA in the global cellular milieu of U-87 astrocytoma cells compared to primary astrocytes.
Bar charts display the relative number (-log(p-value)) of mRNAs with a fold change >2.5 and were considered significant (p<0.01). RISC-IP mRNA were indicated with dark blue bars and the global mRNA were indicated with light blue bars. The threshold (yellow lines) were set at p<0.01 and were calculated using Fischer's exact p-value test using IPA software.
Figure 10.
Biological pathway analysis showing the links between miRNA and mRNAs IP from RISC of U-87 astrocytoma cells compared to primary astrocytes using IPA software.
All miRNA and mRNA were significantly expressed (p<0.01). MiRNA had a log2 fold change >3 and mRNA had a log2 fold change >2. Some key canonical pathways are indicated by labels.
Table 5.
Specific messenger RNA fold change linked to the increased levels of miR-34a in U-87 astrocytoma RISC.
Table 6.
Specific messenger RNA fold change linked to increased levels of miR-195 in U-87 astrocytoma RISC.