SUPPLEMENTARY TEXT
Molecular signature of miR-34a overexpression.
To gain more insight into target genes either directly or indirectly affected by miR-34a
overexpression over a temporally defined time course of human neurodevelopment, the
miR-Ctrl and miR-34a overexpression lines were differentiated for 8 weeks with total
RNA samples collected at t=0, 2, 4, 8 weeks in duplicate and subject to NanoString
assay with a probe set consisting of a total of 131 genes, and the data merged with that
from the t=6 weeks sets that were done in triplicates (Table S9). Using a cut off of a
change greater than +/- 1.5-fold with miR-34a overexpression compared to the matched
miR-Ctrl control, along with the requirement that at least two of the replicates showed
concordant results for the particular time period, a total of 70 transcripts (53%) were
identified at one more time point over the neurodevelopmental time course as showing
differential expression (Table S10). To help visualize pathways within the set of
differentially affected genes, using DAPPLE we built a direct and indirect (through other
proteins) interaction network amongst the set of 70 input seed genes, with edges
between the nodes representing protein-protein interaction data from the InWeb
database as described by Lage et al.1 (Figure 4B). Within-degree, node-label
permutation methods were then used to assess the statistical significance of the
connectivity of individual proteins to other seed proteins, as well as a set of global
network connectivity parameters. The resulting network was composed of a total of 719
interactions between 299 unique proteins, with both the observed mean associated
protein direct connectivity of the seed proteins (1.8; p=0.007) and the observed mean
associated protein indirect connectivity of the seed proteins (29.8; p=0.001) highly
significant with 5,000 permutations. Analysis of the 21 seed proteins with at least one
direct interaction amongst the other seed proteins revealed three main modules of
multiple interacting proteins with known function, including cell adhesion and
cytoskeletal regulation (CNTN1, CNTN2, NFASC, ANK3, ANK2, CNTNAP1, CNTNAP2,
TNC, TNR, TNIK), calcium channels (CACNA1C, CACNB1, CACNB3, CACNA2D), and
glutamatergic synaptic transmission (GRIN1, GRIN2A, GRIN2B, GRIN2D) amongst
others (Figure 4B). In total, there were 21 proteins within the network that had
statistically significant (p<0.05) connectivity after 5,000 within-degree, node-label
permutations.
SUPPLEMENTARY REFERENCES
1.
Lage K, Karlberg EO, Storling ZM, Olason PI, Pedersen AG, Rigina O et al. A
human phenome-interactome network of protein complexes implicated in genetic
disorders. Nature biotechnology 2007; 25(3): 309-316.