Supplementary Information
Figure S1 Ingenuity Pathway Analysis (IPA) summary. To investigate possible interactions of
differently regulated genes, datasets representing 182 genes with altered expression profile
obtained from the illumina microarray were imported into the Ingenuity Pathway Analysis Tool
and the following data is illustrated: (a) The top five toxicology functions as calculated by IPA
based on differentially expressed genes are highlighted along with their respective p-value. (b)
X-axis represents the top canonical pathways identified by IPA based on differentially expressed
genes whereas y-axis (log value) represents the number of genes from the dataset that map to the
pathway divided by the number of all genes ascribed to the pathway. The yellow line represents
the threshold of p<0.05 as calculated by Fischer's test. (c) Taqman assay for mature miR-128 in
HEK293T cells showing overexpression of miR-128-2 in cells transfected with 4g p(128). n = 3
± SEM; *p < 0.05. U = Untransfected, p(128) = cells transfected with 4μg p(128), N= negative
control. (d) Annexin V-staining shows that miR-128 induces apoptosis in HEK293T cells.
Representative of three independent experiments has been shown with similar results (n=3,
P<0.05). (e) Schematic of Cholesterol Homeostasis. Cholesterol homeostasis is governed by
complex processes of cholesterol uptake and synthesis and cholesterol efflux. Two key
transcription factors SREBPs and LXRs regulate these complex processes. SREBP1 target genes
involved in fatty acid metabolism and SREBP2 is specific to cholesterol synthesis. Under low
cholesterol
conditions,
SREBP2
activates
cholesterogenic
genes.
During
cholesterol
accumulation, LXR activates its target genes involved in cholesterol efflux after
heterodimerization with RXR.
Figure S2 MiR-128-2 regulates SREBPs. (a-b) Real-time PCR analysis of SREBP2 (a) SREBP1
(b) in p(128) transfected HepG2, MCF7 and HEK293T cells. 18s rRNA was used for the
normalization. (c-f) Western blot analysis for cleaved SREBP2 and SREBP1 expression after
overexpression of p(128) (c-d), after overexpression of SIRT1 siRNA (e) and after
overexpression of SIRT1ORF (f) in HEK293T cells . (g-h) Western blot analysis for SIRT1 in
the presence of SIRT1 siRNA (at 100 nM dose) / scrambled siRNA/ SIRT1 ORF / negative
control (N) in HepG2, MCF7 and HEK293T cells. Tubulin was used as a loading control and
numbers represent normalized densitometric values.
U = Untransfected, p(128) = cells
transfected with 4μg p(128), N= negative control. n = 3 ± SEM; *p < 0.05 for all experiments.
Figure S3 MiR-128-2 regulates genes of cholesterol/lipid homeostasis. (a-h) Real-time PCR
analysis of HMGCS1 (a), LDLR (b), HMGCR (c), LXR (d), LXR (e), RXR (f), ABCA1
(g) and ABCG1 (h) mRNAs in p(128) )/ SIRT1 siRNA/ SIRT1ORF transfected HepG2, MCF7
and HEK293T cells. 18s rRNA was used for the normalization. U = Untransfected, p(128) =
cells transfected with 4μg p(128), N= negative control. n = 3 ± SEM; *p < 0.05 for all
experiments.
Figure S4 Fatty acid synthesis genes are regulated by miR-128-2. (a-c) Real-time PCR analysis
of ACACA, FASN, SCD1 after overexpression of p(128) in HEK293T cells. 18s rRNA was used
for the normalization. U = Untransfected, p(128) = cells transfected with 4μg p(128), N=
negative control. n = 3 ± SEM; *p < 0.05 for all experiments.
Figure S5 Conservation of binding sites. The binding site of miR-128-2 in ABCA1 3’UTR (a)
and ABCG1 (b) is well conserved among different species (Hsa, human; Ptr, chimpanzee; Rno,
rat; Cpo, guinea pig; Mmu, mouse; Dno, armadillo; Eca, horse; Cfa, dog; Eeu, hedgehog; Ocu,
rabbit, Mml, rhesus; Oga, bushbaby). (c-d) Real-time PCR analysis of ABCA1 (c) and ABCG1
(d) after overexpression of p(128)/ anti-miR in HEK293T cells. 18s rRNA was used for the
normalization. U = Untransfected, p(128) = cells transfected with 4μg p(128), N= negative
control, AM=antimiR, scrambled anti-miR (SAM), n = 3 ± SEM; *p < 0.05 for all experiments.
(e) Evolutionary conservation of the predicted miR-128-2 binding site in the 3’ UTR of RXR.
Figure S6 (a-b, e-f) Western blot analyses of ABCA1 (a-b) and ABCG1 (e-f) protein expression
in MCF7 cells after 24 h of transfection of p(128) or anti-miR-128 in a dose dependent manner.
Tubulin served as a loading control. Bar diagram represents the integrated densitometric values
normalized to Tubulin. U = Untransfected, 2 = cells transfected with 2μg p(128), 4 = cells
transfected with 4μg p(128), N= negative control, AM 100 = cells transfected with 100nM antimiR-128, AM 200 = 200nM anti-miR-128, SAM = Scrambled anti-miR. (c-d,g-h) Real-time
PCR analysis analyses ABCA1 (c-d) and ABCG1 (g-h) mRNA in p(128) or anti-miR-128
transfected MCF7 cells. 18s rRNA was used for the normalization. n = 3 ± SEM; *p < 0.05 .
Figure S7 (a) Evolutionary conservation of mature-miR-128-2 among different species. (b)
Bioinformatic analyses show presence of SREBP transcription factor in the promoter region of
ARPP21 gene encoding miR-128-2. (c) Real time PCR of SREBP2 in simvastatin (10M)
treated HepG2 cells at different time points (at 0, 3, 6, 12, 24 h). 18s rRNA was used for the
normalization.
Supplementary Table S1: Sequence of primers of different metabolic genes of human used for
real time PCR in a 5’-3’ direction. (FP-Forward Primer, RP-Reverse primer).
LXRα FP
GTTATAACCGGGAAGACTTTGCCA
LXRα RP
GCCTCTCTACCTGGAGCTGGT
LXRβ FP
CGTGGACTTCGCTAAGCAAGTG
LXRβ RP
GGTGGAAGTCGTCCTTGCTGTAGG
ABCA1 FP
GCACTGAGGAAGATGCTGAAA
ABCA1 RP
AGTTCCTGGAAGGTCTTGTTCAC
ABCG1 FP
CAGGAAGATTAGACACTGTGG
ABCG1 RP
GAAAGGGGAATGGAGAGAAGA
PPARα FP
GGCGAGGATAGTTCTGGAAGC
PPARα RP
CACAGGATAAGTCACCGAGGAG
PGC-1α FP
TGCCCT GGATTGTTGACATGA
PGC-1α RP
TTTGTCAGGCTGGGGGTAGG
APOE FP
CTGCTCAGCTCCCAGGTC
APOE RP
TTGTTCCTCCAGTTCCGATT
SREBP2 FP
AGGAGAACATGGTGCTGA
SREBP2 RP
TAAAGGAGAGGCACAGGA
SREBP1 FP
GCAAGGCCATCGACTACATT
SREBP1 RP
GGTCAGTGTGTCCTCCACCT
ABCG5 FP
ACCCAAAGCAAGGAACGGGAA
ABCG5 RP
CAGCGTTCAGCATGCCTGTGT
LDLR FP
GTCTTGGCACTGGAACTCGT
LDLR RP
CTGGAAATTGCGCTGGAC
Supplementary Table S2: Sequence of primers of different metabolic genes of mouse used for
real time PCR in a 5’-3’ direction. (FP-Forward Primer, RP-Reverse primer).
ABCA1 FP
GGTTTGGAGATGGTTATACAATAGTTGT
ABCA1 RP
CCCGGAAACGCAAGTCC
ABCG1 FP
TCACCCAGTTCTGCATCCTCTT
ABCG1 RP
GCAGATGTGTCAGGACCGAGT
GAPDH FP
AACTTTGGCATTGTGGAAGG
GAPDH RP
ACACATTGGGGGTAGGAACA
SREBP2 FP
GCGTTCTGGAGACCATGGA
SREBP2 RP
ACAAAGTTGCTCTGAAAACAAATCA
ARPP21 FP
CCCTACCTCAACCTCACAGC
ARPP21 RP
GACTGGCTGGTAACCTGCTT