supplementary information - media.nature.com · the expression levels of mir-151, mir-548d is...

s u p p l e m e n ta ry i n f o r m at i o n

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DOI: 10.1038/ncb2039

Figure S1 One hundred Twenty-nine Potential Chromosomal Breakpoints and associated microRNAs in hepatocellular carcinoma. Alignment of human miRNAs with chromosomal instability sites in hepatocellular carcinoma (HCC).

Solid triangles indicate the common deletion chromosome regions in HCC. Solid circle indicates the common amplification chromosome regions in HCC. The miRNAs genes are marked on the right at their approximate positions.

Chromosome 1 Chromosome 4 Chromosome 6 Chromosome 7 Chromosome 8 Chromosome 9 Chromosome 10

Chromosome 11 Chromosome 13 Chromosome 16 Chromosome 17 Chromosome 19 Chromosome 20 Chromosome 22

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© 2010 Macmillan Publishers Limited. All rights reserved.


2 www.nature.com/naturecellbiology

Figure S2 Representative results of genomic DNA contents and the expression of some aberrant miRNAs in HCC. (a) Representative results of genomic real-time PCR screening for analysis of miRNA DNA copy numbers in HCC. Quantitative real time-PCR analysis of the genomic DNA copies for miR-151, miR-550-2, miR-486 and miR-138-2 respectively in HCC tissues were performed by using SYBR Premix Ex Taq assays. β-actin was used for normalization. Data are shown in triplicate with means (n=41 HCC samples and

n=7 normal liver tissues). (b) The expression of 12 aberrant miRNAs in HCC tissues and corresponding noncancerous liver tissues. MicroRNA expression was determined by TaqMan qRT-PCR in HCC and adjacent noncancerous liver tissue samples (NT) (n=50). Box plots describe the relative expression of miRNAs. The ends of the boxes define the 25th and 75th percentiles, a line indicates the median, and bars define the 5th and 95th percentiles. Individual outliers are also shown. Statistical analysis was performed by paired t-test.

T1 T2 T3 T4 T5 T6 T7 T8 T9T10T1

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T1 T2 T3 T4 T5 T6 T7 T8 T9T10T11T12T13T14T15 T16T17T18T19T20T21T22T23T24T25T26T27T28 T29T30T31T32T33T34T35T36T37T38T39T40T41 N1 N2 N3 N4 N5 N6 N7

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Figure S3 The correlations of the expression levels of miR-151, miR-548d and miR-153 with their genomic DNA contents or intrahepatic metastasis of HCC. (a) Relative expression levels of miR-151, miR-548d and miR-153 in patient samples with genome copy gains or losses. Box plots describe the relative expression of miRNAs. Statistical analysis was performed by Student’s t-test. The expression levels of miR-151, miR-548d is higher in HCC tissues with genomic amplification than those without, whereas the expression level of miR-153 is lower in HCC tissues with genomic deletion than those without (n = 33). (b) Relative expression level of miR-548d in patient samples with

intrahepatic metastasis or not. Box plots describe the relative expression level of miR-548d-1. Statistical analysis was performed by Student’s t-test. The expression of miR-548d-1 has no significant difference between HCC patients with intrahepatic metastasis and those without (n = 46). (c) Relative expression levels of miR-151-5p in patient samples with intrahepatic metastasis or without. Box plots describe the relative expression levels of miR-151-5p in HCC patients with intrahepatic metastasis or without. Statistical analysis was performed by Student’s t-test. The expression level of miR-151-5p is significantly correlated with intrahepatic metastasis of HCC (n = 46).

miR-151

0

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Normal Gain

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M(-) M(+)0.0

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Intrahepat ic Metastas is

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M(-) M(+)

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miR-151-5p

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a

b c




Figure S4 The expression levels and genomic DNA copies of miR-151 and FAK in HCC cell lines and HCC tissues. (a) The expression levels of miR-151 and FAK in various HCC cell lines. MiR-151 expression was determined by TaqMan real time PCR in various HCC cells. The miR-151 level was normalized to U6 snRNA. FAK expression was examined by quantitative real-time PCR in various HCC cells. The FAK expression level was normalized to β-actin. Data are shown in triplicate with means. The correlation between expression levels of mature miR-151 and FAK in various HCC cells were performed using Pearson’s correlation. (b) Amplification status of FAK/miR-151 genomic locus in HCC cell lines. The status of the FAK/miR-151 genomic locus was examined by Affymetrix SNP 6.0 array in Huh-7, SMMC-7721, MHCC-LM3 cells and a normal liver tissue (served as control). Raw data was normalized by adapter-type-normalization and converted to CNCHP file by Affymetrix-Power-Tool. FAK/miR-151 locus is amplified in MHCC-LM3 cells (about two folds), while it has no change in both Huh-7 and SMMC-7721 cells. (c) The expression levels of miR-151 and FAK with the

corresponding genomic content of the FAK/miR-151locus in HCC tissues. The data for the genomic content of the region of miR-151 (Supplementary Fig. S2a), the expression levels of miR-151 (Fig. 1a), and the expression levels of FAK (Fig. 1e, for comparison, the relative expression value of FAK was multiplied by 10) in 33 overlapped HCC tissues were integrated. Statistical analysis was performed by Student’s t-test. The expression levels of FAK and miR-151 are higher in HCC samples with genomic amplification than those without amplification (p=0.0101 and p=0.029, respectively). (d) Knockdown of p53 by siRNA increases the mRNA expression levels of FAK and miR-151 in HepG2 cells. The scheme of FAK promoter with p53 binding sites is drawn as reported by Golubovskaya VM, et al. BBA. 2004; 1678: 111-25 and Mol Carcinog. 2008; 47: 373-82; the expression levels of p53, FAK and miR-151 in HepG2 cells were determined after transfected with siRNA against p53 or negative control by SYBR real time PCR or TaqMan real time PCR assays. β-actin and U6 snRNA serve as the internal controls. A representative experiment is shown in triplicate along with s.e.m (n = 4).

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10Genomic DNA copyFAK expression(normalized)miR-151 expression

FAK(none.) vs FAK(Amp.) P=0.0101miR-151(none.) vs miR-151(Amp.) P=0.029

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LM3.CNCHP: Log2Ratio (-2, 2)

LN1.CNCHP: Log2Ratio (-2,2)

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chr8 (q24.3) 23.1 8p22 8p12 21.3 24.3

Scalechr8:

100 kb141700000 141750000 141800000 141850000 141900000 141950000 142000000

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p53 FAK miR-1510

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Figure S5 miR-151 has no significant effect on SMMC-7721, Huh-7 and MHCC-LM3 cell growth. (a) The expression levels of miR-151 in the stable cell lines infected with miR-151 lentivirus. The relative expression of mature miR-151 was determined by ABI TaqMan miRNA assay in Huh-7 or SMMC-7721 cells infected with either miR-151-expressing lentivirus or control lentivirus. U6 snRNA serves as an internal control. A representative experiment is shown in triplicate with means (n = 4). (b) Cell proliferation assays of SMMC-7721 and Huh-7 cells were performed

after infected with miR-151-expressing lentivirus or control lentivirus. The effect of miR-151-3p inhibitor, miR-151-5p inhibitor or control in cell proliferation was assessed in MHCC-LM3 cells by Cell Counting Kit-8 (CCK-8) assays. These assays were performed every day for 5 days. (c) Cell proliferation assays were performed by CCK-8 analysis. 1,000 cells per well were cultured in 96-well plates coated with Matrigel. This assay was performed every day for 3 days. Data are presented as means±s.e.m of triplicate expresiments in b and c (n = 3).

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c




Figure S6 miR-151 down-regulates RhoGDIA expression by directly targeting its 3’ UTR in SMMC-7721 cells. (a) Luciferase activity assays of luciferase reporters with wild type or mutant RhoGDIA 3’ UTR were performed after co-transfected with miR-151 expressing plasmids, vector control, miR-151-3p mimics, -5p mimics or NC control in SMMC-7721 cells. The luciferase activity of each sample was normalized to the Renilla luciferase activity. The normalized luciferase activity of vector and NC transfection in each experiment was set as relative luciferase activity=1. A representative

experiment is shown in triplicate with means (n = 3). (b) The protein levels of RhoGDIA were determined by western blot analyses after infected with miR-151 or control lentivirus, or transfected with miR-151-3p mimics, -5p mimics or NC control in SMMC-7721 cells. (c) The mRNA levels of RhoGDIA were determined by quantitative real-time PCR analyses after transfected with miR-151-3p mimics, -5p mimics or NC control in SMMC-7721 cells. β-actin serves as an internal control. A representative experiment is shown presented in triplicate as means ± s.e.m (n = 4).

0.0

0.5

1.0

1.5Fo

ld c

hang

es

Vector

miR-151

NC

miR-151-3p

miR-151-5p

+ - - - - + - - - -

- + - - - - + - - -

- - + - - - - + - -

- - - + - - - - + -

- - - - + - - - - +

Wildtype Mutant

SMMC-7721

a

b c

Vector

miR-15

1 NC

miR-15

1-3p

miR-15

1-5p

0.0

0.1

0.2

0.3P=0.0096

P=0.0068

Rel

ativ

e ex

pres

sion

leve

lof

Rho

GD

IA m

RN

A

Vector

miR-15

1

RhoGDIA

β-actin

RhoGDIA

β-actin

NC miR-15

1-3p

miR-15

1-5p




Figure S7 The activity and protein levels of Rac1, cdc42 and Rho GTPases in Huh-7 cells were determined after transfected with siRNA against RhoGDIA or negative control. The results showed that knockdown of

RhoGDIA can increase the activities of Rac1, Cdc42 and Rho GTPases, whereas does not have any effects on the expression levels of these proteins.

NC siRhoGDIARac1-GTP

Cdc42-GTP

Rho-GTP

Rac1-total

Cdc42-total

Rho-total

β-actin




Figure S8 Selected representative full scans. (a) Fig. 4c and Fig. S6b, showing western blot (WB) analyses of RhoGDIA after infected with miR-151 or control lentivirus in Huh-7 and SMMC-7721 cells. (b) Fig. 5e, showing WB analyses of RhoGDIA after transfected with miR-151-5p inhibitor, siRNA against RhoGDIA, or negative control in MHCC-LM3 cells. (c) Fig. 6b,

showing WB analyses of Rac1, Cdc42 and Rho GTPases for the activities of these RhoGTPases after infected with miR-151, FAK or control lentivirus in Huh-7 cells. (d) Fig. 6e, showing WB analyses of RhoGDIA and FAK proteins after transfected with miR-151-5p inhibitor, siRNA against FAK, or negative control in MHCC-LM3 cells.

Vector

Lenti

-miR

-151

Vector

Lenti

-miR

-151

SMMC-7721 Huh-7

MW(kDa)3025

46

WB:

RhoGDIA

β-actin

RhoGDIA

β-actin

MW(kDa)

3025

46

NC Anti-m

iR-15

1-5p

siRho

GDIA

Anti-m

iR-15

1-5p

siRho

GDIA+

WB:

Vector

Lenti

-miR

-151

Lenti

-FAK

Lenti

-miR

-151+

FAK

MW(kDa)

25

17

25

17

25

17

Pull down/WB:

Rac1

Cdc42

Rho

MW(kDa)

30

25

175

80

46

WB:

RhoGDIA

β-actin

FAK

NC Anti-m

iR-15

1-5p

siFAK

Anti-m

iR-15

1-5p

siFAK+

Fig.S6b and Fig.4c Fig.5e

Fig.6b Fig.6e

a b

c d


supplementary information - media.nature.com · the expression levels of mir-151, mir-548d is...

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