NEAT1 is Required for the Expression of the Liver Cancer Stem Cell Marker CD44

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Table S1. Clinical characteristics of HCC patients.

Number of patients

Age (y)

Gender (male / female)

Etiology (HBV / HCV / nonB·nonC)

Child-Pugh score (5 / 6 / ≥7)

Number of tumors (1 / ≥ 2)

Tumor size (cm)

TNM stage (IA / IB / II / IIIA / IIIB / IVA)

Survival period (y)

† mean ± standard deviation

§ median [interquartile range]

92

67.2 ± 10.7†

79 / 13

40 / 23 / 29

67 / 19 / 6

80 / 13

5.3 ± 4.1†

8 / 30 / 38 / 7 / 7 / 2

4.2 [2.8 - 7.3]§

Table S2. OligoDNAs used in the present study

Reverse transcription primers

hNEAT1v1-specific oligo(dT) primer (hV1-dT13)

mNeat1v1-specific oligo(dT) primer (mV1-dT14)

random nanomer ligoDNA primer

TTTTTTTTTTTTTTCTAATG

TTTTTTTTTTTTTTGAAGCT

NNNNNNNNN

(N: A, G, C, T)

qPCR primers

human

β-actin

CD133

CD44

EPCAM

HPRT1

total NEAT1

NEAT1v2

NOTCH3

mouse

total Neat1

Neat1v2

sense

antisense

GATGCAGAAGGAGATCACTGC

AATTCACCAGCAACGAGTCC

AAGGTGGAGCAAACACAACC

ATAACCTGCTCTGAGCGAGTG

TGGCGTCGTGATTAGTGATG

TGCCACAACGCAGATTGATG

AGGGTTCTGTTGCTAACACG

AGCCATGCTGATGTCAATGC

TGATCCACATCTGCTGGAAG

AATCCATTCCCTGTGCGTTG

TCGACTGTTGACTGCAATGC

AACGCGTTGTGATCTCCTTC

ATCTCGAGCAAGACGTTCAG

ACAAGAAGGCAGGCAAACAG

TGCCGATGAAGCAACAAAGC

TTTTGAGCAGGGCCAAAGTG

AGCAGGCACCCTTTTTCATG

ATCAGGTTTGGCAGCCAATG

TGCGCAGACGTTAAAGTGTC

CAGGCAACAAAAGCAAACGC

AflII-linker oligoDNA

Up

TTAACGCCAGTCGGATCCAGCGAT

Down

TTAAATCGCTGGATCCGACTGGCG

psgNEAT1a/b construction

sgNEAT1a

sgNEAT1b

Up

CACCgTCCCTCCCTGTCGCTAACTC

CACCgACAGGGAGGGATGCGCGCCT

Down

AAACGAGTTAGCGACAGGGAGGGAc

AAACAGGCGCGCATCCCTCCCTGTc

pTarget-hNEAT1 construction

5'-homology arm

3'-homology arm

Forward

gcagcttaagTGTCCCTCGGCTATGTCAGA

gtcgtctagaTGGGTGTAGTTGTGGGGGAG

Reverse

ggtcccgcggCGCTAACTCCGGCTGGACTT

ccagctcgagCACATTCACTCCCCACCCTC

NEAT1 knock-in check primers

5'-knock-in

3'-knock-in

Forward

AGACTGATGGGGCAGATCCT

AGGGGATCCGCTGTAAGTCT

Reverse

CCATGTTATCCTCCTCGCCC

ACCACGGTCCATGAAGCATT

Spheroids

monolayer

HuH7

HepG2

HLE

HLF

HuH6

PLC/PRF/5

100 µm

Figure S1 Spheroid culture of HCC cell lines. A, Representative images of spheroids of HCC cell lines.

(Figure S1, continue)

total hNEAT1

PLC/PRF/5

HuH6

HLF

HLE

16

12

10

HuH6

PLC/PRF/5

HLF

HLE

PLC/PRF/5

HepG2

HuH6

HuH7

HLF

HLE

PLC/PRF/5

HuH6

14

HLF

EPCAM

18

HLE

CD133

HepG2

HuH7

CD44

100

HepG2

HuH7

HuH6

HLE

HepG2

HuH7

PLC/PRF/5

HLF

relative mRNA levels / HPRT1

monolayer culture

spheroid culture

90

80

70

60

50

40

30

20

10

HepG2

10

NEAT1v2

HuH7

relative mRNA levels / HPRT1

12

(Figure S1, continue) B, mRNA expression levels of CSC markers relative to HPRT1 in monolayer cells (blue

columns) and in spheroids (orange columns) (n = 3). The data are the same as those shown in Figure 1, but without

normalization to the expression levels in the monolayer cells.

mouse Neat1 (GRCm38/mm10: chr19: 5,824,708 – 5,845,480)

3.2 kb

mNeat1v1 (NR_003513)

mNeat1v2 (NR_131212)

20.8 kb

human NEAT1 (GRCh38/hg38: chr11: 65,422,798 – 65,445,540)

3.8 kb

hNEAT1v1 (NR_028272)

hNEAT1v2 (NR_131012)

22.7 kb

percent identity (hNEAT1/mNeat1)

Alignment tools

variant 1

variant 2

BLASTN

69%

69%

MAFFT

65.3%

55.6%

Kalign

64.4%

53.8%

Clustal Omega

55.2%

55.5%

mouse Neat1

mNeat1v1

mNeat1v2

v1 v2

mNeat1

HuH7

v1 v2

mNeat1

HepG2

1.0

0.8

CTRL (C)

mNeat1v1

mNeat1v2

0.2

v1 v2

mNeat1

HuH7

< 0.001

0.4

< 0.001

0.6

< 0.001

< 0.001

1.0

mRNA/β-actin

1.5

< 0.001

mRNA/β-actin

2.0

0.5

v2 primers

1.2

< 0.001

total primers

2.5

v1 v2

mNeat1

HepG2

Figure S2 Establishment of HCC cell lines expressing mouse Neat1 variant 1 (mNeat1v1) and Neat1 variant 2

(mNeat1v2). A, Genomic structure of mouse (upper) and human (lower) NEAT1. Identity of the mouse and human

NEAT1 gene is summarized in the table. hNEAT1v1, human NEAT1 variant 1; hNEAT1v2, human NEAT1 variant

2. B, The expression levels of mNeat1 in HCC cells stably transfected with mock (open columns), mNeat1v1 (gray

columns) or mNeat1v2 (black columns) plasmids. Total primers (left) amplify both mNeat1v1 and mNeat1v2, while

v2 primers (right) amplify only mNeat1v2. Their locations are shown. (n = 4).

(Figure S2, continue)

mouse Neat1

AAAAA....

mNeat1v1 RNA

mNeat1v2 RNA

(no polyA tail)

total primers

v2 primers

RT primers:

mV1-dT14 (mNeat1v1-specific oligo(dT) primer)

N9 (random N9 oligoDNA primer)

HuH7

6.0

4.0

2.0

0.0

1.5

3.0

2.0

1.0

0.0

0.5

0.0

1.0

1.0

0.8

0.8

RT–/RT+

RT–/RT+

mV1-dT14

mNeat1v2

(v2 primers)

0.6

0.4

0.6

0.4

0.2

0.0

0.0

N9

mV1-dT14

N9

mV1-dT14

N9

mV1-dT14

1.0

RT–/RT+

1.0

RT–/RT+

N9

0.5

mV1-dT14

0.2

HepG2

mV1-dT14

1.0

total mNeat1

(total primers)

HuH7

N9

1.5

0.0

N9

RT–

overexpressing cells

CTRL (C)

mNeat1v1

mNeat1v2

1.0

mV1-dT14

mRNA/β-actin (N9)

mRNA/β-actin (N9)

N9

HepG2

mNeat1v2

(v2 primers)

mRNA/β-actin (N9)

RT+

mRNA/β-actin (N9)

total mNeat1

(total primers)

TTTTTTTTTTTTTTGAAGCT

NNNNNNNNN

0.8

0.6

0.4

0.2

0.8

0.6

0.4

0.2

0.0

0.0

N9

mV1-dT14

(Figure S2, continue) C, The expression levels of mNeat1v1 in stably transfected with mock (open columns),

mNeat1v1 (gray columns) or mNeat1v2 (black columns) plasmids. Following DNase treatment, the total RNA were

reverse-transcribed with mNeat1v1-specific oligo(dT) (mV1-dT14) or random N9 oligoDNA (N9) primers by

ReverTra Ace (TOYOBO, Japan) (RT+). The data sown as RT+ were normalized by the expression levels of β-actin

that was reverse-transcribed by the N9 primer. RT–; total RNA incubated in the same reaction mixture without ReverTra Ace. The ratio of RT– to RT+ was shown to indicate the negligible contamination of genomic DNA. (n = 4).

Figure S3 Establishment of NEAT1-KO HCC cell lines. We used two different sgRNAs (sgNEATa and sgNEATb),

which were designed to target sites proximal to the transcription start site of the NEAT1 gene. Genomic DNA PCR

with primers indicated in this figure demonstrated that both alleles were deleted in HuH7 clone #4 obtained by

sgNEATa and clone #10 obtained by sgNEAT1b, and HepG2 clone #4 obtained by sgNEATb. HepG2 clone #1 had

single allele deletion.

Figure S4 Involvement of P21CDKN1A in spheroid formation of HCC cell lines. A, P21CDKN1A expression in HCC cell

lines expressing mNeat1v1 (v1) and mNeat1v 2 (v2). B, P21CDKN1A expression in NEAT1-KO HCC cell lines. C,

Confirmation of P21CDKN1A knockdown in HCC cell lines. GAPDH was used as an internal control. D, Spheroid

formation ability of HCC cell lines transfected with a negative control siRNA (siNC) or siRNAs targeting P21CDKN1A

(si1 and si2). *P < 0.05 vs. siNC; Dunnett’s test (n = 4).

Figure S5 Cell cycle analysis of NEAT1-KO HCC cell lines. A, Representative histograms of flow cytometry

analysis of NEAT1-KO cell lines.

(Figure S5, continue) B, The cell cycle fraction of NEAT1-KO HCC cell lines. (n = 4–5)

Figure S6 Relationship between NEAT1 expression and prognosis in HCC patients. A, (left) total NEAT1 and

NEAT1v2 expression levels in HCC tumor (T) or non-tumor liver (NT) tissues. Bars indicate average. P values were

calculated by Student’s paired t-test. (right) Regression analysis between total NEAT1 and NEAT1v2 expression in

tumor tissues. Patients above the regression line were considered as total NEAT1 dominant (NEAT1v1 dominant),

while the others were NEAT1v2 dominant. B–D, Overall survival (left) and recurrence-free survival (right) rates of

all HCC patients (B), NEAT1v1 dominant patients (C), and NEAT1v2 dominant patients (D) stratified according to

the median values of total NEAT1 or NEAT1v2 expression. P values were determined by Gehan’s Wilcoxon test.

Figure S7 Correlation between CD44 and NEAT1 expression in HCC tumor tissues. A–C, Regression plots between

NEAT1 total and NEAT1v2 (left), between NEAT1 total and CD44 (middle), and between NEAT1v2 and CD44 (left)

in all HCC patients (A), NEAT1v1 dominant patients (B), or NEAT1v2 dominant patients (C) stratified according to

median values of NEAT1 total or NEAT1v2 expression. P values were determined by Spearman’s rank correlation

test.

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