HEP-11-1477 Revised Supporting Information
1
Supporting Information
Supporting Materials and Methods
Plasmids Construction. An intact mature hsa-miR-7 target site was obtained by annealing the following DNA
oligonucleotides (5’-GGAGATCTGCTAGCCAACAAAATCACTAGTCTTCC A-3’and
5’-GGAGATCTGATATCTGGAAGACTAGTGATTTTGTTG-3’)，in which contained BglII, NheI and EcoRV
sites for cloning. The reporter plasmid phEFW-luc/miR-7-target was generated by inserting BglII-digested
fragment above into phEFW-luc plasmid, which was similar to what we described in our previous work [1]. The
3’UTR (2078bp) of PIK3CD was amplified by PCR using the following primers: 5’-TCTGCTAGCACATTGGT
CCTAAAGGGGCT-3’ and 5’-GGT GATATCGTTTCCCTTCGCAGATGTGT-3’. The 3’UTR was
double-digested with NheI/EcoRV and inserted into the plasmid phEFW-luc/miR-7-target to replace the intact
mature hsa-miR-7 target site, and the resultant reporter plasmid was designated as phEFW-luc/PIK3CD 3’UTR.
Similarly, the reporter plasmids phEFW-luc/PIK3CD -A, -B, -C and -D were constructed by replacing the mature
miR-7 target site in phEFW-luc/miR-7-target with oligonucleotides corresponding to 3587-3626, 4104-4143,
4440-4479, and 4758-4797nt of the 3’UTR (GeneBank accession number NM_005026.3) respectively. The
putative miR-7 target sites A, B, and C were integrated into target E, and the resultant reporter plasmid named as
phEFW-luc/ PIK3CD -E. All above putative miR-7 target sites (A, B, C and D) were enrolled in wild type (WT)
and mutant (MT) counterparts simultaneously. Likewise, the putative miR-7 target site counterparts (WT and MT)
in the 3’UTR of mTOR (GeneBank accession number NM_004958.3, nt.8311-8360), and p70S6K (GeneBank
accession number NM_003161.2, nt. 2911-2960 and 3541-3590 respectively) were also used to construct
corresponding reporter plasmids, those are phEFW-luc/mTOR -WT, phEFW-luc/Mtor -MT, and
phEFW-luc/p70S6K -WT and phEFW-luc/p70S6K -MT respectively. Plasmid pEGP-miR-7 was constructed to
overexpress miR-7 in HCC cell lines by inserting pri-miR-7 fragment (110bp, GeneBank accession number
NR_029605.1) into a pEGP-miR-null vector (Cell BioLabs, USA). Plasmid pEGP-miR-NC (Cell BioLabs) was
employed as a non-effective control vector containing a 304bp mmu-let7a-1 precursor sequence. All of the
plasmids were confirmed by sequencing and purified with the Endo-free Plasmid Midi Kit (QIAGEN, USA) for
transfections.
HEP-11-1477 Revised Supporting Information
2
Establishment of miR-7-overexpression Stable Cells. The miR-7 expression vector pEGP-miR-7 and the other
two control vectors were transfected into QGY-7703 cell respectively using Lipofectamine 2000 (Invitrogen). 48
hrs after transfection, transfected cells were selected with 1.5 μg/ml puromycin (Sigma-Aldrich, USA).
Quantitative RT-PCR. The miR-7 precursor molecules, PIK3CD siRNAs (PIK3CD stealth, Invitrogen), mTOR
siRNAs (FRAP stealth, Invitrogen) and relevant controls were first transfected into QGY-7703 cells respectively,
then total RNA was extracted using TRIZOL (Invitrogen) 24 hours after transfection and subjected to reverse
transcription for investigating the expression of target genes using the cDNA-specific primers (Supporting Table
1). For qRT-PCR assay, aliquots of cDNA were amplified using SYBR-Green mixture (TOYOBO, Tokyo, Japan)
and ABI Prism7000 Sequence Detection System (Applied Biosystems). For analysis of miR-7 expression, reverse
transcription and qRT-PCR were carried out using TaqMan miRNA assay kits (Applied Biosystems) with
has-miR-7 (Applied Biosystems, Part # 4373014) and U44 (internal control, Applied Biosystems, Part # 4373384)
specific reverse transcription primers respectively according to the manufacturer’s instructions.
Western Blot. Cells were harvested and lysed as what we described in our previous work [1]. All the samples
were then subjected to electrophoresis using 10% SDS-PAGE and transferred to a polyvinylidene difluoride
membrane (Amersham Biosciences, USA). The membrane was first probed with relevant antibodies and
sequentially hybridized with HRP-labeled secondary antibodies for visualization using ImmobilonTM Western
HRP substrate kit (Millipore, USA). Antibodies to p110α, p110β and p110δ were purchased from Santa Cruz
Biotechnology. Antibodies to p85, AKT, phospho-AKT, mTOR, phospho-mTOR p70S6K, phosphor-p70S6K,
4EBP1，phosphor-4EBP1 and GAPDH were purchased from Cell Signaling Technology.
Wound Healing Assay. A scraped area was created using a 200-μl pipette tip in order to assess cell migration by
measuring the mobility of cells into the acellular area. The speed of wound closure was assessed at 24 and 48 hrs
using an IX70 microscope (Olympus, Tokyo, Japan).
HEP-11-1477 Revised Supporting Information
3
Supporting Reference
1. Fang YX, Zhang XB, Wei W, Liu YW, Chen JZ, Tian L Development of chimeric gene regulators for
cancer-specific gene therapy with both transcriptional and translational targeting. Mol Biotechnol 2010; 45:71-81.
HEP-11-1477 Revised Supporting Information
Supporting Figures and Tables
Figure S1. Down-regulation of the PIK3CD expression induces cell cycle arrest of HCC cells. (A) The
expression of miR-7, PIK3CD mRNA and p110δ protein was tested in transiently transfected QGY-7703 cells.
PIK3CD siRNA#3 was identified as the most effective silencer and was selected as a positive control for the
following experiments. (B) Transient transfection with either miR-7 precursor or PIK3CD siRNA#3 resulted in a
G0/G1 phase arrest. The proportion of cells in G0/G1 phase (top), S phase (middle) or G2/M phase (bottom) was
measured every 4 hrs from 48 to 72 hrs post-transfection. All of the experiments were repeated in triplicate. ＊
indicates a significant difference from the control (p < 0.01).
4
HEP-11-1477 Revised Supporting Information
Figure S2. Transient transfection of miR-7 arrests cell cycle progression without triggering apoptosis. The
proportion of cell numbers was measured at 72 hrs post-transfection by PI staining and flow cytometry analysis.
No apoptosis was observed in either miR-7 precursor or PIK3CD siRNA#3 transfected QGY-7703 cells.
5
HEP-11-1477 Revised Supporting Information
6
Figure S3. Stable expression of miR-7 inhibits cell growth without triggering apoptosis. (A) The expression
level of miR-7 was detected in stably expressed subclones by TaqMan MicroRNA Assay (means±SE, n=3). (B)
Stable expression of miR-7 did not affect the expression of the p110 alpha, p110 beta or their corresponding
regulatory subunit, p85. (C) Cell growth was suppressed by overexpressing miR-7 without triggering apoptosis.
Original magnification: 100×. ＊indicates a significant difference from the QGY-null subclone control (p < 0.01).
HEP-11-1477 Revised Supporting Information
Figure S4. Down-regulation of the PIK3CD expression induces cell growth inhibition. Cell growth was
suppressed by PIK3CD siRNA#3 without triggering apoptosis (means ± SE, n=3). Original magnification: 100×.
＊indicates a significant difference from the control (p < 0.01).
7
HEP-11-1477 Revised Supporting Information
Figure S5. Stable expression of miR-7 decreases cell motility in HCC cells. Using wound healing assay, the
cell motilities of QGY-null, QGY-miR-NC and QGY-miR-7 subcloned cells were observed at 0, 24 and 48 hrs
following wounding by a pipette tip. Original magnification: 100×.
8
HEP-11-1477 Revised Supporting Information
9
Figure S6. Stable expression of miR-7 reduces the invasive capacity of HCC cells. Using the matrigel invasion
assay, the invasion capabilities of QGY-null, QGY-miR-NC and QGY-miR-7 subcloned cells were assessed by
counting the number of invasive cells after a 24 hr incubation. The results demonstrate a limited effect of miR-7
on invasiveness. Original magnification: 400×.
HEP-11-1477 Revised Supporting Information
10
Figure S7. Down-regulation of the PIK3CD expression inhibits the invasion and migration of HCC cells.
The invasion and migration assays showed that PIK3CD repression had a limited effect on invasiveness but could
significantly inhibit the migration of QGY-77003. Original magnification: 400×. ＊indicates a significant
difference from the control (p < 0.01).
HEP-11-1477 Revised Supporting Information
11
Figure S8. The inhibition of PIK3CD expression by PIK3CD siRNA#3 alters the expression of key
components in the PI3K/Akt pathway. The expression profile changes of Akt, mTOR, p70S6K and 4EBP1
genes were measured at the post-transcriptional level (left). The values were measured by qRT-PCR and
normalized to beta actin mRNA (means±SE, n=3). The changes were also measured at the translational level
(right). PIK3CD repression significantly inhibited the expression of Akt, mTOR and p70S6K proteins but
increased the expression of 4EBP1 protein. ＊indicates a significant difference from the control (p < 0.01).
HEP-11-1477 Revised Supporting Information
Figure S9. Overexpression of miR-7 reduces the expression of PI3K/Akt pathway components in tumor
tissues and reverses metastasis in vivo. (A) Tumorigenesis in a subcutaneous xenograft of stable subclones in
vivo. A typical example of each tumor on Day 30 is shown here. (B) Identification of the expression of PIK3CD
and PI3K/Akt pathway components in tumor samples. The expression of PIK3CD and downstream components
was detected by western blot. (C) GFP fluorescence imaging for detecting cancer cell distribution in situ.
miR-7-overexpressing QGY-7703 stable subclones or null controls were injected intravenously into nude mice
(n=5). Imaging was performed on Week 8 after injection. Typical examples of QGY-null mice (left) or
QGY-miR-7 mice (right) are shown.
12
HEP-11-1477 Revised Supporting Information
13
Figure S10. Overexpression of miR-7 inhibits the metastasis of HCC cells in vivo. The nodule generation was
obviously observed on the surface of the lung in all QGY-null injected mice but almost not in QGY-miR-7 ones.
No obvious nodules were observed on the surface of the liver in both groups while local inflammation and
necrosis existed in one of the QGY-null generated liver (shown in arrow).
HEP-11-1477 Revised Supporting Information
14
Figure S11. Overexpression of miR-7 reduces the expression of PIKK3CD and PI3K/Akt pathway
components in nude mouse model. (A) The expression of miR-7, PIK3CD and PI3K/Akt pathway components
was detected in the liver of both QGY-null and QGY-miR-7 injected mouse by TaqMan MicroRNA assay,
qRT-PCR and western blot analysis respectively. (B) Analysis of the expression of miR-7, PIK3CD and PI3K/Akt
pathway components in lung-metastatic nodules. The expression of miR-7, PIK3CD and downstream components
was detected by TaqMan MicroRNA Assay, qRT-PCR and western blot respectively. (C) Histological studies for
the in vivo confirmation of metastasis. H&E staining was performed on serial sections of metastatic tumors (M)
and normal tissues (N) derived from the liver. Original magnification: 400×. ＊indicates a significant difference
from the QGY-null-generated tumor controls (p < 0.01).
HEP-11-1477 Revised Supporting Information
15
Figure S12. Repression of miR-7 up-regulates the expression of PIK3CD and PI3K/Akt pathway components. The expression of PIK3CD, Akt and mTOR
was detected by western blot in 10 pairs of HCC samples. PIK3CD and PI3K/Akt pathway components were found to be up-regulated in 7 out of 10 HCCs. P,
Patient; N, Normal tissue; T, HCC tumor.
HEP-11-1477 Revised Supporting Information
16
Figure S13. The expression of miR-7 and PIK3CD is negatively correlated in clinical HCC specimens.
Correlation analysis was performed by Pearson χ2 test in 10 pairs of clinical HCC specimens, and a negative
correlation might exist between the expression of miR-7 and PIK3CD.
HEP-11-1477 Revised Supporting Information
17
Figure S14. Down-regulation of the mTOR expression induces cell growth inhibition of HCC cells. (A)The
expression of mTOR was repressed by mTOR siRNAs in transient transfected QGY-7703 cells. The expression of
mTOR mRNA and protein was detected by qRT-PCR and western blot analysis respectively. mTOR siRNA#3 was
found to have the best effect on repressing the mTOR expression and was employed for the following experiments.
(B) Cell growth was suppressed by mTOR siRNA#3 without triggering apoptosis (means ± SE, n=3). Original
magnification: 100×. ＊indicates a significant difference from the control (p < 0.01).
HEP-11-1477 Revised Supporting Information
18
Figure S15. Down-regulation of the mTOR expression reduces the invasive migratory capability of HCC
cells. Invasion and migration assay showed that mTOR repression had a limited effect on invasiveness but could
significantly inhibit the migration of QGY-77003. Original magnification: 400×. ＊indicates a significant
difference from the control (p < 0.01).
HEP-11-1477 Revised Supporting Information
19
Figure S16. miR-7 action in QGY-7703 and L-02. (A) The expression assay of miR-7, PIK3CD mRNA and
p110δ protein in QGY-7703 and L-02. The endogenous expression of miR-7, PIK3CD mRNA and p110δ protein
was compared between QGY-7703 and L-02. (B) The alignment assay of PIK3CD 3’UTR from both cell lines
with standard sequence. PIK3CD 3’UTR cloned from either QGY-7703 or L-02 was aligned with standard
sequence by BLAST assay and no mutations was found in the miR-7 target sites.
20
HEP-11-1477 Revised Supporting Information
Supporting Table 1: Primers for qRT-PCR assay
Gene
Forward
Reverse
PIK3CD
5’-TCGCCAACATCCAACTCAAC-3’
5’-CACACAATAGCCAGCACAGG-3’
Akt
5’-CTTCTTTGCCGGTATCGTGT-3’
5’-TGTCATCTTGGTCAGGTGGT-3’
mTOR
5’-AGGGTTCGAGATAAGCTCAC-3’
5’-TAGCACTGGCAGAGGTTTTC-3’
4EBP1
5’-ATTCCTGATGGAGTGTCGGA-3’
5’-CCGCTTATCTTCTGGGCTAT-3’
p70S6K
5’-CACCTCGAAGATTTATTGGC-3’
5’-GCTCTATGCCACTTGTTTCC-3’
beta actin
5’-CATCCTCACCCTGAAGTACCC-3’
5’-AGCCTGGATAGCAACGTACATG-3’
21
HEP-11-1477 Revised Supporting Information
Supporting Table 2: Clinical and pathologic features of patients with HCC
Patient
Gender
(M/F)a
Age
P1
M
61
Liver
Cirrhosis
(Y/N)b
Y
Etiology
Tumor
Numbers
HBV
1
Tumor
Size
(cm)
9
1210
ChildPugh
Score
A
Microvessel
Invasion
(Y/N)b
Y
Vessel
Invasion
(Y/N)b
Y
AFP
(μg/L)
TNM
Staging
Grade of
Differenti
ation
PFS
(month)
OS
(month)
IIIA
Ⅲ
3
4.6
P2
M
42
Y
HBV
>4c
2
2.13
B
Y
N
II
Ⅲ
2
2
P3
M
56
Y
HBV
1
7.5
261.37
B
Y
N
IIIA
Ⅲ
27.6
27.6
P4
M
51
Y
HBV
1
10
2.65
A
Y
N
IIIA
Ⅱ
4.8
4.8
P5
M
22
N
HBV
10
9
1210
A
Y
N
IIIA
Ⅱ
3.1
18
P6
M
56
N
HBV
6
2
6.33
A
N
II
Ⅱ
16.3
16.3
P7
M
40
Y
HBV
>4c
4.8
97.91
B
N
II
Ⅱ
24.3
24.3
P8
M
65
Y
HBV
1
4.5
113.3
B
N
I-II
Ⅱ
12.4
12.4
P9
M
46
Y
HBV
2
6
200.02
B
N
Data
missing
Data
missing
Y
Y
IIIA
Ⅰ
16.1
16.1
P10
M
56
Y
HBV
2
6
228.2
B
N
N
IIIA
Ⅰ
31.6
31.6
a: M = male; F = female b: Y = yes; N = no
Survival.
c: the accurate number is not determinant. AFP, alpha-fetoprotein; PFS, Progression-Free Survival; OS, Overall