Interleukin-22 promotes papillary thyroid cancer cell
migration and invasion through direct downregulation of
Sox17 expression mediated by miR-595
Zhidan Meia; Li Zhoub; Youhua Zhua; Kejia Jiea; Daqing Fana ; Jian Chena; Xiguo Liua;
Liang Jianga; Qike Jiaa; Wei Lia*
Supplementary Figure 1. IL-22 can not affect the proliferation of these thyroid
cancer cells. TPC-1 cells (A) and KAT-5 cells (B) were treated with rhIL-22 for 48
hour at the indicated concentrations. The proliferation of the cells were quantified by
MTT assay. Bar graphs represent means ± SD, n = 3 (**P < 0.01; *P < 0.05).
Supplementary Figure 2. IL-22 promotes the migration and invasion of KAT-5
cell.
Experiments were performed as in Fig 1 except KAT-5 cells were performed.
Bar graphs represent means ± SD, n = 3
Supplementary Figure 3. MicroRNA-595 is involved in IL-22 induced migration
and invasion of KAT-5 cells. (A) KAT-5 cells were transfected with pre-miR-595 or
control for 24 h. Then, cells were treated with or without rhIL-22 (50 ng/ml, 24 h) and
allowed to migrate or invade towards serum for 24 hours. (B) The experiments were
performed as in (A), except the indicated anti-miR-595 or anti-miR control were used.
The images shown are representative images from three independent experiments, and
a statistical analysis was performed. Bar graphs represent means ± SD, n=3 (**P <
0.01; *P < 0.05).
Supplementary Figure 4. MiR-595 up-regulated the migration and invasion of
TPC-1 cells. (A) TPC-1 cells were transfected with pre-miR-595 or control for 48 h
at the indicated concentrations. The proliferation of the cells were quantified by MTT
assay. (B) Experiments were performed as in A, except the indicated anti-miR-595 or
anti-miR control were used. (C, D) TPC-1 cells were transfected with pre-miR-595 or
control for 48 h at the indicated concentrations prior to transwell migration (C) or
invasion (D) analyses. (E, F) Experiments were performed as in C and D, except the
indicated anti-miR-595 or anti-miR control were used. Bar graphs represent means ±
SD, n = 3
Supplementary Figure 5. Sox17 is a target of miR-595. (A, B) KAT-5 cells were
co-transfected with either WT or MUT Sox17 3’-UTR reporter plasmids and either
pre-miR-595 (A) or anti-miR-595 (B) for 48 h prior to luciferase activity analyses. (C)
KAT-5 cells were transfected with pre-miR-595 (left panel) or anti-miR-595 (right
panel) for 48 h. Sox17 mRNA was quantified by real-time PCR. Bar graphs represent
means ± SD, n= 3 (**P < 0.01; *P < 0.05).
Supplementary Figure 6. The role of Sox17 in the migration and invasion of
KAT-5 cells. (A) KAT-5 cells were transfected with pCMV-Sox17 or control vector
for 48 h prior to transwell migration and invasion. (B) Experiments were performed as
in (A), except cells were transfected with shRNA-control or Sox17 shRNA. The
images shown are representative images from three independent experiments, and a
statistical analysis was performed. Bar graphs represent means ± SD, n=3 (**P < 0.01;
*P < 0.05).
Supplementary Figure 7. MiR-595 inhibition of KAT-5 cell migration and
invasion is mediated by Sox17. Experiments were performed as in Fig 5 except
KAT-5 cells were performed. Bar graphs represent means ± SD, n = 3
Supplemental Table 1: Correlation of IL-22, miR-595 and Sox17
expression with clinicopathologic features in papillary thyroid cancers
(PTC).
Clinicopatholo
Cas
IL-22
P
miRNA-595
P
Sox17
P
gic parameters
e
expression (folds)
value
expression
value
expression
value
no.
(folds)
(folds)
Age
≤50
18
170.18±10.36
＞50
30
177.31±18.24
ns
630.19±64.25
635.47±60.87
ns
13.29±7.48
11.18±3.34
ns
Tissue type
Normal tissue
33
8.67±4.76
<0.01
Carcinoma
48
175.04±16.58
Male
18
183.2±21.43
Female
30
169.47±17.26
≤5cm
29
108.33±24.56
＞5cm
19
242.58±45.26
Ⅰand Ⅱ
27
93.64±18.24
Ⅲ and Ⅳ
21
257.18±32.17
Negative
26
91.24±14.65
Positive
22
259.47±37.81
Negative
26
64.21±17.18
Positive
22
286.14±40.59
18.49±9.79
<0.01
633.54±73.16
36.42±9.09
<0.01
12.79±6.59
Sex
ns
618.53±57.89
ns
12.08±5.21
649.24±55.61
ns
13.14±3.47
Tumor size
<0.01
426.59±49.36
<0.01
18.64±
840.27±61.69
<0.01
7.36±
TNM stage
<0.05
387.45±47.61
<0.01
879.17±67.26
20.83±5.97
<0.05
5.17±1.37
Lymph
nodemetastasis
<0.01
354.28±34.18
<0.01
912.08±74.36
21.75±7.27
<0.01
4.25±1.18
Distant
metastasis
<0.01
284.36±30.79
982.17±77.37
<0.01
22.83±8.31
<0.01
3.17±1.28
Supplementary Table 2: Primers Used in Real-time PCR
5’primer
Gene
3’primer
name
GAPDH 5’-GGAAGGTGAAGGTCGGAGTCAA 5’-CTCGCTCCTGGAAGATGGTGATG
Sox17
CGG-3’
GG-3’
5’-GGATACGCCAGTGACGACC-3’
5’-ACGACTTGCCCAGCATCTT-3’
Supplementary Table 3: The target sequence of shRNAs
shRNAs
shRNA-Sox17#1
target sequence (5’ to 3’)
5’-GCATCGCTGGGTTTAAGATAAAGGA-3’
shRNA-Sox17#2
5’-CACGGAATTTGAACAGTATCTGCAC-3’
shRNA-Sox17#3
5’-CCTGGAGGAGCTAAGGAAATCCTCA-3’
shRNA-control
5’-CAACTATCCTGACGTGTGACAGGTC-3’
Supplementary Table 4: Identification of miRNAs differentially
expressed in IL-22 treated TPC-1 cells.
miRNA
hsa-miR-155
hsa-miR-184
hsa-miR-711
hsa-miR-563
hsa-miR-595
hsa-miR-210
hsa-miR-93
hsa-miR-150
hsa-miR-409-3p
Fold-change
3.12
4.17
3.34
7.36
5.24
6.17
3.46
0.26
0.49
P-valve
0.015
0.029
0.041
0.008
0.026
0.001
0.028
0.013
0.007