01.12.2017 | Research article | Ausgabe 1/2017 Open Access

Amplification and up-regulation of MIR30D was associated with disease progression of cervical squamous cell carcinomas
- Zeitschrift:
- BMC Cancer > Ausgabe 1/2017
Electronic supplementary material
Background
Methods
Patients and tissue collection
Characteristics
|
Information
|
|
---|---|---|
Age
|
Average age
|
49.8
|
Range
|
18 ~ 71
|
|
Risk factors
|
Multiple sex partners
|
55
|
Multiparous or pregnant at youth
|
26
|
|
First sex before 18
|
19
|
|
None of above
|
36
|
|
FIGO Staging
|
I
|
35
|
II
|
78
|
|
III
|
16
|
|
IV
|
7
|
|
HPV infection
|
High risk (HPV16, 18 or both)
|
99
|
Other HPVs
|
37
|
|
Lymph node metastasis(LNM)
|
With LNM
|
33
|
Without LNM
|
103
|
|
Smoking history
|
Yes
|
21
|
No
|
115
|
RNA extraction and quantitative real-time PCR (qPCR)
Gene
|
Forward
|
Reverse
|
---|---|---|
ATG12
|
TTGTGGCCTCAGAACAGTTG
|
GAGAGTTCCAACTTCTTGGTCTG
|
GNPDA1
|
TGCCTGTTTGAAGCTACTGC
|
ACCAAACATAGCCCTTAGGC
|
CASP3
|
AGGACTCTAGACGGCATCCA
|
TGACAGCCAGTGAGACTTGG
|
CCNE2
|
TAATAAGGCTTAGATGAACATGGTG
|
AGTTAGGAAGGAGCCACAGC
|
GALNT1
|
TTGTGCCTAAGAATGTTTCCA
|
CCCATGTGCTTGATGTTGAT
|
GNAI2
|
GACCATCTGCTTCCCTGAGT
|
TGGTGTCTTTGCGCTTATTC
|
FOXO3
|
TGATTTGAAGCACCTCATCC
|
TTAAGAAAGGCGGCAGAGTT
|
SNAI1
|
TCTGGTTCTGTGTCCTCTGC
|
GACAGGCCAGCTCAGGAAT
|
SOCS1
|
CGACTACCTGAGCTCCTTCC
|
AACACGGCATCCCAGTTAAT
|
SPRR2D
|
CTGTAGTACACATCACTTGTGGC
|
ACTTGCATCCCAGGACAGAT
|
GAPDH
|
TCCAAAATCAAGTGGGGCGA
|
TGATGACCCTTTTGGCTCCC
|
ACTB
|
GACCTGACTGACTACCTCATGAAGAT
|
GTCACACTTCATGATGGAGTTGAAGG
|
DNA extraction and quantification of copy numbers
Fluorescence in situ hybridization (FISH) analysis
Cell culture and proliferation assay
Cell invasion assay
Retroviral transduction
Generation of the in vivo xenograft model
Gene expression studies
Statistical analysis
Results
Enhanced expression of miR-30d in CSCCs was correlated with tumor progression
Gene copy number gains of MIR30D in CSCC samples
Samples
|
n
|
Copy number
|
P
(vs. HNC)
|
||||
---|---|---|---|---|---|---|---|
Deletion
|
Amplification
|
||||||
0
|
1
|
2
|
3
|
>3
|
|||
HNC
a
|
168
|
1
|
7
|
155
|
3
|
2
|
---
|
ANT
a
|
136
|
2
|
1
|
127
|
4
|
2
|
0.747
|
Population
|
Number
|
Copy numbers
|
P (vs. ANT)
|
P (vs. early-stage CSCC)
|
||
---|---|---|---|---|---|---|
Deletion or Unalteration
|
Amplification
|
|||||
≦2
|
>2
|
|||||
Total
|
ANT
|
136
|
130
|
6
|
---
|
---
|
CSCC
|
105
|
31
|
9.79E-06
|
---
|
||
Early-stage (0-IIa)
|
ANT
|
82
|
80
|
2
|
---
|
---
|
CSCC
|
68
|
14
|
2.83E-3
|
---
|
||
Stage 3–4 (IIb-IV)
|
ANT
|
54
|
50
|
4
|
---
|
---
|
CSCC
|
38
|
17
|
8.92 E-4
|
0.038
|
Population
|
Number
|
Copy numbers
|
P (vs. ANT)
|
P (vs. early-stage CSCC)
|
||
---|---|---|---|---|---|---|
Deletion or Unalteration
|
Amplification
|
|||||
≦2
|
>2
|
|||||
Total
|
ANT
|
136
|
130
|
6
|
---
|
---
|
CSCC
|
105
|
31
|
9.79E-06
|
---
|
||
Without LNM
|
ANT
|
103
|
99
|
4
|
---
|
---
|
CSCC
|
84
|
19
|
1.95E-3
|
---
|
||
With LNM
|
ANT
|
33
|
31
|
2
|
---
|
---
|
CSCC
|
21
|
12
|
6.73E-3
|
0.0327
|
Results from real-time PCR analysis
|
Case number
|
Stage
|
CSCC
|
ANT
|
MIR30D gene amplification Ratio (T/N)
|
||
---|---|---|---|---|---|---|---|
Gains on 8q24 (MIR30D)
|
Gains on 8q
|
Gains on 8q24 (MIR30D)
|
Gains on 8q
|
||||
Unaltered MIR30D copy number
|
1
|
2
|
−
|
−
|
−
|
−
|
0.97
|
2
|
3
|
−
|
−
|
−
|
−
|
1.03
|
|
3
|
3
|
−
|
−
|
−
|
−
|
0.92
|
|
4
|
3
|
−
|
−
|
−
|
−
|
1.15
|
|
5
|
4
|
−
|
−
|
−
|
−
|
1.02
|
|
MIR30D amplification
|
6
|
2
|
+
|
−
|
−
|
−
|
16.75
|
7
|
3
|
+
|
−
|
−
|
+
|
8.48
|
|
8
|
4
|
+
|
+
|
−
|
−
|
3.62
|
|
9
|
4
|
+
|
−
|
−
|
−
|
22.43
|
|
10
|
4
|
+
|
+
|
−
|
−
|
9.67
|
Positive correlation between amplifications of MIR30D gene and miR-30d up-regulation in CSCCs
miR-30d plays an oncomiric role in CSCC cells
SiHa (mm
3)
|
HeLa (mm
3)
|
|
---|---|---|
Control (
n = 15)
|
892.037 ± 329.843
|
496.234 ± 219.317
|
miR-30d suppressed (
n = 15)
|
342.902 ± 152.035
|
204.298 ± 98.953
|