miR-409 directly targets RDX in GC
MicroRNA-409 Suppresses Tumour Cell Invasion and
Metastasis by Directly Targeting Radixin in Gastric Cancers
Biqiang Zheng1,*, Linhui Liang2,*, Shenglin Huang2,, Ruopeng Zha2,, Li Liu2,, Deshui
Jia2,, Qi Tian2,, Qifeng Wang1,, Chunmong Wang1,, Ziwen Long1,, Ye Zhou1,, Xi Cao1,,
Chunyan Du1,, Yingqiang Shi1,†, Xianghuo He1,2,†
1, Department of Gastric Cancer and Soft Tissue Sarcomas, Fudan University Shanghai Cancer
Center; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032,
China;
2, State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai
Jiao Tong University School of Medicine, Shanghai 200032, China;
*
†
These authors contributed equally to this study.
Contacts ： Dr. Yingqiang Shi, Department of Gastric Cancer and Soft Tissue
Sarcomas, Fudan University Shanghai Cancer Center; Department of Oncology,
Shanghai Medical College, Fudan University, Shanghai 200032, China. Tel/Fax:
86-21-64430130; Email: [email protected] and Dr. Xianghuo He, State Key
Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai
Jiao Tong University School of Medicine, Shanghai 200032, China. Tel/Fax:
86-21-64436539; E-mail: [email protected].
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miR-409 directly targets RDX in GC
Supplementary Table and Figures
Supplementary Table S1 The candidate targets for miR-409-3p in GC cells
EXT1
REPS1
SPRED1
RDX
ZFAND5
ZMIZ1
exostoses (multiple) 1
RALBP1 associated Eps domain containing 1
sprouty-related, EVH1 domain containing 1
radixin
zinc finger, AN1-type domain 5
zinc finger, MIZ-type containing 1
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miR-409 directly targets RDX in GC
Supplementary Figure S1
Supplementary Figure S1 miR-409-3p inhibits GC cell migration and invasion.
Transwell migration (A) and invasion (B) assays of HGC-27 cells transfected with
miR-9 mimic, miR-126 mimic, miR-151-3p mimic, miR-151-5p mimic, miR-199b-3p
mimic, miR-199b-5p mimic, miR-218 mimic, miR-409-3p mimic or NC. *P<0.05;
**
P<0.01.
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miR-409 directly targets RDX in GC
Supplementary Figure S2
Supplementary Figure S2 Stable expression of miR-409-3p in gastric cancer cell
lines. (A) The expression level of miR-409-3p was determined by TaqMan real-time
PCR in various GC cells and immortal gastric epithelial cells (GES-1). U6 served as
an internal control. The value of miR-409-3p expression (miR-409-3p/U6) was
calculated as fold change relative to GES-1. (B) Successful overexpression of mature
miR-409-3p was confirmed by TaqMan real-time PCR after infection with
miR-409-expressing or control lentivirus. The value of miR-409-3p expression
(miR-409-3p/U6) was calculated as fold change relative to the vector control.
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miR-409 directly targets RDX in GC
Supplementary Figure S3
Supplementary Figure S3 miR-409 has no effect on GC cell proliferation in vitro
and in vivo.
(A) miR-409 expression has no correlation with Ki-67 level in 62 GC samples
analysed. Correlation was performed with Spearman correlation analysis. (B) Cell
proliferation assays in the miR-409 stably expressing cell lines were performed by
Cell Counting Kit-8. (C) The effect of miR-409-3p mimic, miR-409-5p mimic or NC
on cell proliferation was assessed in SGC-7901 and HGC-27 cells. Statistical analysis
was performed by one-way ANOVA. (D) Cell proliferation assays of MKN-45 cells
after transfection with miR-409-3p inhibitor, miR-409-5p inhibitor or NC inhibitor
were performed. Statistical analysis was performed by one-way ANOVA. (E)
SGC-7901 cells infected with miR-409 or mock vector were subcutaneously injected
into mice respectively. Tumor volumes were calculated for each week. (F) After four
weeks, the mice were sacrificed. The tumour weights were measured and analysed.
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miR-409 directly targets RDX in GC
Supplementary Figure S4
Supplementary Figure S4 Luciferase assays of potential targets of miR-409-3p.
Luciferase activity assays of luciferase vectors with 3′UTR of EXT1, REPS1, RDX,
SPRED1, ZFAND5 or ZMIZ1 were performed after cotransfection with miR-409-3p
mimic or NC. The luciferase activity was normalised to Renilla luciferase activity.
The normalised luciferase activity of vector and NC transfection was set as relative
luciferase activity 1.
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miR-409 directly targets RDX in GC
Supplementary Figure S5
Supplementary Figure S5 the mRNA expression levels of RDX in regulated by
miR-409-expressing GC cells.
The mRNA level of RDX was determined by quantitative real-time PCR in the
miR-409 stably expressing cell lines (A) or in SGC-7901 and HGC-27 cells after
transfection with miR-409-3p mimic, miR-409-5p mimic or NC (B). β-Actin served
as an internal control.
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miR-409 directly targets RDX in GC
Supplementary Figure S6
Supplementary Figure S6 the relationship between miR-409-3p and RDX
expression.
A.The expression levels of RDX mRNA had no significant reverse correlation with
those of miR-409-3p in GC samples examined in this study. B. The expression levels
of miR-409-3p had no significant reverse correlation with RDX protein levels in GC
samples examined in this study.
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