Oncogene (2012) 31, 4509 --4516 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc

ORIGINAL ARTICLE MicroRNA-409 suppresses tumour cell invasion and metastasis by directly targeting radixin in gastric cancers

B Zheng1,2,4, L Liang3,4, S Huang3, R Zha3, L Liu3, D Jia3, Q Tian3, Q Wang1,2, C Wang1,2, Z Long1,2, Y Zhou1,2, X Cao1,2,CDu1,2, Y Shi1,2 and X He1,2,3

Emerging evidence has shown that aberrantly expressed microRNAs (miRNAs) are highly associated with tumour development and progression. However, little is known about the potential role of miRNAs in gastric cancer (GC) metastasis. In this study, miR-409-3p was found to be downregulated frequently in human GCs, and its expression was significantly associated with tumor-node-metastasis (TNM) stage and lymph node metastasis. Enforced expression of miR-409 in GC cells significantly reduced their migration and invasion in vitro and their capacity to develop distal pulmonary metastases and peritoneal dissemination in vivo. Moreover, we found that miR-409 exerted its function predominantly through the mature miR-409-3p, but not miR-409-5p. Microarray and bioinformatics analysis identified the pro-metastatic gene radixin (RDX) as a potential miR- 409-3p target. Further studies confirmed that miR-409-3p suppressed the expression of RDX by directly binding to its 30- untranslated region. Silencing of RDX by small interfering RNAs phenocopied the effects of miR-409 overexpression, whereas restoration of RDX in miR-409-overexpressed GC cells reversed the suppressive effects of miR-409. Taken together, these results demonstrate that miR-409 suppresses GC cell invasion and metastasis by directly targeting RDX and that patients with downregulated miR-409-3p are prone to lymph node metastasis.

Oncogene (2012) 31, 4509--4516; doi:10.1038/onc.2011.581; published online 19 December 2011 Keywords: gastric cancer; metastasis; microRNA-409; radixin

INTRODUCTION infection, multidrug resistance, disease diagnosis and prognosis 13,14 15 Gastric cancer (GC) is the fourth most common human malignant of GC. However, only a few miRNAs, including miR-218, miR- 16 17 18 disease and the second leading cause of cancer-related death 516a-3p, miR-107 and Let-7f, are known to be involved in the worldwide, with particularly high incidence in East Asia.1 Despite metastasis of GC. These miRNAs may have the potential to act as the significant achievements that have been made in the either biomarkers or therapeutic targets for advanced GC. We treatment of early GC, the long-term survival rate for advanced focused on miR-409 to further explore the novel mechanism GC is still quite low.2 The major obstacle in the treatment of miRNAs have in advanced GC, because miR-409-3p is highly 19 advanced GC is the presentation of lymphatic, peritoneal or distal correlated to the metastatic status of GC patients. However, the organ metastases, which simultaneously predict poor outcome for biological function of miR-409 in GC metastasis remains unknown. these patients.3,4 Although it has been reported that a large In this study, we demonstrated that miR-409-3p was frequently number of oncogenes and tumour suppressors are involved in the downregulated in human GC and was significantly associated with development of gastric carcinomas,5--7 the molecular mechanisms lymph node metastasis in an independent GC cohort. Functional underlying the invasion and metastasis of advanced gastric investigation indicated that miR-409 might act as a novel anti- carcinomas are still poorly understood. metastatic miRNA. Moreover, the potentially prometastatic gene MicroRNAs (miRNAs) are small non-coding RNAs that function radixin (RDX) was identified as a direct, functional target of miR-409. as negative regulators for protein coding genes at the post- transcriptional level.8 miRNAs can bind to complementary sequences in the 30-untranslated regions (30-UTR) of their target RESULTS mRNAs and induce mRNA degradation or translational repres- miR-409-3p is downregulated in GC and is associated with lymph sion.9 An increasing number of studies indicate that miRNAs are node metastasis involved in many important biological processes, including A previous microarray study has shown that miRNAs are proliferation, apoptosis, differentiation, angiogenesis and immune differentially expressed in GC and that some miRNAs are response, with deregulation leading to aberrant gene expression correlated with the depth of invasion, lymph node metastasis in various diseases.10 In the last 5 years, compelling evidence has and peritoneal metastasis;19 however, the functional role of these shown that miRNAs have important roles in the initiation and miRNAs in the metastasis of GC remained unexplored. Cell progression of cancers, and the molecules have thus become a migration and invasion have an important role in the process of hotspot in cancer research.11,12 Currently, many miRNAs are cancer metastasis. In this study, we first selected several miRNAs reported to be involved in proliferation, Helicobacter pylori (miR-9, miR-126, miR-151, miR-199b, miR-218 and miR-409-3p)

1Department of Gastric Cancer and Soft Tissue Sarcomas, Fudan University Shanghai Cancer Center, Shanghai, China; 2Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China and 3State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiao Tong University School of Medicine, Shanghai, China. 4These authors contributed equally to this work. Correspondence: Dr Y 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. E-mail: [email protected] or Dr X He, State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China. E-mail: [email protected] Received 22 June 2011; revised 7 November 2011; accepted 10 November 2011; published online 19 December 2011 miR-409 targets RDX expression in GC B Zheng et al 4510

Figure 1. Downregulation of miR-409-3p in human GC associates with advanced clinical stage and lymph node metastasis. (a) Expression of miR-409-3p in 90 pairs of human GC and their corresponding non-tumorous (NT) samples. Expression level of miR-409-3p was determined by TaqMan real-time PCR and normalised against an endogenous control (U6 RNA). Statistical analysis was performed by paired t-test. (b) Paired comparison of miR-409-3p expression level between primary GC and their corresponding NT samples. (c) miR-409-3p expression level in different clinical stages. Patients were staged in accordance with the 7th Edition of the American Joint Committee on Cancer TNM Classification. (d) miR-409-3p is differentially expressed in the lymph node metastasis negative group (LN-Negative) compared with the positive group (LN-Positive).

that exhibited both correlations with depth of invasion and lished miR-409 stably expressing HGC-27 and SGC-7901 cells, peritoneal dissemination in GC patients. These miRNAs were then which have a low basal level of miR-409 expression. The subjected to cell migration and invasion assays. Among them, we overexpression of miR-409-3p in the stable cell lines was found that miR-409-3p significantly suppressed GC cell migration confirmed by quantitative real-time PCR (Supplementary Figure and invasion (Supplementary Figure S1). However, to the best of S2B). The expression of miR-409-3p exhibited no relationship with our knowledge, the biological roles of miR-409-3p in human Ki-67 level (Supplementary Figure S3A) or tumour size (Table 1) in cancers have not been characterized, so we next sought to GC tissues. Correspondingly, overexpression of miR-409 had no determine the roles of miR-409 in GC. effect on the growth of the GC cells (Supplementary Figure S3B). To further confirm the correlation between miR-409-3p expres- miR-409 encodes two mature sequences, miR-409-3p and sion and metastasis in GC, we first determined the expression of miR-409-5p. We determined the individual effect of miR-409-3p miR-409-3p in 90 paired GCs and their corresponding non- and miR-409-5p on GC cell growth. As shown in Supplementary tumorous tissue, using quantitative reverse-transcription PCR. We Figure S3C, neither miR-409-3p nor miR-409-5p had an influence found that miR-409-3p expression was significantly downregu- on GC cell growth. In line with these findings, the growth of GC lated in GC tissues when compared with the corresponding non- cells was not affected by the addition of miR-409-3p or miR-409- tumorous samples (Figures 1a and b). The correlation between 5p inhibitors (Supplementary Figure S3D). Intriguingly, over- miR-409-3p expression level and the clinicopathological charac- expression of miR-409 significantly suppressed the migration teristics of GC were summarised in Table 1. The expression of miR- and invasion of SGC-7901 and HGC-27 cells compared with the 409-3p in GC patients did not significantly correlate with age, control (Figures 2a and b). gender, tumour size, location, differentiation or local invasion. However, miR-409-3p expression was significantly associated with Overexpression of miR-409 suppresses distal pulmonary tumor-node-metastasis (TNM) stage and lymph node metastasis metastases and peritoneal dissemination in vivo status. We found that miR-409-3p expression was gradually miR-409-3p has been shown to be associated with the depth of decreased in stage I, II, III and IV GC (Figure 1c); however, the invasion and peritoneal dissemination in GC patients,19 so we only significant difference (P ¼ 0.03) was between stage II and III. examined the effects of miR-409 on distal pulmonary metastases Interestingly, it was found that miR-409-3p expression was and peritoneal dissemination in vivo. For pulmonary metastasis markedly lowered in GC patients with lymph node metastasis assays, SGC-7901 cells stably expressing miR-409 were injected (n ¼ 52) than in those without lymph node metastasis (n ¼ 38; into nude mice through the lateral tail vein. Six weeks later, the Figure 1d), suggesting that miR-409-3p might have a suppressive mice were killed. The lungs were removed and subjected to role in lymph node metastasis. In addition, the expression of miR- histological and pathological examination. Intriguingly, the 409-3p in various human GC cell lines was determined. miR-409- numbers and size of the metastatic nodules in the lung samples 3p had a lower basal level of expression in HGC-27 and SGC-7901 were significantly reduced in miR-409 groups when compared cell lines, both of which originated from lymph node metastasis with the vector controls (Figure 3a). For peritoneal dissemination sites of GC (Supplementary Figure S2A). assays, the miR-409 expressing cells of SGC-7901, along with the control cells, were implanted into the peritoneal cavity of mice. Overexpression of miR-409 has no effect on GC cell proliferation, After 5 weeks, the results showed that miR-409 had the capability but suppresses GC cell migration and invasion in vitro to reduce the peritoneal dissemination, as the number of To better understand the biological functions of miR-409, we first macroscopic nodules in miR-409 groups was much lower than constructed a lentivirus vector expressing miR-409, and estab- the vector controls (Figure 3b). Of note, we also examined the

Oncogene (2012) 4509 --4516 & 2012 Macmillan Publishers Limited miR-409 targets RDX expression in GC B Zheng et al 4511 influence of miR-409 on the local growth in vivo. The data further miR-409-3p, but not miR-409-5p, suppresses GC cell migration and confirmed that miR-409 did not affect GC cell proliferation invasion (Supplementary Figures S3E and F). Taken together, these results To determine which mature sequence of miR-409 is involved in GC support the role of miR-409 in the suppression of GC metastasis. cell migration and invasion, we introduced the mimic of miR-409- 3p and miR-409-5p into SGC-7901 cells individually. We found that miR-409-3p, but not miR-409-5p, inhibited GC cell migration and Table 1. The correlations of miR-409-3p expression with various invasion (Figure 4a). To further support these findings, MKN-45 clinicopathological features of GC cells with a relatively high baseline expression of miR-409 were transfected with the miR-409-3p inhibitor and miR-409-5p inhibitor, respectively. The results showed that miR-409-3p Clinicopathological Number Median P-value inhibitor significantly increased the migration and invasion of features of cases expression MKN-45 cells, whereas miR-409-5p inhibitor had no effect on the of miR-409-3p migration and invasion of MKN-45 cells (Figure 4b). These results Age (years) indicate that miR-409-3p, not miR-409-5p, inhibits the migration X60 44 0.10±0.01 0.29 and invasion of GC cells. o60 46 0.09±0.01 miR-409-3p downregulates RDX expression by directly targeting Gender its 30UTR Male 68 0.10±0.01 0.62 Female 22 0.09±0.01 A miRNA usually exerts its function by suppressing the expression of target genes. Therefore, our next aim was to investigate the Diameter (cm) targets of miR-409 that contributed to its anti-metastatic function. X5 37 0.09±0.01 0.60 We examined the gene expression profile of miR-409 over- o5 53 0.10±0.01 expressing and control cells using an mRNA microarray. The results revealed that 476 genes were downregulated by miR-409 Location (data not shown). When the result was compared with the Distal third 55 0.09±0.01 0.28 Middle and proximal 35 0.11±0.02 predicted targets from the bioinformatics algorithms of TargetS- third can (www.targetscan.org), six genes (EXT1, REPS1, RDX, SPRED1, ZFAND5 and ZMIZ1) were identified as the potential targets of Degree of differentiation miR-409-3p (Supplementary Table S1). To determine whether the Well and moderately 45 0.09±0.01 0.82 six genes were regulated by miR-409-3p through direct binding to Poorly 45 0.10±0.01 their 30-UTR, we constructed vectors containing the 30-UTR of each gene, which was individually fused directly downstream of the Local invasion firefly luciferase gene. The luciferase assays showed that miR-409- ± T1+T2 22 0.12 0.03 0.09 3p could reduce the luciferase activities of the 30-UTR of REPS1 T3+T4 68 0.09±0.01 and RDX (Supplementary Figure S4). However, the luciferase TNM stage activity of RDX showed a more significant decline than REPS1, and I+II 44 0.12±0.02 0.01 only RDX has been reported to be associated with cancer 20,21 III+IV 46 0.07±0.01 metastasis, so we focused on RDX for further research. We constructed a vector containing mutant 30-UTR of RDX fused Lymph node metastasis directly downstream of the firefly luciferase gene (Figure 5a). For No 38 0.13±0.02 0.00 the luciferase assays, the wild-type or mutant vector was Yes 52 0.07±0.01 cotransfected into HEK293T cells with a negative control, miR- Abbreviation: GC, gastric cancer. 409-3p or miR-409-5p mimic. As shown in Figure 5b, miR-409-3p, but not miR-409-5p, sharply decreased the relative luciferase

Figure 2. miR-409 suppresses GC cell migration and invasion in vitro. Transwell migration (a) and invasion (b) assays of SGC-7901 and HGC-27 cells performed after transduction with a miR-409-expressing or vector-control lentivirus.

& 2012 Macmillan Publishers Limited Oncogene (2012) 4509 --4516 miR-409 targets RDX expression in GC B Zheng et al 4512

Figure 3. miR-409 suppresses metastasis and peritoneal dissemination in vivo.(a) Representative haematoxylin and eosin-stained sections of lung tissues collected from SGC-7901-vector and SGC-7901-miR-409 groups. The arrowhead points to the tumour focus formed in the lung. The number of metastases in the lung were counted and analysed. (b) Representative tumour nodules in the abdominal cavity were shown from SGC-7901-vector and SGC-7901-miR-409 groups. The number of macroscopic nodules were counted and analysed.

activity of the wild-type 30-UTR of RDX, whereas the luciferase expression. Moreover, silencing of RDX significantly inhibited the activity of the mutant 30-UTR was not significantly changed, migration and invasion of GC cells (Figures 6c and d), which indicating that such regulation was sequence-specific. In addition, phenocopied the migration and invasion-inhibitory effects of miR- western blot analysis demonstrated that the stably expressed miR- 409. In addition, we introduced a construct expressing RDX into 409 significantly inhibited the endogenous protein level of RDX miR-409 stably expressing cell lines of SGC-7901. The restoration (Figure 5c). Furthermore, transient transfection with miR-409-3p of RDX expression in the stable cell lines was confirmed by mimic in SGC-7901 cells resulted in a clear attenuation of RDX western blot (Figure 6e). Restoration of RDX significantly increased protein expression, whereas no distinct changes were detected the migratory and invasive capabilities of GC cells. Importantly, we after transfection with miR-409-5p mimic (Figure 5d). We also showed that restoration of RDX could significantly reverse the found that inhibition of miR-409-3p, but not miR-409-5p, could migration and invasion suppression imposed by miR-409 increase the expression of RDX in GC cells (Figure 5d). Interest- (Figure 6f). Collectively, our data suggested that inhibition of ingly, the mRNA level of RDX was significantly decreased by miR- RDX expression by miR-409 was, at least in part, responsible for 409-3p, but not miR-409-5p, in SGC-7901 cells, whereas no miR-409 suppression of cell migration and invasion and, conse- significant change was observed in HGC-27 cells when transfected quently, lymph node metastasis in human GC. with either miR-409-3p or miR-409-5p mimic (Supplementary Figures S5A and B). These results suggested that miR-409 regulates RDX expression both through induced-mRNA degrada- DISCUSSION tion and translational repression, which was consistent with a The majority of cancer-related deaths are caused by metastasis. recent report showing that the repression of mRNA by miRNAs Therefore, developing an effective intervention targeting meta- 22,23 was cellular-context dependent. Next, we examined both static disease should improve the mortality of cancer patients. the mRNA and protein expression of RDX in GC tissues, which had Mounting evidence suggests an important role for miRNAs in the been used to detect the expression of miR-409-3p; however, regulation of cancer metastasis, including miR-31,20 miR-151,26 we found that RDX had no significant reverse relationship with miR-34a,27 miR-146a17 and miR-92a.28 However, the functional miR-409-3p expression (Supplementary Figures S6A and B). role of miRNAs in GC metastasis is still largely unknown. In this study, we demonstrated that miR-409-3p was frequently down- RDX promotes the migration and invasion of GC cells and the regulated in human GC and that its downregulation was enhanced expression of RDX can block miR-409-induced significantly associated with clinical stage and lymph node migration and invasion suppression metastasis. Enforced expression of miR-409 could suppress the RDX is a member of the ezrin--RDX--moesin family that has an migration, invasion and metastasis of GC cells in vitro and in vivo. important role in cancer progression and metastasis.20,24,25 To We further identified RDX, a putative pro-metastatic gene,20,25 as a determine whether downregulation of RDX was involved in miR- direct functional target of miR-409. To our knowledge, this is the 409-mediated suppression of migration and invasion, we first first study to explore the role of miR-409 in cancer, and our results analysed the effects of RDX in GC cells. As shown in Figures 6a indicate that miR-409 acts to suppress metastasis in GC. and b, transfection of a specific small interfering RNA against RDX Although the expression and functions of some miRNAs have into SGC-7901 and HGC-27 cells led to sharply decreased RDX been characterised in GC,29 --31 studies on the relationship

Oncogene (2012) 4509 --4516 & 2012 Macmillan Publishers Limited miR-409 targets RDX expression in GC B Zheng et al 4513

Figure 4. miR-409-3p, but not miR-409-5p, suppresses GC cell migration and invasion. (a) Transwell migration and invasion assays of SGC-7901 cells transfected with miR-409-3p mimic, miR-409-5p mimic or negative control (NC). (b) Transwell migration and invasion assays of MKN-45 cells transfected with miR-409-3p inhibitors, miR-409-5p inhibitors or NC control.

Figure 5. RDX is a direct downstream target for miR-409-3p. (a) Model of the construction of wild-type or mutant pLUC--RDX-- 30-UTR vectors. The wild-type or mutant (underlined) binding site of RDX--30-UTR for miR-409-3p is shown. nt, nucleotides. (b) Luciferase activity assays of luciferase vectors with wild-type or mutant RDX--30-UTR were performed after cotransfection with miR-409-3p mimic, miR-409-5p mimic or negative control (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. (c) Western blot assays of RDX protein in SGC-7901 and HGC-27-cells after infection with miR-409 or control lentivirus. (d) Western blot assays of RDX protein after transfection with miR-409-3p mimic, miR-409-5p mimic or NC in SGC-7901 cells and with anti-miR-409-3p, anti-miR-409-5p or anti-NC in MKN-45 cells. b-actin served as an internal control.

& 2012 Macmillan Publishers Limited Oncogene (2012) 4509 --4516 miR-409 targets RDX expression in GC B Zheng et al 4514

Figure 6. RDX restoration blocks miR-409-induced suppression of migration and invasion. (a, b) Knockdown of RDX by small interfering RNA was confirmed by western blot in SGC-7901 and HGC-27-cells. (c, d) Migration and invasion assays of SGC-7901 and HGC-27-cells were performed after transfection with si-RDX or negative control (NC). (e) SGC-7901-vector and SGC-7901-miR-409 cells were infected with RDX-expressing or mock lentivirus, respectively. RDX protein level was confirmed by western blot assays. b-actin served as an internal control. (f) Migration and invasion assays of SGC-7901 stable cell lines were performed. *Po0.05; **Po0.01; ***Po0.001.

between miRNAs and lymph node metastasis are still rare. metastasis is important for clarifying the complicated mechanisms Metastasis to lymph nodes is a frequent event in early GC underlying GC metastasis. In this study, we found that miR-409 metastasis, and it is an important prognostic indicator for patient could reduce the GC cell migration and invasion in vitro. Moreover, outcome.4,32 In our study, we found that downregulation of miR- we demonstrated that miR-409 possessed the capacity to 409-3p was significantly associated with lymph node metastasis in suppress distal pulmonary metastasis and peritoneal spreading GC patients. Interestingly, SGC-7901 and HGC-27 cell lines, which in vivo, without affecting cell proliferation. Taken together, these both originate from the lymph node metastatic locus of GC findings suggested that miR-409 acts as a new anti-metastatic patients, had relatively lower miR-409-3p levels compared with miRNA in GC and that miR-409 may have therapeutic potential in other cell lines not originating from lymph node metastatic sites. GC patients with metastasis. Consistent with our results, an elegant microarray analysis19 in RDX belongs to the ezrin--RDX--moesin family, which act as GC tissues showed that miR-409-3p was associated with membrane--cytoskeletal crosslinkers and signal transducers in clinicopathological metastasis-related parameters such as perito- mediating cytoskeletal remodelling.36 The ezrin--RDX--moesin neal dissemination, depth of tumour invasion and lymph node family members can regulate multiple cell functions,37,38 such as metastasis. Recently, it was reported that miR-409 may be cell shape maintenance, formation of microvilli, cell adhesion, subjected to epigenetic modulation in GC, which in turn may be cell migration and cell polarity. Mounting evidence suggests that responsible for miR-409-3p downregulation in GC.33 Taken the ezrin--RDX--moesin family members have an important role together, these results suggest that reduced miR-409-3p is a in tumour metastasis.21 RDX was originally identified as a frequent event in human GC and that patients with down- constituent of adherence junctions in the livers of rats,36 and it regulated miR-409-3p are prone to lymph node metastasis. has more recently been implicated in cancer progression and The capability of cell migration and invasion is considered to be metastasis.20,24,25,39 It has been reported that RDX may cooperate one of the important determinants in the process of cancer with ITGA5 and RhoA to affect three distinct steps in the invasion- metastasis. Although a large number of coding genes are reported metastasis cascade in vivo, local invasion, early post-intravasation to be involved in the course of this disease, the molecular events and metastatic colonisation.25 Silencing of RDX has also mechanisms are still largely unknown. Recently, compelling been found to suppress the migration and invasion of GC cells.40 evidence has shown that miRNAs exert significant impacts on Together with our findings, these results suggested that RDX the migration, invasion and metastasis of human cancers, and could act as a pro-metastatic factor in various cancers. Our study even demonstrates that miRNAs have the therapeutic potential.34 indicates that miR-409 can regulate the expression of RDX in GC. However, only a few miRNAs (miR-218,15 miR-516a-3p,16 miR- Another report indicates that the anti-metastatic miRNA miR-31 107,35 Let-7f18) have been reported to be involved in the inhibits breast cancer metastasis by targeting RDX and that metastasis of GC. So identifying more miRNAs related to GC re-expression of RDX could partially reverse the miR-31-imposed

Oncogene (2012) 4509 --4516 & 2012 Macmillan Publishers Limited miR-409 targets RDX expression in GC B Zheng et al 4515 invasion defects. Together, these results suggest that RDX is modification) and small interfering RNA against RDX were synthesised by regulated by miRNAs and that downregulation of these miRNAs in Ribobio (Guangzhou, China). The sequence of the small interfering RNA cancer may facilitate the expression of RDX, resulting in enhanced targeting RDX is 50-GCCAGAGATGAAACCAAGAAA-30. Oligonucleotide metastasis of the cancer. transfection was performed with Lipofectamine 2000 reagents according In summary, we have identified an important anti-metastatic to the manufacturer’s instructions. For proliferation assays, cells were miRNA, miR-409, the mature 3p sequence of which is frequently trypsinised 24 h after transfection. For migration, invasion and western blot underexpressed and associated with lymph node metastasis in assays, cells were collected 48 h after transfection. GC. Re-expression of miR-409 suppresses GC cell invasion, metastasis and peritoneal dissemination through negative regula- Cell migration and invasion assays tion of RDX expression. Expanding on the role miRNAs have in GC 4 lymph node metastasis will improve our understanding of the For the migration assays, 5 Â 10 cells in serum-free media were placed into the upper chamber of an insert (8-mm pore size; BD Bioscience, complicated mechanisms underlying GC progression, and may 5 promote the development of new therapeutic regimens for Franklin Lakes, NJ, USA). For the invasion assays, 1 Â 10 cells in serum-free treating advanced GC. media were placed into the upper chamber of an insert coated with Matrigel (BD Bioscience, Woburn, MA, USA). Media containing 10% foetal bovine serum were added to the lower chamber. After several hours of MATERIALS AND METHODS incubation, the cells remaining on the upper membrane were removed Human samples with cotton wool, whereas the cells that had migrated or invaded through Human GCs and their corresponding non-tumorous gastric samples were the membrane were stained with 20% methanol and 0.1% crystal violet, collected at the time of surgical resection from the Shanghai Cancer Center imaged, and counted using an IX71 inverted microscope (Olympus, Tokyo, of Fudan University from 2008 to 2009. Written informed consent was Japan). Experiments were independently repeated three times. obtained before collection. Samples were immediately frozen in liquid nitrogen and stored in a À80 1C freezer. Use of human tissues was Luciferase assays approved by the Clinical Research Ethics Committee of Fudan University HEK293T cells of 8 Â 103 were plated into 96-well plates. After 24-h Shanghai Cancer Center. incubation, a mixture of 100-ng pLUC--30-UTR, 5-pmol negative control , miR-409-3p mimic or miR-409-5p mimic was cotransfected with 20 ng Cell culture Renilla into HEK293T cells using Lipofectamine 2000. Forty-eight hours SGC-7901, HGC-27, AGS, MGC-803, NCI-N87 and HEK293T cells were after transfection, firefly and Renilla luciferase activities were measured purchased from the Shanghai Institute of Biochemistry and Cell Biology at with a Dual-Luciferase Reporter System (Promega, Madison, WI, USA). The the Chinese Academy of Sciences. MKN-45 and SV40-transformed transfection efficiency was normalised by cotransfection with a Renilla immortal gastric epithelial-1 cells were obtained from the Shanghai Ruijin reporter vector. Hospital of Shanghai Jiaotong University School of Medicine. SGC-7901, HGC-27, MGC-803, NCI-N87, MKN-45 and gastric epithelial-1 cells were In vivo metastasis assays maintained in RPMI1640, AGS in F12 and HEK293T in Dulbecco’s modified For in vivo pulmonary metastasis assays, 1 Â 106 SGC-7901 cells infected Eagle’s medium media. Media was supplemented with 10% foetal bovine with miR-409 or mock vector, respectively, were suspended in 200 ml serum. The cells were incubated at 37 1C in a humidified chamber phosphate-buffered saline for each mouse. Nude mice (six per group, containing 5% carbon dioxide. Cells were in the logarithmic phase of 5-week-old BALB/c-nu/nu) were inoculated through the lateral tail vein. growth for all experiments. After 6 weeks, the mice were killed. Their lungs were dissected and fixed with phosphate-buffered neutral formalin before paraffin embedding. The RNA extraction and quantitative real-time PCR tissue was then sectioned and stained in haematoxylin and eosin . For Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, peritoneal dissemination assays, we performed intraperitoneal injection in USA). cDNA was synthesised with the PrimeScript RT reagent Kit (TaKaRa, mice (nine per group, 1.5 Â 106 cells for each mouse). Five weeks later, the Dalian, China). Quantitative RT--PCR assays to detect mRNA expression mice were killed. The macroscopic nodules in abdominal cavity were were conducted using SYBR Premix Ex Taq (TaKaRa), with b-actin as an counted; the fused nodules were calculated as a single nodule. To test the internal control. TaqMan miRNA assays (Applied Biosystems, Foster City, tumourigenicity, SGC-7901 cells infected with miR-409 or mock vector, CA, USA) were used to quantitate the expression level of mature miR-409- respectively, were subcutaneously injected into mice (six per group, 3p according to the manufacturer’s protocol, with U6 small nuclear RNA as 3 Â 106 cells for each mouse). Four weeks later, the mice were killed. Mice an internal control. were manipulated and housed according to protocols approved by the Shanghai Medical Experimental Animal Care Commission. Vector constructs The pri-miR-409 sequence was amplified from normal human genomic Western blot analysis DNA and inserted into the pWPXL vector (a generous gift from Dr T Didier). Proteins were separated on SDS--polyacrylamide gel electrophoresis and The RDX expression vector was generated by inserting its open reading transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The frame into the pWPXL vector. The wild type and mutated 30-UTR of RDX membrane was blocked with 5% non-fat milk and incubated with rabbit were cloned downstream of a cytomegalovirus promoter-driven firefly anti-RDX monoclonal antibody (1:1000; Cell Signaling Technology, luciferase cassette in a pCDNA3.0 vector. Danvers, MA, USA) and mouse anti-b-actin monoclonal antibody (1:10 000; Sigma). The proteins were detected with enhanced chemilumi- Lentivirus production and transduction nescence reagents (Pierce, Rockford, IL, USA). pWPXL-miR-409 was co-transfected with the packaging plasmids psPAX2 and pMDG2 into HEK293T cells using Lipofectamine 2000 (Invitrogen). Statistical analysis Virus particles were collected 48 h later and added to the media of SGC- Data were presented as mean±s.e.m. Unless otherwise noted, Student’s 7901 and HGC-27 cells with 6 mg/ml Polybrene (Sigma, St Louis, MO, USA). t-test was used for comparisons, with Po0.05 considered significant.

Oligonucleotide transfection miR-409-3p and miR-409-5p mimics were synthesised by Genepharma CONFLICT OF INTEREST (Shanghai, China). miR-409-3p inhibitor, miR-409-5p inhibitor (20-O-methyl The authors declare no conflict of interest.

& 2012 Macmillan Publishers Limited Oncogene (2012) 4509 --4516 miR-409 targets RDX expression in GC B Zheng et al 4516 ACKNOWLEDGEMENTS 20 Valastyan S, Reinhardt F, Benaich N, Calogrias D, Szasz AM, Wang ZC et al. A We are most grateful for Dr T Didier’s gifts of the pWPXL, psPAX2 and pMD2.G lenti- pleiotropically acting microRNA, miR-31, inhibits breast cancer metastasis. Cell virus plasmids. 2009; 137: 1032 --1046. Author contributions: BZ, LL, YS, and XH designed the experiments, interpreted the 21 Yu H, Zhang Y, Ye L, Jiang WG. The FERM family proteins in cancer invasion and data and wrote the manuscript. BZ, LL, SH, LL, DJ and QT performed experiments. RZ metastasis. Front Biosci 2011; 16: 1536 --1550. and QW conducted the animal experiments. CW, ZL, YZ, XC and CD collected the 22 Doench JG, Sharp PA. Specificity of microRNA target selection in translational human samples and clinical data. repression. 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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene (2012) 4509 --4516 & 2012 Macmillan Publishers Limited