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Carcinogenesis vol.33 no.11 pp.2147–2154, 2012 doi:10.1093/carcin/bgs259 Advance Access publication August 2, 2012 MiR-196a binding-site SNP regulates RAP1A expression contributing to esophageal squamous cell carcinoma risk and metastasis Kai Wang1,†, Juan Li1,†, Hong Guo1, Xueqing Xu1, Gang Xiong2, Xingying Guan1, Botao Liu1, Junxia Li1, Xuedan Chen1, Kang Yang2 and Yun Bai1,* 1 Department of Medical Genetics, Third Military Medical University, Chongqing 400038, China and 2Department of Thoracic and Cardiac Surgery, Southwest Hospital, Third Military Medical University, Chongqing 400038, China * To whom correspondence should be addressed. Tel: +86 23 68752258; Fax: +86 23 68752224; Email: [email protected] Correspondence may also be addressed to Kang Yang. Tel/Fax: +86 23 68754183; Email: [email protected] Polymorphisms in 3′ untranslated region (UTR) of cancer-related genes might affect regulation by microRNA (miRNA) and contribute to carcinogenesis. In this study, we screened several single nucleotide polymorphisms (SNPs) in 3′UTR of cancer-related genes and investigated their effects on the risk of esophageal squamous cell carcinoma (ESCC). First, we used SNaPshot assay to genotype seven 3′UTR SNPs in 537 ESCC cases and 608 normal controls in a Chinese Han population and found that SNP rs6573 in 3′UTR of RAS-related proteins (RAP1A) was significantly associated with ESCC risk [P = 0.02, odds ratio (OR) = 0.43; 95% confidence interval (CI): 0.21–0.91] and pathologic stage (P = 0.03, OR = 1.89; 95% CI: 1.06–3.36). A putative binding site for miRNA-196a (miR-196a) exists in the 3′UTR of RAP1A, and the genetic variant, rs6573 A→C, is present in this binding region. We confirmed that miR-196a regulated the expression of RAP1A by luciferase reporter assay and that the regulation was affected by the RAP1A genotype. SNP rs6573 A to C change interfere in the interaction of miR-196a binding to RAP1A 3′UTR, resulting in higher constitutive expression of RAP1A. Moreover, we observed that RAP1A was overexpressed in the majority of ESCC tissues and correlated with RAP1A genotype and lymph node metastasis. In vitro study indicated RAP1A might function as a promoter for esophageal cancer cell migration and invasion through matrix metalloproteinase 2. Our study highlights RAP1A and SNP rs6573 functioning as potential personal diagnostic and prognosis markers for ESCC. Introduction Esophageal cancer is one of the most aggressive cancers worldwide and the incidence rate is significantly increasing in recent years. Overall survival of this cancer is <10% and the 5 years survival rate is 5–20% after surgery (1). The major reason for this poor prognosis is that most patients have already had distant metastasis at the time of diagnosis. After complete surgical removal of primary tumor, the 5 years survival rate is 50–80% for stage I disease, 10–40% for stage II disease and 10–15% for stage III disease. Patients with distant metastatic recurrence (stage IV) who are treated with palliative chemotherapy have a median survival of <1 year (2). Therefore, more efforts are needed to identify the mechanism underlying esophageal cancer metastasis. The molecular mechanisms leading to metastasis of esophageal squamous cell carcinoma (ESCC) have not been fully elucidated, although several molecules are known to be involved. The expression of mucin 1 and matrix metalloproteinase 13 (MMP13) are strongly correlated to lymph node metastasis (3). MicroRNAs (miRNAs) are non-proteincoding RNA molecules that can function as tumor suppressors and/or oncogenes (4). They play key roles in regulating the translation and degradation of messenger RNA (mRNA) genes by sequence complementarity (5–7). It is estimated that about 30% of human genes are regulated by miRNAs (8). MiRNA-375 inhibits tumor growth and metastasis in ESCC through repressing insulin-like growth factor 1(9). MiRNA92a promotes lymph node metastasis of human ESCC via decreasing E-cadherin (10). Single nucleotide polymorphisms (SNPs) located in miRNA binding sites are probably to affect the expression of miRNA target genes and may contribute to the susceptibility of humans to common diseases (11), including cancers (12–16). Therefore, we hypothesized that SNPs in potential miRNA binding sites might affect the susceptibility and progression of ESCC. For this purpose, seven miRNA-binding site SNPs, namely rs2239680 in baculoviral IAP repeat containing 5, rs1476215 in fibroblast growth factor 2, rs6573 in RAS-related proteins (RAP1A), rs473698 in transforming growth factor, rs1057035 in ribonuclease type III, rs1049931 in collagen, type IV, α 2 and rs3757 in DiGeorge syndrome critical region gene 8 (DGCR8), were chosen by bioinformational tools. An association study between SNPs in the potential miRNA binding sites and ESCC was performed in the Chinese Han population. We found that SNP rs6573 in the 3′ untranslated region (UTR) of the RAP1A gene was significantly associated with ESCC risk [P < 0.05, odds ratio (OR) = 0.43; 95% confidence interval (CI): 0.21– 0.91) and pathologic stage (P = 0.03, OR = 1.89; 95% CI: 1.06–3.36). RAP1A is a member of the Ras oncogene family of small G proteins, which play important roles in a variety of cellular processes, including proliferation, adhesion and cancer progression (17). Recent studies have indicated that abnormal RAP1A activation can contribute to malignancy via distinct biological effects in different cell types (18). RAP1A activation promotes epidermal growth factor receptordependent pancreatic carcinoma cell metastasis (19). Activation of RAP1A promotes prostate cancer metastasis (20). Prior findings suggested that abnormal RAP1A activation can contribute to malignancy via distinct signal pathways, but the regulation of RAP1A mRNA levels involved in carcinogenesis remains undefined. In this study, we used a case-control study to demonstrate that the SNP rs6573 is an important genetic variant for esophageal squamous cell cancer risk. We also validated that SNP rs6573 was related to RAP1A expression through affecting miR-196a binding to RAP1A 3′UTR. Finally, we provided evidence that RAP1A gene was an important regulator of esophageal cancer cell invasion and migration through MMP2. Our results will provide a new insight into ESCC tumorigenesis and metastasis by resolving the genetic background. Materials and methods Abbreviations: CI, confidence interval; ESCC, esophageal squamous cell carcinoma; GAP1, GTPase-activating protein 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; miRNA, microRNA;NC, negative control; OR, odds ratio; RAP1A, RAS-related proteins; RT-PCR, reverse transcription–polymerase chain reaction; siRNA, small interfering RNA;SNP, single nucleotide polymorphism; UTR, untranslated region † These authors contributed equally to this work. Study population and tissue samples All people were genetically unrelated Han Chinese from Chongqing city of southwest China. The ESCC patients were histopathologically diagnosed and confirmed at the Southwest Hospital, the Third Military Medical University. The exclusion criteria included metastasized cancer and previous radiotherapy or chemotherapy. The controls were healthy individuals who participated in a physical examination in Chongqing, who had no history of cancer and who were frequency matched to cases based on age, sex and residential area. Informed consent was obtained from the subjects, and the study was performed with the © The Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 2147 K.Wang et al. approval of the ethical committee of Third Military Medical University. The interviewers collected the demographic data and environmental exposure history using a questionnaire. An individual who smoked >100 cigarettes in his or her lifetime was defined an ever smoker. Former smokers were those who had quit smoking at least 1 year before diagnosis (for cases) or enrollment in this study (for controls). Recent quitters were those who had quit within 1 year of diagnosis (for cases) or enrollment in this study (for controls). After the interview, about 5 ml of venous blood was collected from each participant. Tumors and adjacent non-tumor tissues were collected from patients who underwent surgery at the Thoracic and Cardiac Surgery of Southwest Hospital. Esophageal cancer cell lines and plasmids Human esophageal cancer cell lines EC109 and TE-1 were purchased from Cell Bank of Chinese Academy of Sciences, Shanghai, China. KYSE150 were obtained from Cancer Institute and Hospital, Chinese Academy of Medical Sciences, Beijing, China. The GTPase-activating protein 1 (RAP1GAP1) plasmid was kindly provided by Dr P.J.Stork (Vollum Institute, Oregon Health and Science University, Portland, OR) and the enhanced green fluorescent protein (EGFP)-RAP1A plasmid was kindly provided by Dr M.R.Philips (New York University, New York, NY). The plasmids pcDNA3.1 and pEGFPC1 were purchased from Invitrogen (Carlsbad, CA). The pMIR-REPORT luciferase miRNA expression reporter vector was purchased from ABI (Foster City, CA). SNP selection First, we used database and literature searches to screen cancer-related genes. A 3′UTR dataset and a miRNA target dataset of human genes were obtained from the UCSC Genome Browser (http://genome.ucsc.edu/cgi-bin/hgTables). The miRNA target dataset, developed by Krek et al., contains the human genes. We used target-scan (http://www.targetscan.org/), miRBase (http:// www.mirbase.org/), miRSNP (http://compbio.uthsc.edu/miRSNP/) and PicTar (http://pictar.mdc-berlin.de/) to choose the SNPs in the miRNA targeting sites. We only choose SNPs with a minor allele frequency >5% in the Chinese population based on the HapMap CHB database. The selected SNPs are listed in Supplementary Table 1, available at Carcinogenesis Online. Genotyping We used the SNaPshot assay to genotype the SNPs. The SNaPshot PCR was run in a 10 µl volume containing 3 µl PCR product mix, 5 µl SNaPshot multiplex kit (ABI), 1 µl primer and 1 µl H2O. The PCR protocol entailed 25 cycles at 96°C for 10 s, 50°C for 5 s and 60°C for 30 s. These samples and Liz120 (ABI) were separated by capillary electrophoresis using Genetic Analyzer 3130 instrument (ABI). The data were analyzed using the Genemapper software version 4.0 (ABI). PCR and SNaPshot primers are shown in Supplementary Table 2, available at Carcinogenesis Online. Genotyping of select SNPs in esophageal cell lines was performed by direct sequencing. Luciferase reporter assay The 3′UTR of the RAP1A gene containing different alleles was amplified and cloned into the SacI/HindIII site of the pMIR-REPORT Firefly luciferase reporter vector (Ambion, Grand Island, NY) using standard DNA techniques. The accuracy of the two plasmid DNA constructs, shown as pMIR-A (for A allele in 3′UTR of RAP1A) and pMIR-C (for C allele in 3′UTR of RAP1A), was further identified by sequencing. For luciferase activity analysis, 293FT and EC109 cells were co-transfected with 100 ng of luciferase reporter constructs 5 ng of the β-gal control plasmid and 10 pmol of miRNAs with 1 µl lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, NY). The miRNA mimics that were transfected into the cells were purchased from GenePharma (Shanghai, China), including the miR-196a mimics and its negative control (NC). After incubation for 48 h, we carried out the luciferase assay using the luciferase reporter assay system (Promega, Madison, WI) according to the manufacturer’s protocol. Measurements of luminescence and absorbance of β-gal were performed on a luminometer (Glomax 20/20; Promega) and enzyme-linked immunosorbent assay (Bio-rad, Hercules, CA) individually. Three independent experiments were performed in triplicate. RT-PCR Total RNA was isolated using the RNAiso Plus Kit (TaKaRa, Otsu, Shiga Japan). Then, 100 ng RNA from each sample was reverse-transcribed into complementary DNA and subjected to conventional PCR (TaKaRa). The PCR primers sequences were as follows: forward 5′-TGTCTCACTGCACCTTCA-3′ and reverse 5′-GACTTCCCAACGCCTCCT-3′. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization. The analysis was performed in triplicate. Western blotting Western blotting was performed using antibodies directed against RAP1 (1:500; Abcam, Cambridge, UK), MMP2 (1:1000; Epitomics, Burlingame, 2148 CA). GAPDH was used for normalization. Quantity One software was used to compare the intensity of bands on the western blot. RNA interference To silence RAP1A expression, we transfected small interfering RNA (siRNA) targeting RAP1A, using lipofectamine 2000 (Invitrogen, NY). RAP1A siRNAs were synthesized by GenePharma. The siRNA sequence was 5′-GCAAGACAGTGGTGTAACT-3′ (RAP1A siRNA). A non-related, scrambled siRNA was used as a control. Cell proliferation assay The proliferation of EC109 cells was determined using Cell-Light EdU DNA Cell Proliferation Kit (RiboBio Co. Ltd, Guangzhou, Guangdong, China) according to manufacturer’s instructions. Briefly, 5 × 103 cells/well were seeded in a 96 well plate, grown at 37°C for 24 h. Subsequently, cells were transfected with RAP1A GAP, EGFP-RAP1A, siRNA or their controls in the presence of 10% fetal bovine serum for 48 h. And then, EdU (50 µM) was added to the wells. After fixation in 4% paraformaldehyde for 30 min and a series of rinses in phosphate-buffered saline, the cells were then incubated with the EdU reaction cocktail for 30 min, followed by rinses in phosphate-buffered saline with 0.2% Triton X-100. Finally, after 5 min of incubation with Hoechst 33342, the cells were observed under a fluorescent microscope (Olympus, Tokyo, Japan). Cell migration and invasion assays For the invasion assay, 24 well Millicell (8 µm Polyethylene Terephthalate, PET; Millipore, Bedford, MA) was coated with a 50 µl ECMgel (Sigma, St Louis, MO). For migration assays, the ECMgel was not needed. After transfection with RAP1A siRNA, EGFP-RAP1, RAP1GAP or their NCs, cells were incubated in serum-free medium for starvation and then cells were starved and 1 × 105 or 5 × 104 cells in 200 µl Dulbecco’s modified Eagle’s medium were placed into the upper chamber of the Milliwell for invasion and migration assay, respectively. Dulbecco’s modified Eagle’s medium containing 20% fetal bovine serum was added to the bottom well. After incubation for 48 h at 37°C in a CO2 incubator, cells in the top well were removed with cotton swabs. Membranes were then fixed in 4% paraformaldehyde and then stained with gentian violet. Finally, the cells on the membranes were counted using a phase-contrast microscope. Six randomly selected fields were counted for each membrane. Statistical analysis Differences between cases and controls were evaluated by the Student’s t-test for continuous variables and the χ2 test for categorical variables. The association between SNPs and ESCC risk was estimated by the OR and 95% CI using the general genetic model. The potential gene–environment interaction was evaluated by logistic regression analysis and tested by comparing changes in deviance (−2log likelihood) between the models of main effects with or without the interaction term. The χ2 test for Hardy–Weinberg equilibrium was applied to the SNPs among controls. Comparisons between groups were analyzed by the t test (two-sided). All statistical analyses were performed using Statistical Package for Social Sciences software (SPSS version 13.0, Chicago, IL). P-values <0.05 were considered statistically significant. Results Association study between SNPs and ESCC To identify candidate genes that contribute to ESCC, we performed an association study between seven SNPs located in the potential miRNA binding site of cancer-related genes and ESCC. The characteristics of patients and controls are shown in Supplementary Table 3, available at Carcinogenesis Online. There was no difference between patients and controls in regard to age, sex and drinking status (P > 0.05). However, about 66.5% of cases were smokers, which is much higher than the controls (P < 0.01). In the clinical stage, most patients were divided into stage II (71.8%) and stage III (24.6%). In the association study, we found that there were no significant differences between these SNPs except rs6573 in the RAP1A 3′UTR. Table I shows the allele frequencies and genotype distributions of these SNPs in the cases and controls. Multivariate logistic regression analysis of the SNP rs6573 showed that subjects carrying the CC genotype had an increased risk for ESCC than carrying CA (OR = 0.31; 95% CI: 0.23–0.41) or AA genotype (OR = 0.43; 95% CI: 0.21–0.91), indicating that C allele is a risk factor in ESCC. In addition, we performed stratification analyses according to TNM (Tumor Node and Metastasis) stages. The risk of ESCC in relation to Metastasis biomarker and personalized medicine Table I. Main effect of SNPs on ESCC Genotype Cases, N RAP1A rs6573 CC AC AA BIRC5 rs2239680 TT CT CC FGF2 rs1476215 AA AT TT TGFA rs473698 GG GC CC DICER rs1057035 AA AG GG COL4A2 rs1049931 TT CT CC DGCR8 rs3757 GG AG AA Controls, N OR (95% CI)a Pa 444 82 11 368 219 21 1.00 (reference) 0.31 (0.23–0.41) 0.43 (0.21–0.91) P < 0.01 P = 0.02 325 184 14 381 204 14 1.00 (reference) 1.06 (0.82–1.35) 1.17 (0.55–2.49) P = 0.7 P = 0.7 467 51 16 531 39 25 1.00 (reference) 1.49 (0.96–2.30) 0.73 (0.38–1.38) P = 0.08 P = 0.33 304 195 31 290 232 45 1.00 (reference) 0.80 (0.62–1.03) 0.66 (0.40–1.07) P = 0.08 P = 0.09 207 323 3 222 373 6 1.00 (reference) 0.93 (0.73–1.18) 0.54 (0.13–2.17) P = 0.55 P = 0.30 78 306 148 83 323 184 1.00 (reference) 1.01 (0.71–1.42) 0.86 (0.59–1.25) P = 0.96 P = 0.42 75 449 10 97 484 12 1.00 (reference) 1.20 (0.86–1.66) 1.08 (0.44–2.63) P = 0.27 P = 0.87 BIRC5, baculoviral IAP repeat containing 5; COL4A2, collagen, type IV, α 2; DGCR8, DiGeorge syndrome critical region gene 8; DICER, ribonuclease type III; FGF2, fibroblast growth factor 2; TGFA, transforming growth factor. a Adjusted for age, sex and pack-years of smoking. Significant p-values (<0.05) are in bold. this SNP was examined with stratification by clinical stages (Table II). An association between RAP1A genotypes and ESCC clinical status was detected (P = 0.03, OR = 1.89; 95% CI: 1.06–3.36). SNP rs6573 affects miR-196a binding to the 3′UTR of RAP1A SNP rs6573 was significantly associated with ESCC development and clinical status. MiRNA regulates gene expression by binding to the 3′UTR of its target gene. In silico analysis showed that SNP rs6573 lay within a putative binding site of RAP1A for miR-196a. In addition, the A allele matches the predicted seed region of miR196a, whereas the C allele represents a C:U mismatch base pairing (Figure 1A). To determine whether miR-196a could regulate the expression of RAP1A, we performed a luciferase reporter assay. First, we constructed two different reporter vectors containing RAP1A 3′UTR with the AA or CC genotype, named pMIR-A and pMIR-C, respectively (Figure 1B). Then, we transfected the two reporter plasmids into 293FT or EC109 cell lines along with a miR-196a mimics or its NC. We found that luciferase activity significantly decreased in the presence of the pMIR-A plasmids but not with the pMIR-C plasmids in both the 293FT and EC109 cell lines (Figure 1C). These data indicated that miR-196a could suppress luciferase activity with the A allele, but not with the C allele. Thus, SNP rs6573 may affect miR-196a binding to the RAP1A 3′UTR. Table II. Association of rs6573 genotypes and ESCC stage in cases Genotype I/II (n = 396), n (%) III/IV (n = 141), n (%) CC AC/AA 319 (80.5) 77 (19.5) 125(88.7) 16(11.3) a Adjusted for age, sex and smoking. Pa ORa (95% CI) P = 0.03 Reference 1.89 (1.06–3.36) SNP rs6573 affects inhibition of RAP1A expression induced by miR-196a MiR-196a can inhibit a reporter gene driven by the RAP1A 3′UTR and that the regulation was affected by the RAP1A genotype. To determine if miR-196a can regulate the expression of RAP1A in esophageal cancer cells, reverse transcription–polymerase chain reaction (RT-PCR) and western blot analysis were used to detect RAP1A expression after transfecting the miR-196a mimics or NC into esophageal cancer cells or normal cells with different genotypes. First, we genotyped cell lines by direct sequencing. We found that EC109 and KYSE150 cells are CC homozygote, whereas TE-1 and 293FT are AC heterozygote (Figure 2A). Then, miR-196a mimics or NC were transfected into these cells. After 48 h of transfection, we measured RAP1A mRNA and protein levels. In TE-1 and 293FT cells, we found that miR-196a significantly reduced the expression of RAP1A compared with the NC (Figure 2B). However, in EC109 and KYSE150 cells, there was no obvious reduction in RAP1A expression after miR-196a overexpression (Figure 2C). Taken together, these data showed that miR-196a could regulate the mRNA and protein expression of RAP1A, and SNP rs6573 might affect this regulation. RAP1A expression in ESCC tissues Because SNP rs6573 was significantly associated with ESCC in the Chinese Han population, we next examined RAP1A protein levels in tumor tissues and corresponding adjacent non-tumor tissues from 35 surgical patients by western blot (Figure 3A). We found that cancerous tissues exhibited significantly higher RAP1A protein levels compared with corresponding adjacent normal tissues (23/35, 65.7%, Figure 3B), which led us conclude that RAP1A expression was increased in ESCC. In addition, the result showed that the expression of RAP1A in CC homozygote patients was higher than that in AC heterozygote and AA homozygote patients (P = 0.006, Figure 3C). These data indicate that RAP1A might play an oncogenic role in the development of ESCC and SNP rs6473 related to 2149 K.Wang et al. Fig. 1. Luciferase reporter gene expression assays with constructs containing pMIR-A and pMIR-C. (A) A schematic shows the potential binding site of RAP1A 3′UTR. M: SNP rs6573 A/C. (B) Schema of the constructs harboring different alleles of miRNA binding sites. (C) MiR-196a mimics or its NC was co-transfected with the pMIR-REPORT constructs containing A allele or C allele into EC109 or 293FT cell lines. Data shown are the mean fold increase ± SD from two independent experiments. **P < 0.01. RAP1A expression in ESCC tissues, which is consistent to in vitro experiment. We next analyzed RAP1A expression in patients with different clinical features and found that expression of RAP1A in patients with lymph node metastasis was higher than that in patients without lymph node metastasis (P = 0.003, Figure 3D). This further suggested that RAP1A might involve in the regulation of ESCC metastasis. RAP1A promotes esophageal cancer cell migration and invasion in vitro Migration and invasion of cancer cells are essential steps of cancer metastasis. Because we found that SNP rs6573 was associated with the pathologic stage of ESCC and the expression of RAP1A correlated to ESCC lymph node metastasis, the impact of RAP1A on esophageal cancer cell migration and invasion was further investigated using transwell assay. Three approaches were used to change RAP1A status in EC109 cells: (i) downregulating RAP1A expression by siRNA; (ii) inhibiting RAP1A activation by RAP1GAP and (iii) increasing RAP1A levels through expression vectors (EGFP-RAP1A). After 48 h of transfection, cells were performed with transwell migration and invasion assay. Compared with the controls, RAP1A siRNA decreased RAP1A protein levels and EGFP-RAP1A transfection increased the expression of RAP1A (Figure 4A). Also, transfection of RAP1A siRNA resulted in a reduced number (~22%) of migrated cells and RAP1A overexpression caused an increase in cell migration. EC109 cells transfected with RAP1GAP showed a relative lower ability of 2150 migration (Figure 4B). In the invasion assay, transfected cells were seeded into transwell Millicell precoated with ECMgel, which imitates the extracellular matrix. Cells migrating through the matrix were stained. The ratio of cells invading through matrigel was 40% less in siRNA-transfected cells than NC cells. In RAP1GAPtransfected cells, number of invasive cells was reduced by ~21%. And EGFP-RAP1A vector overexpression of RAP1A increased esophageal cell invasion by 40% (Figure 4C). These results indicated that RAP1A might have a role in promoting esophageal squamous cancer cell migration and invasion. RAP1A promotes esophageal cancer cell migration and invasion through MMP2 Remodeling of the extracellular matrix is essential for tumor cell invasion and metastasis. Degradation of this matrix requires several matrix metalloproteinases (MMPs). MMP2 as a downstream molecule of RAP1A plays a critical role in esophageal cancer cell migration and invasion. We detected the expression of MMP2 after transfecting of miR-196a mimics, RAP1A siRNA or EGFP-RAP1A into esophageal cancer cells. MiR-196a significantly reduced the expression of MMP2 compared with the NC (Supplementary Figure 1A, available at Carcinogenesis Online). RAP1A siRNA and RAPGAP also reduced MMP2 expression. Otherwise, EGFP-RAP1A increased the expression of MMP2 compared with the control vector (Supplementary Figure 1B, available at Carcinogenesis Online). These results indicate that RAP1A promoted esophageal cancer cell invasion and metastasis, most probably via MMP2. Metastasis biomarker and personalized medicine Fig. 2. RT-PCR and western blotting were used to detect mRNA and protein expression of RAP1A after transfection of miRNA-196a into cells with different genotypes. (A) Using sequencing technology to genotype the SNP in EC109, KYSE150 (CC homozygote) and TE-1, 293FT (AC heterozygote). (B) Cells with AC heterozygote transfected with miRNA-196a mimics or its NC. After 48 h of transfection, the cells were harvested for measurements of mRNA and protein expression of RAP1A using RT-PCR and western blot analysis, respectively. GAPDH served as a control. (C) Cells with CC homozygote transfected with miRNA-196a mimics or NC. After 48 h of transfection, cells were harvested for measurement of mRNA and protein expression of RAP1A using RT-PCR and western blot analysis, respectively. GAPDH served as a control. *P < 0.05. Discussion Because ESCC is one of the most aggressive cancers with very poor prognosis, a clear understanding of the genetic factors for the development and metastasis of this cancer is very important. In this study, we screened genetic variations in the 3′UTR of cancer-related genes by a population-based association study approach and identified SNP rs6573 located in the binding site of miR-196a for RAP1A was significantly associated with susceptibility and metastasis of ESCC in a Chinese Han population. Though there have been many ESCC association studies, including the genome wide association study (21,22). SNPs involved in ESCC development and metastasis have remained unclear. Our research suggested a novel potential marker for predicting the susceptibility and malignancy of ESCC. To our knowledge, this is the first study evaluating the relationship between SNP rs6573 and human disease. RAP1A has been reported to implicate in a wide range of biological processes, from cell proliferation, differentiation to cell mobility. Numerous reports have implied that abnormal RAP1A activation contributed to the tumorigenic processes. Expression of RAP1 at high levels can morphologically transform Swiss 3T3 fibroblasts and formed tumors when injected into nude mice (23). In our study, we found that overexpression of RAP1A existed in most ESCC tissues and expression of RAP1A was higher in patients with lymph node metastasis than those without. In vitro study indicated RAP1A might function as a promoter for esophageal cancer cell migration and invasion. However, we have not detected any role for RAP1A in regulating cell proliferation of esophageal cancer cells (Supplementary Figures 2A–C). Thus, we suggest that RAP1A might play an oncogenic role in the development of ESCC. Abnormal activation of RAP1A has also been observed in other type of cancer, such as melanoma (24), cervical cancer (25), papillary thyroid cancer (26), squamous cell carcinoma (27), thyroid cancer (28), colon cancer (29) and pancreatic cancer (30). However, the impact of RAP1A on cancer cell migration and metastasis was not consistent. Inhibition of RAP1A reduced invasion of pancreatic cancer cells (19) and thyroid cancer (31). Activation of RAP1A was shown to promote prostate cancer metastasis (20). Nevertheless, inhibition of RAP1A has been reported to correlate with increased invasion of squamous cell carcinoma in an in vitro study (32). Our results showed that RAP1A promoted esophageal cancer cell migration and invasion. These opposing findings indicate that activation of RAP1A may play different roles in different cancers. MMP2 plays an important role in esophageal cancer metastasis (33). Previous studies have demonstrated that 2151 K.Wang et al. Fig. 3 Expression of RAP1A protein in cancer tissues and adjacent non-tumor tissues of 35 ESCC patients. (A) Western blotting was used to measure the protein expression of RAP1A in esophageal cancer patients and adjacent non-tumor tissues. Ca: cancer tissues. N: non-tumor tissues. (B) The relative expression level of RAP1A was measured by western blotting. C (cancer)/N (non-cancer) relative protein expression ratios between normalized values were calculated. + and – indicate that the expression is higher or lower, respectively, in the cancer tissue than in the respective adjacent non-cancer tissue. (C) Gray scale of the western blot bands indicates the expression of RAP1A related to GAPDH in different genotypes. (D) Gray scale of the western blot bands indicates the expression of RAP1A related to GAPDH in non-lymph node metastasis (N0) and lymph node metastasis (Nx) patient tissues. GAPDH served as a control. Fig. 4. RAP1A promotes esophageal cancer cell migration and invasion. (A) RAP1A expression was measured by western blot analysis after transfection with EGFP-RAP1, control vector EGFP and RAP1A siRNA into the EC109 cell line. GAPDH served as a control. (B) Migration assays of RAP1 siRNA-treated, RAP1GAP-treated and EGFP-RAP1-treated EC109 cells. (C) Invasion assays of RAP1 siRNA-treated, RAP1GAP-treated and EGFP-RAP1-treated EC109 cells. Experiments were performed in duplicate and at least six fields were counted. *P < 0.05. 2152 Metastasis biomarker and personalized medicine RAP1GAP promotes invasion through MMP2 and MMP9 secretion, which is associated with poor survival in squamous cell carcinoma (34). Our results also demonstrate that RAP1A promotes esophageal cancer cell migration and invasion, potentially by regulating MMP2 protein level. Therefore, we first showed the role of RAP1A during the carcinogenesis of esophageal squamous cell cancer that RAP1A probably function as a promoter of metastasis of ESCC. MiRNAs regulate gene expression by mRNA degradation or by reducing the translation of target mRNAs. Our study has added RAP1A mRNA as one more target of miR-196a, which has also been implicated in targeting HOX gene family (35). Further study provided confirmation of its role in regulation of HOX gene expression (36). The elevated expression of HOX genes in leukemogenesis is very well confirmed and this oncogene had myeloid differentiation capacities (37). In this study, we found that miR-196a could inhibit the expression of RAP1A. Although RAP1A was related to increase MMP2 levels and promote invasive ability of ESCC cells. This suggests miR196a might be prone to suppress the development of ESCC, which was similar to the role of miR-196a during leukemogenesis. However, there were also some studies showing that increase of miR-196a levels was the character of breast cancers (38), esophageal adenocarcinoma (39) and pancreatic adenocarcinoma (40). As a single miRNA can target many genes, the functions of any miRNA as oncogenic or tumor suppressive might be cancer type-specific and dependent on the target genes whose expressions it may be regulating. In previous study, Maru et al. found that miR-196a is a potential marker of progression during Barrett’s metaplasia–dysplasia–invasive adenocarcinoma sequence in esophagus (41). In our previous study, a SNP in the pre-miR-196a was associated with susceptibility of ESCC risk in this Chinese Han population (42). These data provided some evidence that miR-196a could play some roles in esophageal cancer. SNP rs6573 located in 3′UTR of RAP1A flanking the binding site for miR-196a and SNP A to C change interfere in the interaction of miR-196a binding to RAP1A 3′UTR, resulting in higher constitutive expression of RAP1A. Because we suggest RAP1A probably function as an oncogene in the development of ESCC, SNP A to C change would be expected to promote the development of ESCC, which is consistent with the results of association study that individuals carrying the rs6573 C allele had an increased risk for the development and metastasis of ESCC. Conclusion In conclusion, we found that SNP rs6573 in the miR-196a binding site of RAP1A is associated with the risk of ESCC. This SNP C allele may prevent miR-196a from binding to RAP1A mRNA, resulting in altered regulation of RAP1A expression and increased ESCC risk and metastasis. RAP1A could promote esophageal cancer cell migration and invasion through MMP2 (Supplementary Figure 3, available at Carcinogenesis Online). Our results provide a new insight into ESCC tumorigenesis and metastasis and have potential implications in individual treatment and prevention of this disorder. 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