The MicroRNAs MiR-31 Promotes Esophageal Squamous Carcinoma Invasion And Metastasis

1. Purpose:Esophageal squamous cell carcinoma is an aggressive malignancy. ESCC is well-known for its metastasis. The understanding of the molecular pathways of metastasis would be helpful in improving diagnosis, therapy, and prevention of the disease. MicroRNAs are endogenously expressed non-coding RNAs that have important biological and pathological functions. miR-31 was found markedly up-regulated in several other malignancies. However, miR-31 expression was also down-regulated in the metastasis process of breast carcinoma. The aim of this study was to evaluate the miR-31 expression patterns in esophageal squamous cell carcinoma.2. Materials and Methods.2.1 Cell culture:Human EC9706 esophageal squamous cell carcinoma cell line was established and maintained in our laboratory two other human esophageal squamous cell carcinoma cell lines KYSE150 and KYSE510 were generous gifts from Dr. Shimada Y (First Department of Surgery, Faculty of Medicine, Kyoto) all three Cell lines were maintained in Dulbecco's modified Eagle's medium(DMEM) with 10% newborn calf serum and incubated at 37℃and 5% CO2.2.2 MicroRNA transfection:SiRNA duplexes homologous in sequence to microRNA 31 (i.e.miR-31 mimics) and its inhibitors were synthesized and purified by Shanghai Gene-Pharma Co. (Shanghai, China). The sequences of these inhibitors are the exact antisense copy of the mature miRNAs and all the nucleotides in the inhibitors contain 29-OMe modifications at each. SiRNA duplexes with non-specific sequences were used as a negative control (NC). MiRNA duplexes or their inhibitors were transfected into EC9706, KYSE150, KYSE510 cell lines by Lipofectamine 2000 reagent (Invitrogen Corp. CA, USA) with the details that:the cultured cells were seeded into 24 well plates at a density of 5×104 cells/well one day prior to transfection, then cells were transfected with miR-31 mimics, inhibitors or non-specific sequences at a concentration of 150nM in each well.2.3 Trans-Well Cell Migration Assay:Cell migration was examined with TranswellR Permeable Supports (Corining, Inc. Corining, NY). Cells after miR-31 or inhibitors transfection for 24hours, was dissociated to 2×104 cells in 100μL of serum-free DMEM and added to the 6.5mm diameter Transwell inserts(8μm pore size), while the lower plate well filled with 600μL 10% serum DMEM. The cells were allowed to migrate at 37℃in a 5% CO2 humidified incubator for 24 hours. Nonmigrating cells on the upper surface were carefully removed with a cotton swab. The filters were then fixed in methanol for 10 minutes and stained with 0.4% crystal violet solution for 10 minutes. Migrated cells on the membrane were counted under a microscope. Migration was quantified by counting the migrated cells in 10 random high-powered fields per filter. Each well set up three parallel controls.2.4 Cell-matrix adhesion assay:Cells with 24hour siRNA or inhibitor transfection were seeded into a 96-well plate which had been pre-coated with Matrigel (5μg/well). After incubation, non adherent cells were washed off using PBS. The adhered cells were fixed, stained and then counted. Respectively in 30 minutes,60 minutes,90 minutes and 120minutes after cell seeding to take out for MTS (cellTiter 96R AQ ueous One Solution Reagent, Promega Corp. USA) test.Patient and samples:2.5 Study populationA total of 120 consecutive patients with pathologically squamous esophageal carcinoma diagnosed from March 2008 to March 2010(in the Tumor Hospital of Henan province, China) and 121 age-matched healthy subjects as controls were enrolled in this study. All the patients involved in our study were not undergoing chemotherapy or radiotherapy before blood and tissue sampling. Clinicopathologic findings were based on the tumor-node-metastasis classification for esophageal carcinoma by the American Joint Committee on Cancer (AJCC) and the International Union for Cancer Control (UICC).2.6 Blood sampleWe collected 120 pairs of blood samples with ESCC patients (with pre-operation) and 121 pairs normal healthy controls, as well as 65 blood samples with post-operation from the previous patient population. For blood sampling, up to 8 ml of whole blood from each participant was collected in EDTA tube. Blood samples were centrifuged at 1,200×g for 10 min at 4℃to spin down the blood cells, and the supernatant was transferred into microcentrifuge tubes, followed by second centrifugation at 12,000×g for 10 minutes at 4℃to completely remove cellular components. Plasma was then aliquoted and stored at -80℃until use.2.7 Tissue sampleMatched esophageal carcinoma tissue samples were obtained from 45 patients. Fresh samples were dissected manually to remove connective tissues and stored in liquid nitrogen immediately. The corresponding normal tissues were obtained from the distant edge of resected esophagus without carcinoma cell invasion in pathological diagnosis.2.8 miR extraction:Total RNA was extracted from cell lines, tissues and blood samples using miRNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. miR extraction from blood sample was as Enders KO Ng et.al described. After DNase treatment (Qiagen) was carried out to remove any contaminating DNA, RNA samples was quantified by SmartSpecTM spectrophotometer (BIO-RAD, USA), only OD260/280 ratio range from 1.7-2.1, is acceptable. The concentration of RNA extracted from plasma range from 20 to 100 ng/μL, and RNA from Tissues range from 310 to 590 ng/μL.2.9 MicroRNA quantification by Real-time quantitative RT-PCR(qRT-PCR)For reverse transcription step, first 50 ng of plasma RNA,500ng of tissues or cell line RNA containing miRNA were polyadenylated by E.coli Poly(A) Polymerase(NEB, USA) and then reverse transcribed to cDNA by PrimeScriptTM RT kit (TaKaRa) all according to the manufacturer's instructions, meanwhile using 5'GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTCG-3'.as RT primerFor qPCR step, we used our certain designed primer(5'-GCGAGCACAGA真度ATTAATACGAC-3') as universal reverse primer; and the forward primers were using the miRNA-specific primer sequences, each of them were 5'-ACGCAAATTCG TGAAGCGTT-3'for RNU6B; 5'-TAGCAGCACGTAA ATATTGGCG-3'for has-miR-16;5'-AGGCAAGATGCTGGCATAGCT-3'for has-miR-31; miRNA sequences obtained from the miRBase database (http://microrna. sanger.ac.uk/). And all the sequences were synthesized in Invitrogen Crop.Real-time qPCR was performed using SYBRR Premix Ex TaqTM PCR kit (TaKaRa) in ABI PRISM 7300 Real-time PCR system (Applied Biosystems). Each reaction was performed in a final volume of 25μL containing 2μL of the cDNA,5μM of each primer and 1x SYBR Green PCR Master mix (Qiagen). The amplification profile was:denaturation at 95℃for 10sec, followed by 40 cycles of 95℃for 5sec,60℃for 20s. At the end of the PCR cycles, melting curve analyses were performed as well as electrophoresis of the products on 3.5% agarose gels in order to validate the specific generation of the expected PCR product. Each sample was run in triplicates. After reaction, the CT data were determined using default threshold settings and the mean CT was determined from the duplicate PCRs. The ratio of cancer sample miRNA to normal sample miRNA was calculated by using the equation 2-⊿CT, in which⊿CT=CT cancer-CT normal. The expression levels of miR-31 were directly normalized to RNU6B or miR-16.2.10 Statistical analysis:All statistical analysis was performed using the SPSS 16.0 software. The two sample t-test was used for normally distributed data. Fisher's exact test was used for analyzing immunohistochemical staining in prostate tissues. Differences were considered to be statistically significant at P<0.05. Expression levels of plasma miRNAs were compared using the Mann-Whitney U test or the Kruskall-Wallis test. Multivariate logistic regression model was established and leave-one-out cross validation to find the best logistic model. Receiver-operating characteristics (ROC) curves were established to evaluate the diagnostic value of plasma miRNAs for differentiating between tumors and controls. A p value of less than 0.05 was considered statistically significant.3. Results:3.1 The relationship between expression of miR-31 and invasion and metastasis of esophageal squamous cell carcinoma1) The expression of miR-31 and the metastatic capacities of three kinds of Human ESCC cell lineThe expressions of miR-31 in ESCC cell line EC9706, KYSE150, KYSE510 were 0.025,0.004 and 0.001 (relatively normalized level), respectively, the expressions of EC9706 and KYSE150 were higher than KYSE510.2) miR-31 over-expression and suppression of the three types of esophageal squamous cell carcinoma cell lines in vitro migration, invasion and adhesionTransWell cell migration assay, cell invasion assay, cell-matrix assay showed that miR-31 transfection resulted in a significant increase of migration, invasion and adhesion than the control group, the cell numbers through the basement membrane were increased more than two times after miR-31 transfection; anti-miR-31 transfection resulted in a significant decrease of migration, invasion and adhesion than the control group, vice versa.3.2 The expressions of miR-31 in ESCC tissues and normal tissues1) The expressions of miR-31 in ESCC tissues and adjacent normal tissueThe expression levels of miR-31 increased in cancer tissues than matched normal tissues was 75.5%(36/45). The expressions of miR-31 in cancer tissues(n= 45) were significantly higher than matched normal tissues (n =45) (P=0.0441).2) The relationship between expression of miR-31 and lymph node metastasisThe expressions of miR-31 in cancer tissues with metastasis (n=21) of miR-31 were significantly higher than those without metastasis (n=24)(P=0.0243)3) The relationship between the expressions of miR-31 and TNM Stage of ESCC There were significant differences among the expressions of miR-31 in all TNM stages (P=0.0496), there was significant difference between stageⅠ(n=7) and stageⅢ(n=21) (P=0.0361), there was no significant difference between stage II and stageⅢ(n=14) (P=0.0832).3.3 The expressions of miR-31 in plasma of ESCC patients1) The expression of miR-31 in plasma of ESCC patients and asymptomatic populationThe expression of miR-31 in plasma of ESCC patients (n=120) was significantly higher than the asymptomatic population (n=121) (P<0.0001)2) The expression of miR-31 in plasma of ESCC patients and lymph node metastasisThe expression of miR-31 in plasma of ESCC patients with lymph node metastasis (n=65) was significantly higher than those without lymph node metastasis (n=55) (P<0.0001)3) The expression of miR-31 in plasma of ESCC patients and TNM stageThe expression of miR-31 in plasma of ESCC patients was associated with TNM stage, the expressions of miR-31 in plasma of ESCC patients with stageⅣ(n=28) were significantly higher than in stageⅠ(n=28),Ⅱ(n=31) andⅢ(n=33), P was 0.0001,0.0002,0.0263, respectively, but between stageⅠandⅡ, stageⅡandⅢ, stageⅠandⅢ, there was no significant difference(P>0.05).4) The expression of miR-31 in plasma of ESCC preoperative and postoperative patientsThe expression of miR-31 in plasma of ESCC preoperative patients was significantly higher than postoperative patients (P<0.0001)5) The expression of miR-31 in plasma of ESCC patients and survival timeMiR-16 expression by internal reference standard, postoperative patients with esophageal squamous cell carcinoma Expression of miR-31 miR-16 were higher than their high expression group, the lower of the low expression group. miR-31 2-year survival rate of low-expression group was 80%(16/20), significantly higher than the high expression group (50%,12/24), survival time of low expression group was significantly longer than the high expression group(P=0.034); no recurrence 2 years survival rate of miR-31 low-expression group was 60%(12/20), significantly higher than the high expression group (42%,10/24) (P=0.0234).6) The predictive power for ESCC using plasma miR-31 as a diagnostic markerTo plasma CT miR-31-CT miR 16≤7.4 as a diagnostic criteria for ESCC, the diagnostic sensitivity and specificity of the expression of miR-31 in plasma of ESCC were 86.7% and 84.3%, respectively.4. Conclusions:4.1 The microRNAs miR-31 promoted esophageal squamous carcinoma invasion and metastasis, and might be a potential targets for therapy.1) miR-31 might play an oncogenic role in the invasion and metastasis of ESCC through promoted migration, invasion and adhesion.2) It is theoretically feasible that inhibition of miR-31 induces normalized miR-31 to suppress metastasis of ESCC in vitro.4.2 Plasma miR-31 was a novel biomarker for ESCC.Plasma miR-31 levels were associated with the presence of primary tumor. In patients with ESCC, plasma levels of miR-31 were significantly higher than those in asymptomatic population, and miR-31 levels were significantly reduced after surgical removal of the primary tumors, correlated to metastasis of lymph node tumor and stage of TNM, might be a potential biomarker for ESCC in the diagnosis, prediction of prognosis, relapse and metastasis.4.3 Recur to bioinformatics and literature, targeted genes of miR-31 were predicted as MAPK1, MAPK4, PDE4D and RASA1。Further studies are warranted.