MiR156a Targets Mammalian Junctional Adhesion Molecule-A To Repress Epithelial-mesenchymal Transition In Human Nasopharyngeal Cancer

Background and objectiveThe Brassica genus is cultivated in most parts of the world. It includes various important agronomical crops such as broccoli, cauliflower and Brassica rapa. Broccoli is very popular in large groups of the population due to its flavor and anti-cancer activities in prostate cancer, colon and rectal cancer, and so on.MicroRNAs （miRNAs） are an abundant class of small non-coding RNAs that suppress gene expression at the post-transcriptional level by usually imperfect base-pairing to the3’untranslated region （UTR） of a target mRNA, which results in either mRNA degradation or translation inhibition. miRNAs have been widely shown to modulate various cellular processes, including proliferation, differentiation, cell death and cell mobility. Furthermore, dysregulation of miRNAs has been linked to cancer and other diseases. Recently, the rate of plant miRNA discovery has increased dramatically with the advent of second generation sequencing technology. In dicotyledonous species,337mature miRNAs in A. thaliana and401in Populus trichocarpa have been discovered, and, in monocotyledonous species,384from Arabidopsis lyrata,713from Oryza sativa and43from Brassica rapa （miRBase release20.0, June,2013, http://mirbase.org/）. Although broccoli is an important cereal crop, none miRNAs in broccoli have been reported. Interestingly, a recent report indicated that food-derived exogenous plant MIR168a can pass through the mouse gastrointestinal （GI） track and enter the circulation.Junctional adhesion molecule-A （JAM-A）, also known as JAM-1or F11R, belongs to a family of cell adhesion molecules that localise at intercellular junctions, and mediate several different physiological processes, including intercellular junction assembly and cell polarity, cell morphology, and leukocyte migration. JAM-A is expressed by a number of cell types including epithelial, endothelial cells, and dendritic cells; leukocytes and platelets. In epithelial cells, JAM-A is preferentially concentrated at tight junctions where it is involved in promoting cell-to-cell adhesion. Although JAM-A is expressed at high levels in the enriched hematopoietic stem cell fraction, the roles of JAM-A in tumour growth and dissemination are still debated.Epithelial-to-mesenchymal transition （EMT） is the process of converting epithelial cells into mesenchymal cells, and is a central mechanism responsible for the invasiveness and metastasis of various cancers. EMT is defined by loss of the epithelial phenotype and the acquisition of mesenchymal characteristics, such as migratory capacity, polarity reversal, and loss of cell-to-cell contacts. One of the earliest steps in EMT is the loss of E-cadherin function. Several transcription factors are reported to drive EMT, including members of the Snail family and the basic helix-loop-helix transcription factor Twist. Moreover, it has been reported that the phosphatidylinositol-3kinase （PI3K） downstream effector, protein Kinase B （PKB, also known as Akt）, is indispensable for the upregulation of Twist and Slug in squamous cell carcinoma and liver cancer. In this study, we aimed to investigate the role of broccoli miRNAs in human nasopharyngeal cancer （NPC）.MethodsPart I Identification and characterization of microRNAs in broccoli, using high-throughput sequencing and bioinformatics analysis1. Total RNA extractions from broccoli were performed using TRIzolTM reagent, following the manufacturer’s instructions, and the RNA samples were used as a single RNA pool.2. The purity and integrity of RNA were measured.3. The sRNA fraction was measured by15%denaturing polyacrylamide gel, then, the integrity of RNA were measured by AgilentBioanalyzer.4. The construction sRNA libraries and deep-sequencing were each performed by the Beijing Genomics Institute. Briefly, the sRNA fraction, of length18and30nt, was extracted from a15%denaturing polyacry lamide gel.5. Total RNA was first extracted using TRIzolTM reagent. Then,1μl of the stem-loop RT primer （1μM） and2mg of total RNA were used in first-strand cDNA synthesis with an iScriptTM DNA synthesis kit.6. The sRNAs were then ligated to a pair of Solexa adapters at their5’-and3’-ends. Next, the sRNAs were converted to DNA by RT-PCR. Finally, the purified PCR products were directly sequenced with a HiSeq2000Sequencing System, used according to the manufacturer’s protocol.7. Statistical analysis Statistical analysis was performed with SPSS statistical package （v20.0）. In vitro experiments were repeated three times and data are presented as the mean±standard deviation （SD）. Statistical differences among groups were assessed with one-way analysis of variance （ANOVA）. Part Ⅱ Junctional adhesion molecule-A, an epithelial-mesenchymal transition inducer, correlates with metastasis and poor prognosis in human nasopharyngeal cancer1. Lentiviral vectors construction The stably overexpressing JAM-A cell lines CNE2-pWPI-JAM-A and Hone1-pWPI-JAM-A and their respective control cell lines were transduced with either pWPI encoding human JAM-A or empty pWPI vector, respectively. For JAM-A short hairpin RNA （shRNA） constructs, the pRS （retro-super）-constructs containing JAM-A shRNA were generated by cloning two JAM-A-specific RNAi target sequences into pRS.2. Wound-healing assay For wound-healing assay, the cells reaching90%confluence were then serum-starved for24hours, and similar sized wounds were made by scraping a conventional10-μl micropipette tip across the monolayer.3. Migration and invasion assays Briefly, for transwell and Boy den chamber assays cells with up-or down-regulated JAMA were serum-starved for24hours, then2×104cells were plated into the upper chamber and incubated for22hours.4. Western blotting was used to measure the protein associated with EMT and CSCs.5. JAM-A was detected using rabbit polyclonal antibody. For clinical samples, all slides were evaluated independently by two investigators who were blinded to the patients’ clinical data. We classified the tumours into two major groups based on JAM-A expression: JAM-A-negative and JAM-A-positive.6. Statistical analysis Statistical analysis was performed with SPSS statistical package （v20.0）. In vitro experiments were repeated three times and data are presented as the mean±standard deviation （SD）. Statistical differences among groups were assessed with one-way analysis of variance （ANOVA）. The χ2test and Fisher’s exact test were used to analyze the JAM-A expression with other clinicopathological parameters. Kaplan-Meier analysis and the log-rank test were used to illustrate differences between recurrence-free survival and overall survival rate according to JAM-A expression. Significant variables were further analyzed by multivariate analysis to test for independent prognosis. The P-values of less than0.05considered statistically significant.Part III miR156a targets mammalian junctional adhesion molecule-A to repress epithelial-mesenchymal transition in human nasopharyngeal cancer1. CCK-8assay:A total of800～1000cells were seeded in a96-well plate and then allowed to grow in normal medium for96hours. Cells were incubated in100ul normal+10ul CCK-8. The absorbance in each well was measured at450nm by a microplate reader.2. Bioinformatic analysis Full-length cDNAs of the human genes were obtained from the NCBI Genebank database. A program was developed and implemented to identify MIR168a-matched sites in the entire CDS/UTR of the transcripts. This program used several common criteria to determine whether a transcript was a target for MIR156a. The first criterion for target recognition named "seed rules" was base pairing between the "seed"（the core sequence that encompassed the first2to8bases of the mature miRNA） and the target. Second, the free energy of the hybrid was expected to be within the range of the authentic miRNA-target pairs, typically lower than-17kcal/mol. Third, an optional rule for target prediction required interspecies conservation of the putative binding sites. Using these rules,～40human genes were identified as putative targets of MIR156a.3. The human JAM-A gene （NM₀16946.4） was cloned from CNE2cells by reverse-transcriptase polymerase chain reaction （RT-PCR） using the primers （Forward:5’-GGC TTA ATT AAA TGG GGA CAA AGG CGC AAG;Reverse:5’-GCG GTT TAA ACT CAC ACC AGG AAT GAC GAG-3’）.Then. a JAM-A fragment generated digestion with Pac1and Pmelrestriction enzymes were ligated into a linearised pWPI plasmid.4. Transient or stable transfection of cells with scramble RNAs or siRNAs targeting JAM-A mRNA was performed using Lipofectamine2000, in accordance with the manufacturer’s protocol. The effects of siRNAs were confirmed by three independent transfection experiments.5.3’-UTR luciferase reporter assay DNA fragments of3’UTR of JAM-A that host the predicted complementary sites of miR156a or the mutated sites were digested by NotI-HF and XhoI. Then the DNA fragments were cloned downstream of the Renilla luciferase reporter gene in psiCHECK2dual luciferase reporter plasmid （Promega）.293T, CNE2and Honel cell lines were seeded in96well plates, and co-transfected with luciferase reporters together with miR156a expression plasmid. Cells were lysed and luciferase activities were measured48hours after transfection, according to manufacturer’s instruction using the Panomics Luminometer.6. Statistical analysis Statistical analysis was performed with SPSS statistical package （v15.0）. In vitro experiments were repeated three times and data are presented as the mean±standard deviation （SD）. Statistical differences among groups were assessed with one-way analysis of variance （ANOVA）.ResultsPart I Identification and characterization of microRNAs in broccoli, using high-throughput sequencing and bioinformatics analysis1. Sequence analysis of sRNAs In order to identify the miRNAs in responding to salt stress, we constructed and sequenced sRNA libraries ranging in size from18to30nt from both control and salt-stressed broccoli. Similar to typical size distribution of small RNAs observed in other plants, the most abundant small RNAs in broccoli were24-nt long in the broccoli library, and the second largest population was the 21-nt ones, followed by22and23-nt small RNAs. Genomic location analysis indicated that a majority of small RNAs were almost evenly located in the strand of chromosome from A1to A13.2. Conserved miRNA families are found in many plant species and have important functions in plant. In our study,84known miRNAs were identified. The identified miRNA families have been shown conserved in a variety of plant species. Furthermore, the relatively high expression of miRNA was also confirmed by quantitative RT-PCR. Consistent with the sequencing results, miR156a, miR168a and miR157d had the highest expression in the broccoli, whereas miR165a-3p expressed at the lowest level among the14miRNAs.3.184miRNAs were considered as novel. With an average of130read counts, the putative novel miRNA showed lower expression levels than the conserved miRNAs. These novel miRNAs precursors had negative folding free energies （-18.5to-123kcal mol-1） with an average of about-47.8kcal mol-1according to Mfold4. Interestingly, miR156a also expressed the highest level in flower and stem of cauliflowerPart II Junctional adhesion molecule-A, an epithelial-mesenchymal transition inducer, correlates with metastasis and poor prognosis in human nasopharyngeal cancer1. JAM-A overexpressed vector were sucessefully constructed.2. Ectopic expression of JAM-A induces EMT and enhances the stem cell properties of NPCs in vitro We found that JAM-A overexpression led to an obvious enhancement of invasion and metastasis of NPC cell lines. the migration index of pWPI-JAM-A NPC cells measured by wound-healing assays also increased compared with vector control cells. Western blotting analyses revealed a decrease in the expression levels of a-catenin and E-cadherin, and an increase in the expression levels of the mesenchymal cell markers N-cadherin and Vimentin in JAM-A-overexpressing NPC cell lines.3. Downregulation of JAM-A represses the EMT phenotype Migration and invasion assays revealed that ablation of endogenous JAM-A markedly reduced NPC cell migration and invasiveness.4. High JAM-A expression correlates with metastasis and poor prognosis in NPC High JAM-A protein expression correlated strongly with poorer OS in patients with NPC （Figure5B, P<0.01）. In addition, patients whose tumours had positive JAM-A protein expression were significantly more likely to develop a recurrence within5years compared with patients with negative JAM-A expression （Figure5C; P<0.01）.Part III miR156a targets mammalian junctional adhesion molecule-A to repress epithelial-mesenchymal transition in human nasopharyngeal cancer1. MiR156a represses EMT phenotype and reduces stemness potential of NPC cells in vitro. miR156a significantly suppressed the invasion of CNE2and HONE1cells in Boyden assays and reduced the migration in Transwell assays. Moreover, the migration index decreased significantly in CNE2and Honel cells when miR156a were transfected. A morphological change was also observed in miR156a transfected CNE2cell lines.2. Broccoli miR156a directly targets3’-UTR of mammalian JAM-A and decreases JAM-A protein level in vitro and in vivo. The most highly conserved sequence of a putative binding site among various species is located in3’UTR of the JAM-A. The minimum free energy of binding was calculated to be-27.3kcal/mol. Firstly, we evaluated whether miR156a could negatively regulated JAM-A expression. Compared with negative control group, co-transfection of miR156a mimics with the human JAM-A3’UTR wild-type reporter （psiCHECK2-JAM-A3’UTR-wt） resulted in a highly significant decrease in luciferase activity. Consistently, no decrease in luciferase activity was observed when miR156a mimics or negative control co-transfected with the empty vector or the mutant reporter （psiCHECK-JAM-A-3’UTR-MUT）.3. JAM-A is essential for miR156a induced EMT of NPC cells. Results indicated that after transfection of si JAM-A into CNE2and Honel cell lines, the expression of E-cadherin was increased. Western blotting indicated that JAM-A could partially abrogate the miR156a-repressed EMT in NPC cells via activating Akt. These results strongly support that miR156a regulates the mesenchymal phnotye directly targets3’-UTR of mammalian JAM-A and activates p-Akt.Conclusions1. Lots of of sRNAs were presented in broccoli. the most abundant small RNAs in broccoli were24-nt long in the broccoli library, and the second largest population was the21-nt ones.84conserved miRNA and novel miRNA were discovered.2. miR156a was dected in stems, leaves and flowers of broccoli, however, miR156a was expressed the most in the flower of broccoli.miRNA156a.3. JAM-A induces EMT in NPC in vitro and in vivo.4. Broccoli miR156a targets mammalian JAM-A to repress EMT in human NPC.