Proteinase-activated Receptor 2 Promotes Cancer Cell Migration through RNA Methylation-mediated Repression of miR-125b*

Background: Proteinase-activated receptor 2 (PAR2) correlated with cancer metastasis. Results: Down-regulation of miR-125b, which targets Gab2, mediates PAR2-induced cancer cell migration. Conclusion: PAR2 promotes cancer cell migration through RNA methylation-mediated repression of miR-125b. Significance: This study suggests a novel epigenetic mechanism by which miRNA expression is altered to regulate cancer cell migration. Proteinase activated-receptor 2 (PAR2) participates in cancer metastasis promoted by serine proteinases. The current study aimed to test the molecular mechanism by which PAR2 promotes cancer cell migration. In different cancer cells, activation of PAR2 by activating peptide (PAR2-AP) dramatically increased cell migration, whereas knock down of PAR2 inhibited cellular motility. The PAR2 activation also repressed miR-125b expression while miR-125b mimic successfully blocked PAR2-induced cell migration. Moreover, Grb associated-binding protein 2 (Gab2) was identified as a novel target gene of miR-125b and it mediated PAR2-induced cell migration. The correlation of PAR2 with miR-125b and Gab2 was further supported by the findings obtained from human colorectal carcinoma specimens. Remarkably, knock down of NOP2/Sun domain family, member 2 (NSun2), a RNA methyltransferase, blocked the reduction in miR-125b induced by PAR2. Furthermore, PAR2 activation increased the level of N6-methyladenosine (m6A)-containing pre-miR-125b in NSun2-dependent manner. Taken together, our results demonstrated that miR-125b mediates PAR2-induced cancer cell migration by targeting Gab2 and that NSun2-dependent RNA methylation contributes to the down-regulation of miR-125b by PAR2 signaling. These findings suggest a novel epigenetic mechanism by which microenvironment regulates cancer cell migration by altering miRNA expression.

In the present study, we demonstrated that miR-125b mediates cell migration induced by PAR 2 activation and identified Gab2 (Grb2-associated-binding protein 2) as a novel target gene of miR-125b. Moreover, it was found that PAR 2 decreased the level of miR-125b through NSun2 (NOP2/Sun RNA methyltransferasefamily,member2)-mediatedpre-miR-125b2methylation. Our findings not only reveal a novel epigenetic mechanism underlying promotion of cancer metastasis by PAR 2 but also provide potential target for development of new therapeutic approaches in human cancer.
RNA Isolation and Real-time PCR-Total RNA was isolated from cell lines, human, or animal samples by using Trizol reagent (Invitrogen). After treatment with DNase I, RNA was reverse transcribed into cDNA with Thermo scientific maxima first strand cDNA synthesis kit for mRNA or pri-miR-125b detection, or with Takara TM microRNA transcription kit for mature and pre-microRNA detection. Real-time quantitative PCR was carried out on Bio-Rad S1000 PCR instrument, and each sample was analyzed in triplicate. PCR data were normalized to GAPDH or U6 snRNA expression for mRNA and miRNA, respectively.
For overexpression study, full-length of Gab2 and BMPR1B sequence were purchased from Vigene (Rockville, MD, USA) and subcloned into pENTER expression vector, respectively.
Migration Assay-After starving in serum-free media for 12 h, 5 ϫ 10 4 cells were seeded on the top chamber of transwell (8.0-m pore; Corning, MA) and incubated with 100 M PAR 2 -AP, while 600 l media containing 10% FBS was added to the bottom chamber. Ten hours later, the cells remaining in the upper chamber were removed with cotton swabs, and the cells on the undersurface of transwells were fixed and stained with crystal violet solution. The number of migratory cells was calculated by counting five different fields under a phase-contrast microscope of each transwell filter.
Gab2 3Ј-UTR Luciferase Reporter-3Ј-UTR sequence of Gab2 containing the putative miR-125b target sites was amplified from A549 genomic DNA using Gab2 sense, 5Ј-CGC TAG CCG TTC TTC CTC CCA TCC AC-3Ј and antisense, 5Ј-CTC TAG ACC ACT GCT GAG CCT CCG TC-3Ј and cloned immediately into the downstream of the luciferase reading frame in the plasmid PIS0-Report-vector. To generate the 3Ј-UTR fragments mutating the miR-125b recognition elements by KOD-plus-mutagenesis kit (TOYOBO Co., Ltd. Japan), the following primer pairs were used: 5Ј-TGA CAA CTG CGT CCC TGA ACC CTT CCC CTG GGA GGT G-3Ј and antisense, 5Ј-AGT GCT TTG AAG GCT GAG AC-3Ј. The sequence of PCR product was confirmed by DNA sequencing.
Transient Transfection and Luciferase Assay-The cells in 24-well plates were transfected with or without miR-125b mimic (100 nM) together with wild type or mutant Gab2 3ЈUTR reporter (200 ng) and pRL-TK (2 ng). The transfection agent Lipofectamine 2000 (Invitrogen) was incubated with DNA or RNA in serum-free medium for 20 min before being added to cells and then incubating for an additional 2 h. After treatment with PAR 2 -AP, cell lysate was collected for luciferase activity assay, which was performed with a Dual-Luciferase assay kit (Promega). All data were normalized by pRL-TK.
Measurement of m 6 A RNA-To detect M 6 A RNA, cell extract was prepared with EZ-Magna RIP TM Kit (Millipore) according to the manufacturer's instructions. Briefly, after treatment with PAR 2 -AP for a half hour, A549 cells were collected and lysed in RIP lysis buffer. To pull down m 6 A RNA, whole-cell extract was immunoprecipitated with anti-m 6 A antibody (Synaptic Systems) conjugated with magnetic beads. After incubation with proteinase K, RNA was recovered followed by treatment with DNase I to avoid DNA contamination. The RNA samples were then subjected to RT and real-time quantitative PCR to detect pri-miR-125, pre-miR-125b and miR-125b. Normal mouse IgG was used as negative control. Sample without immunoprecipitation was used as input and positive control.
Human Subjects Tissue Specimen-All the colorectal cancer tissues and the paired normal tissues (Ն5 cm from cancer tissue) were obtained from Cancer hospital, Chinese Academy of Medical Sciences, Beijing, China. All specimens were classified according to the tumor node metastasis (TNM) classification system. No patients received chemotherapy or radiotherapy before resection. Tissues were submerged in RNA later TM (Ambion) for 24 h and then stored at Ϫ70°C until use. The use of human tissues was approved by the Institutional Review Board of the Chinese Academy of Medical Sciences Cancer Institute.
Statistical Analysis-Statistical analyses were performed on data collected from at least three independent experiments. Data were presented as means Ϯ S.D. and analysis using GraphPad Prism 5 software. Comparison of Ͼ2 groups was made using ANVOA with Tukey test. Comparison of 2 groups was made using Student's t test for unpaired data. The differences were considered statistically significant when p value less than 0.05.

Results
MiR-125b Mediates PAR 2 -induced Cell Migration-In the first set of experiments, we found that PAR 2 protein was FIGURE 1. PAR 2 activation promotes cell migration through the down-regulation of miR-125b. A, expression of PAR 2 protein was measured with Western blot in different cell lines. B, cell migration was evaluated with transwell assay. Cells were treated with the activating peptide of PAR 2 (PAR 2 -AP, 100 M) for 10 h and then migrated cells were stained and quantified. C, upper panel, Western blot shows the protein level of PAR 2 in SW620 and RKO cells stably transfected with or without shPAR 2 . ShPAR2-1 and shPAR2-2 represent different clones from the same parental cells. Lower panel, cell migration was tested with transwell assay in these cells. D, expression level of miR-125b was measured with real time PCR in different cell lines. E, after the treatment with PAR 2 -AP (100 M) for 6 h, cells were collected and tested for miR-125b expression by real-time PCR. F, level of miR-125b was measured in SW620 and RKO cells stably transfected with or without shRNA-PAR 2 (shPAR 2 ). G, cells were transfected with miR-125b mimic (50 M). For the migration assay, thirty-six hours later, cells were seeded into transwell and treated with PAR 2 -AP for another 10 h. The data showed the means Ϯ S.D. from three independent experiments. expressed in all the human cancer cell lines we tested (Fig. 1A).
To determine whether PAR 2 activation induce cell migration, activating peptide (PAR 2 -AP) was used to selectively activate PAR 2 on cell surface. As shown, activation of PAR 2 by PAR 2 -AP for 10 h significantly stimulated cell migration as measured by the transwell assay in different cancer cell lines (Fig. 1B). And there was no any effect on cell proliferation as examined by MTT assay (data not shown). In addition, knockdown of PAR 2 expression with shRNA remarkably attenuated cell migration compared with that observed in scramble RNA control group in different cancer cells (Fig. 1C). These results strongly suggested that PAR 2 activation promotes migration in different cell lines.
To test whether miR-125b involves in PAR 2 -related migration, we measured the level of miR-125b and found that it negatively correlated with PAR 2 expression in all cell lines we used (Fig. 1D). In addition, the expression of miR-125b was significantly repressed after the stimulation with PAR 2 -AP (Fig. 1E). On the contrary, inhibition of PAR 2 expression dramatically elevated miR-125b expression compared with scramble RNA control group (Fig. 1F). Importantly, over expression of miR-125b blocked cell migration induced by PAR 2 activation in A549 cells (Fig. 1G). Similar results were observed in SW620 cells (data not shown). Taken together, these findings indicate that down-regulation of miR-125b mediates PAR 2 -induced cell migration.
Prediction of miR-125b Target Genes-To inquire into the mechanism by which miR-125b mediates PAR 2 -induced cell migration, we identified the downstream targets of miR-125b. First, the target mRNAs of miR-125b were predicted with different database including Pitsca, TargetScan, miRwalk, and miRDB. There were 259 potential target transcripts by at least two databases. And 124 of them were overlapped with PAR 2related transcriptome, which was conducted with Human Gene Expression Microarray V3.0 in HT-29 cells. Among them, 7 genes were annotated involved in cell migration according to the DAVID Annotation Bioinformatics Database. Furthermore, 3 of them showed significant difference between stably knockdown of PAR 2 group and scramble control group (p Ͻ 0.05) ( Fig. 2A). At last, validating with real-time PCR, Gab2, and BMPR1B were significantly down-regulated with the knock down of PAR 2 in HT29 cells (Fig. 2B).
Since BMPR1B has been reported as the target gene of miR-125b (Ref. 49), we verified the regulation of Gab2 by miR-125b. It was found that forced expression of miR-125b achieved by the transfection with the mimic of miR-125b inhibited Gab2 expression at both mRNA and protein levels (Fig. 2C). In contrast, the inhibitor of miR-125b elevated the expression of Gab2 (Fig. 2D). Furthermore, activation of PAR 2 increased Gab2 expression (Fig. 2E) while disruption of PAR 2 signaling repressed Gab2 (Fig. 2F) at both mRNA and protein levels. All these results strongly suggest that Gab2 is a possible target gene of miR-125b.
Gab2 Was Identified as a Novel Target Gene of miR-125b-To confirm Gab2 is a direct target gene of miR125b, the possible binding site of miR-125 in the Gab2 mRNA was analyzed. TargetScan analysis indicated that Gab2 contains miR-125b binding site within its 3Ј-UTR, and the sequence of the binding site is highly conserved across different species (Fig. 3A). To further validate that Gab2 is regulated by miR-125b through interaction of the complementary 3Ј-UTR region, luciferase reporters containing wild-type or mutant 3Ј-UTR of Gab2 were constructed (Fig. 3B). MiR-125b mimic significantly reduced the activity of the luciferase reporter, which was partially abolished by the mutation of miR-125b binding site in 3Ј-UTR of  Gab2 is a direct downstream target of miR-125b. A, potential binding site of miR-125b in Gab2 3Ј-UTR in different species. B, diagram showed the putative miR-125b-targeting seed sequence on Gab2-3Ј-UTR and illustrated the structure of wild type and mutant luciferase reporter of Gab2 3Ј-UTR. C, A549 cells were transfected with or without miR-125b mimic combined with wild type or mutant luciferase reporter of Gab2-3Ј-UTR. Twenty-four hours later, the cells were collected for luciferase assay. D, after the transfection with wild type or mutant luciferase reporter of Gab2-3Ј-UTR, A549 cells were stimulated with PAR 2 -AP for 6 h and then collected for luciferase assay. E, A549 cells were transfected with miR-125b mimic (mimic-125b) or control RNA. One day after transfection, the cells were treated with PAR 2 -AP for 6 h, and the cell lysates were collected for Gab2 mRNA and protein expression assay. F, SW620 cells with or without stable transfection of shPAR 2 were transfected with inhibitor of miR-125b (Inhibitor-125b) or control RNA. Twenty-four hours later, the cell lysate was collected for Gab2 mRNA and protein expression assay. The data showed the means Ϯ S.D. from three independent experiments. Gab2 (Fig. 3C). Furthermore, PAR 2 activation up-regulated the activity of wild type Gab2 3Ј-UTR-derived luciferase reporter but not the mutated one (Fig. 3D). Consistent with luciferase activity, PAR 2 activation also elevated Gab2 mRNA and protein levels, which were reduced by overexpression of miR-125b (Fig.  3E). Contrarily, inhibition of PAR 2 repressed the levels of Gab2 mRNA and protein, which was recovered by the inhibitor of miR-125b (Fig. 3F). All these results suggest that miR125b partially regulates Gab2 expression through the binding site within 3Ј-UTR of Gab2 mRNA.
Gab2 Mediates PAR 2 -induced Cell Migration-To test whether Gab2 mediates PAR 2 -induced cell migration, specific siRNAs against Gab2 were applied to reduce the expression of Gab2 at both mRNA and protein levels (Fig. 4A). Knockdown of Gab2 significantly blocked PAR 2 -induced cell migration (Fig. 4, C and E). Since BMPR1B, a known target gene of miR-125b, had been changed after PAR 2 activation, we checked the effect of BMPR1B on PAR 2 -induced cell migration. Knockdown of BMPR1B with specific siRNA (Fig. 4B) also blocked cell migration stimulated by PAR 2 activation (Fig. 4, C and E). In addition, overexpression of Gab2 or/and BMPR1B totally reversed the repression of cell migration induced by miR-125b (Fig. 4, D and   E). These findings indicate that repression of miR-125b mediates cell migration induced by PAR 2 through targeting Gab2 and BMPR1B expression in vitro.
Correlation of PAR 2 , miR-125b, and Gab2 in CRC Specimens-To confirm the findings in vivo, we tested the expression of PAR 2 , miR-125b and Gab2 in 40 paired human CRC tissue specimens measured by real-time PCR. As shown in Fig.  5A, both PAR 2 and Gab2 were up-regulated while miR-125b was down-regulated in lymphatic metastasis group compared with those of non-metastasis group. Importantly, in lymphatic metastasis group, the level of PAR 2 was negatively correlated with that of miR-125b and positively correlated with that of Gab2 (Fig. 5B). Furthermore, miR-125b expression was negatively correlated with Gab2 levels (Fig. 5B). In addition, the level of PAR 2 was also positively correlated with Gab2 in gene expression profiling of lung adenocarcinoma (NCBI GEO accession number GSE31546) uploaded by Hamon Center for Therapeutic Oncology Research of UT Southwestern Medical Center, which provided 17 primary lung cancer specimens expression profiling (Fig. 5C). These results strongly suggest that PAR 2 mediates metastasis through miR-125b/Gab2 in human CRC.

PAR 2 Suppresses miR-125b through NSun2-mediated Repression of miR-125b
Processing-Since miR-125b mediate PAR 2induced cell migration both in vitro and in vivo, it is important to understand how PAR 2 signaling regulates the level of miR-125b. Recent studies showed that the methylation state of CpG-rich regions is correlated with the expression pattern of miR-125b in breast cancer (22). To investigate whether hypermethylation of miR-125b promoter contribute to PAR 2induced repression of miR-125b, 5-azacytidine, a demethylation agent, was used. The data showed that pretreatment with 5-azacytidine completely reverse the level of miR-125b reduced by PAR 2 -AP (Fig. 6A). However, there was no obvious change in methylation status of pri-miR-125b1 promoter as measured by MSP after PAR 2 activation (Fig. 6B). Moreover, there was no significant change of primary miR-125b after PAR 2 activation (data not shown).
Actually, mature miR-125b comes from two primary forms, pri-miR-125b1 and pri-miR-125b2. Most recently, NSun2, a RNA methyltransferase, is shown to interfere in the mature processing of miR-125b from pri-and pre-miR-125b2. Interestingly, we found that pri-and pre-miR-125b2 was the predominant form of miR-125 precursor in A549 cells but not pri-and pre-miR-125b1 (Fig. 6C). Furthermore, knockdown of NSun2 with specific siRNA (Fig. 6D) blocked the repression of miR-125b induced by PAR 2 activation (Fig. 6E).
To test whether PAR 2 activation induces methylation of miR-125b and its precursors, methylated RNA was pull down with anti-m 6 A antibody and miRNA and its precursors were measured with qPCR. The data showed that PAR 2 activation induced m 6 A methylation of pre-miR-125b2 (Fig. 6F) but not pri-miR-125b2 (Fig. 6G). Moreover, knockdown of NSun2 completely blocked both PAR 2 -induced m 6 A methylation of pre-miR-125b2 (Fig. 6F) and PAR 2 -induced Gab2 mRNA expression (Fig. 6H). These findings strongly suggest that PAR 2 reduces miR-125b expression through an NSun2-dependent and m 6 A methylation-involved mechanism.

Discussion
Both PAR 2 and its-activating proteinases, including tissue factor, trypsin, KLKs, are aberrantly overexpressed in a variety of malignancies, and shown to be involved in many aspects of cancer biology including cell proliferation, angiogenesis, and metastasis (26,27). In addition, PAR 2 contributes to the tumorigenesis through the induction of Cox-2 (28), MMP 2/9 (29), FIGURE 5. Correlation of PAR 2 , miR-125b and GAB2 in human specimens. A, expression of PAR 2 , Gab2 and miR-125b in human CRC samples was tested with real time PCR. The data are shown as the fold change of tumor sample (T) to paired normal tissue (N). B, Pearson's correlation analysis was conducted for PAR 2 /miR-125b, Gab2/miR-125b and PAR 2 /Gab2. Black line represents linear regression line. C, Pearson's correlation analysis was conducted for PAR 2 /Gab2 in human lung cancer specimens microarray, which provided 17 primary lung cancer specimens gene expression profiling (NCBI GEO accession number GSE31546). and interleukin-8 (30). Inhibition of PAR 2 not only effectively suppresses tumor growth (31) but also prevents tumor formation in mouse model (8). Therefore, PAR 2 is an attractive and potential therapeutic target for tumor. Here, we demonstrated that miR-125b mediates PAR 2 -promoted cell migration by targeting Gab2 in cancer cells. Moreover, PAR 2 suppressed the processing of miR-125b in Nsun2-dependent and miRNA methylation-mediated manner (Fig. 7). Thus, the interaction between PAR 2 signaling and miRNAs provides novel intrinsic molecular mechanism by which PAR 2 participates in tumorigenesis.
In the last decade, miRNAs have emerged as critical regulators in various cancers, including colorectal carcinoma (33,34). The role of miR-125b in tumorigenesis is controversial since it can act as oncogene or tumor suppressor, depending on types of different tumors. Deregulations of miR-125b are observed commonly in breast (22,32), ovarian (36), and liver cancers (21), while its overexpression is implicated in pancreatic (37), and prostate (38) cancers. With regard to the expression of miR-125b in CRC, the evidence is conflicting. A study from Japan shows that high levels of miR-125b expression are associated with reduced survival rates in 89 colorectal tumors specimens (39). However, due to the absence of paired adjacent non-tumor tissue for each CRC sample, it is unclear whether the level of miR-125b in tumor is lower than that in non-tumor tissue. Compared with paired non-tumor tissue, our results clearly showed that miR-125b expression was down-regulated in tumor site, and it was also associated with lymphatic metastasis in CRC specimens (Fig. 5A).
Although deregulation of miR-125b plays a role in tumor development, the regulation of miR-125b expression is not well understood. Promoter methylation is a common mechanism for silencing miRNAs in cancer (40). Hypermethylation of miR-125b-1 promoter is involved in miR-125b down-regulation in FIGURE 6. PAR 2 regulates miR-125b expression through NSun2-dependent mechanism. A, after the pretreatment with 5Ј-azacytidine for 72 h, A549 cells were stimulated with PAR 2 -AP for 6 h. The level of miR-125b was measured by real time PCR. B, A549 cells were treated with PAR 2 -AP for 6 h and then collected for MSP PCR to detect the methylation of miR-125b1 promoter. Upper panel, representative pictures of MSP PCR; lower panel, quantification of three independent MSP PCR. M, methylated form. U, unmethylated form. C, levels of pri-and pre-miR-125b1, pri-and pre-miR-125b2 were measured with PCR. D, Western blot shows the expression of Nsun2 after transfection with control RNA or siRNA against Nsun2 (siNSun2) in A549 cells. E, 40 h after transfection with si-Nsun2, cells were stimulated with PAR 2 -AP for 0.5 or 2 h and then collected to measure miR-125b expression. F and G, after transfection with siNSun2, A549 cells were treated with PAR 2 -AP for 0.5 h. RNA was isolated and pulled down with an anti-m 6 A antibody. Pull down material was collected to measure the levels of (F) pre-miR-125b2 and (G) pri-miR-125b2 and miR-125b with real-time PCR. H, after transfection with siNSun2, A549 cells were treated with PAR 2 -AP for 6 h and then collected to detect Gab2 mRNA by real time PCR. The data showed the means Ϯ S.D. from three independent experiments. invasive breast cancer (22). Although the inhibition of methylation by 5-aza recovered the level of miR-125b (Fig. 6A), the expression of pri-miR-125b-1 and pri-miR125b-2 were not altered by PAR 2 activation (data not shown). Thus, our evidence does not support the transcriptional suppression of miR-125b by PAR 2 .
Except for DNA methylation, RNA methylation is also implicated in the control of miR-125b processing (41). Methylation is a prevalent modification of almost all species of RNA. Methylation of RNA regulates the stability of RNAs and the biogenesis/processing of RNAs (42). Recently, the processing of miRNA-125b was found to be regulated by Nsun2-mediated RNA methylation in mammalian cells (41). NSun2 (NOP2/Sun domain family, member 2 is a nucleolar RNA methyltransferase implicating in cell proliferation, stem cell differentiation, and human cancers (42). NSun2 is highly expressed in cancer (43) and validated as a transcriptional target of the Lef1/␤-catenin complex in hair follicles (44) and c-myc in skin (43). NSun2 methylates the 3Ј-untranslated region (UTR) of p16 mRNA and induces the stabilization of p16 mRNA (45). Methylation by Nsun2 decreases the levels miR-125b and disrupts its function. NSun2 is also shown to methylate precursor of miR-125b, interferes with its processing and reduces the level of mature miR-125b (41). In our study, Nsun2 silencing not only inhibited m 6 A-containing pre-miR-125b-2 ( Fig. 6F) but also rescued the down-regulation of miR-125b induced by PAR 2 (Fig. 6E). These findings indicate that PAR 2 signaling interferes miR-125b processing through miRNA methylation in Nsun2-dependent manner.
Gab2 was identified as a novel target of miR-125b in the current study. Gab2, as a scaffolding protein, integrates and amplifies signals in a variety of sources including growth factors, cytokine, and antigen receptors as well as cell adhesion molecules (46). Recently, Gab2 is emerging as an oncoprotein, and its protein is frequently overexpressed in various cancers (46). Here we showed that Gab2 expression was associated with lymph node metastasis in CRC, and the findings are consistent with other recent reports (46,47). Besides gene amplification, E2F and estrogen signaling have also been reported to transcriptionally up-regulate Gab2 expression (35). In the present study, we provided a novel epigenetic mechanism by which Gab2 was up-regulated by PAR 2 signaling. Our previous study demonstrated that PAR 2 signaling also activates the transcriptional activity of E2F in colon cancer cells (16). Therefore, our results strongly suggest that PAR 2 up-regulates Gab2 expression at both transcriptional and post-transcriptional levels in vivo. Moreover, as a scaffolding protein, Gab2 mediates the crosstalk between various signaling pathways (46). This also provides the basis for the synergistic action of PAR 2 signaling. For example, Gab2 acts as a downstream of EGFR (35,47), which is transactivated by PAR 2 (7). However, further study is needed to test whether Gab2 mediates transactivation of EGFR by PAR 2 activation. Therefore, considering the simultaneous exposure to plenty of stimuli in the real in vivo condition, Gab2 is considered as a key molecule for the crosstalk and integration of PAR 2 signaling and other pathways.
In conclusion, our results indicate that activation of PAR 2 promotes cell migration through the down-regulation of miR-125b via m 6 A-related mechanisms. Our study provides a novel epigenetic mechanism by which microenvironment regulates the level of miRNAs and promotes cancer cell migration.