LncRNA RP11-465B22.8 triggers esophageal cancer progression by targeting miR-765/KLK4 axis

LncRNAs play an important role in tumorigenesis and progression; however, the function and mechanisms of lncRNAs in esophageal cancer (EC) remain largely unclear. In this study, we screened the differentially expressed lncRNAs in EC by using RNA-seq and one of the most upregulated lncRNAs, lncRNA RP11-465B22.8, was further characterized. LncRNA RP11-465B22.8 was upregulated in EC tissues and high lncRNA RP11-465B22.8 expression was associated with poor survival of EC patients. Ectopic expression of lncRNA RP11-465B22.8 enhanced the proliferation, migration, and invasion of EC cells, whereas knockdown of lncRNA RP11-465B22.8 led to the opposite effects. Mechanistically, lncRNA RP11-465B22.8 sponged miR-765 to increase the expression of KLK4. Moreover, LncRNA RP11-465B22.8 could be delivered from EC cells to macrophages via exosomes and subsequently induced M2 macrophage-induced cell migration and invasion. Our findings revealed a novel lncRNA RP11-465B22.8/miR-765/KLK4 pathway in EC and indicated that lncRNA RP11-465B22.8 might be a potential target for EC therapy.


INTRODUCTION
Esophageal cancer (EC) is one of the most common gastrointestinal malignancies. In 2012, approximately 460,000 EC cases and 400,000 deaths occurred in the world [1]. Because of the unspecific symptoms, EC patients usually develop metastases at the time of diagnosis, which makes it difficult to therapy and leads to poor survival. Therefore, efforts are still needed to improve the treatment of EC, and understanding the mechanisms of EC development and progression is one of the important steps to achieve this goal.
Long noncoding RNAs (LncRNAs) are a class of endogenous cellular noncoding RNAs that have more than 200 nucleotides [2]. LncRNAs are essential in a number of biological processes, such as cell proliferation, migration, apoptosis, angiogenesis, inflammation, immune response, etc. [3,4]. Aberration of lncRNAs has been reported to be associated with the pathogenesis of many diseases, such as aortic aneurysm, atherosclerosis, pathogen infection, as well as cancers [4][5][6][7]. For example, lncRNA AFAP1-AS1 has been found to be overexpressed in esophageal adenocarcinoma (EAC) and its silencing suppresses the proliferation, migration, and invasion of EAC cells [8]. Similarly, lncRNA HNF1A-AS1 is upregulated in primary EAC, and knockdown of HNF1A-AS1 represses cell proliferation, anchorage-independent growth, cell migration, and invasion [9].
Accumulating evidence has been demonstrated that lncRNAs trigger cancer progression via their interactions with different molecules, such as DNA, RNA, and proteins [10]. Acting as competing endogenous RNAs (ceRNAs) is one of the key mechanisms of lncRNAs to exert their functions, through which lncRNAs regulate target gene expression by competitively interacting with their common miRNAs [11]. For instance, KRTAP5-AS1 and lncRNA-TUBB2A sponge miR-596 and/or miR-3620-3p to increase the expression of Claudin-4, thus promoting the proliferation and invasion of gastric cancer cells [11]. LncRNA-ATB upregulates ZEB1 and ZEB2 by competitively binding the miR-200 family and induces EMT program and invasion in hepatocellular carcinoma cells [12]. However, the action and mechanisms of a majority of lncRNAs in EC remain unelucidated.
In this study, we screened lncRNAs that were differentially expressed in EC tissues by performing RNA-seq. LncRNA RP11-465B22.8, which was significantly upregulated in EC tissues was selected for further investigation. Our data showed that increased LncRNA RP11-465B22.8 expression was linked to shorter survival of patients with EC. LncRNA RP11-465B22.8 positively regulated KLK4 expression by sponging miR-765 and thus enhanced EC cell proliferation and migration both in vitro and in vivo. Moreover, lncRNA RP11-465B22.8 could be secreted by EC cells via exosomes. Exosomal lncRNA RP11-465B22.8 promoted M2 macrophage polarization, which in turn triggered EC cell migration and invasion. Our results provide evidence that lncRNA RP11-465B22.8 acts as an oncogene in EC, which may serve as a novel target for EC therapy.

RESULTS
LncRNA RP11-465B22.8 expression is increased in EC tissues and linked to poor survival To identify the key lncRNAs involved in EC progression, we performed RNA-seq analysis using three pairs of EC tissues and their matched adjacent normal tissues. A total of 151 lncRNAs were upregulated and 104 lncRNAs were downregulated in tumor samples compared with normal samples (Fig. 1A). Among those differentially expressed lncRNAs, we mainly focused on lncRNA RP11-465B22.8, which is one of the most upregulated lncRNAs and its function is not yet understood. Consistently, quantitative realtime polymerase chain reaction (qRT-PCR) assay showed that the expression of LncRP11-465B22.8 was markedly increased in esophageal tumors compared with normal tissues (Fig. 1B). We also examined lncRNA RP11-465B22.8 expression in several EC cell lines and found that the expression of lncRNA RP11-465B22.8 was higher in three EC cell lines (TE-1, Eca-109, and KYSE-150), compared with the normal endometrial epithelial cell line (HEEC) (Fig. 1C). Moreover, survival analysis revealed that high lncRNA RP11-465B22.8 expression was associated with higher tumor stage and poor survival of patients with EC (Fig. 1D, E, Supplementary Table 2). Together, these results indicate that lncRP11-465B22.8 is a biomarker of poor prognosis in EC patients.
LncRNA RP11-465B22.8 triggers EC metastasis To investigate the effect of lncRNA RP11-465B22.8 on EC cell motility, Transwell migration and invasion assays were performed. Compared with the control cells, the migration and invasion were significantly increased in lncRNA RP11-465B22.8-overexpressed TE-1 and KYSE-150 cells (Fig. 3A, B). In contrast, the motility of TE-1 and KYSE-150 cells was markedly inhibited following lncRNA RP11-465B22.8 knockdown (Fig. 3A, B). The potential function of lncRNA RP11-465B22.8 in angiogenesis was also addressed. Conditioned media were collected from TE-1 cells with lncRNA RP11-465B22.8 overexpression or knockdown and applied to HUVECs. The results showed that the treatment with conditioned medium from TE-1/ RP11-465B22.8 cells significantly increased the tube formation of HUVECs, whereas exposure of conditioned medium from TE-1/si-RP11-465B22.8 cells to HUVECs resulted in a predominant decrease in the tube number (Fig. 3C). It is known that EMT progression plays an important role in cancer metastasis, thus we determined whether lncRNA RP11-465B22.8 could affect the EMT program. Overexpression of lncRNA RP11-465B22.8 in TE-1 and KYSE-150 cells decreased the expression of the epithelial protein (E-cadherin) but increased the expression of mesenchymal proteins (N-cadherin and vimentin) (Fig. 3D). On the contrary, silencing of lncRNA RP11-465B22.8 resulted in the opposite results in TE-1 and KYSE-150 cells (Fig. 3D). To confirmed that lncRNA RP11-465B22.8 could promote cell migration in vivo, a metastasis assay was performed in nude mice via tail vein injection of TE-1 cells overexpressing lncRNA RP11-465B22.8 or the control vector. An increased number of metastasis nodules was observed in lung  tissues of mice injected with TE-1/RP11-465B22.8 cells compared with mice injected with TE-1/Vector cells (Fig. 3E, F). Taken together, these results indicate that lncRNA RP11-465B22.8 can promote EC cell migration both in vitro and in vivo.
LncRNA RP11-465B22.8 enhances KLK4 expression by targeting miR-765 MiRNAs commonly function via the downregulation of target proteins. Therefore, we used TagetScan, miRDB, and miRcode platforms to search the potential targets of miR-765 and found KLK4 as a candidate (Fig. 5A), which has a critical effect on cancer progression [14][15][16]. Luciferase assay showed that transfection with miR-765 mimics decreased the luciferase activity of wild-type KLK4 3′-UTR, but did not alter the activity of KLK4 3′-UTR mutant (Fig. 5B), indicating a direct interaction between miR-765 and KLK4. KLK4 expression was significantly increased in esophageal tumors and cells compared with controls (Fig. 5C, D). Treatment with miR-765 mimics greatly decreased the expression of KLK4, which was consistent with the effect by silencing of lncRNA RP11-465B22.8 (Fig. 5E, F). Moreover, silencing of KLK4 expression decreased the proliferation and migration and increased apoptosis in EC cells (Fig. 5G, H), suggesting that KLK4 functions as a tumor promoter in EC. In addition, transfection of miR-765 mimics reduced the enhancement in KLK4 expression induced by lncRNA RP11-465B22.8 (Fig. 5I). Together, these results indicate that lncRNA RP11-465B22.8 positively regulated KLK4 expression via targeting miR-765, thus contributing to EC progression.
Exosomal lncRNA RP11-465B22.8 triggers M2 macrophagemediated cell migration and invasion The communication between cancer cells and immune cells in the tumor microenvironment is essential for cancer progression. Exosome-mediated delivery of lncRNAs to target cells is one of the most important mechanisms of cancer cells to regulate immune cells. M2 macrophages play a key role in cancer development. Thus, we investigated whether exosomal lncRNA RP11-465B22.8 had an effect on macrophage polarization. Exosomes were isolated from KYSE-150 cells following lncRNA RP11-465B22.8 knockdown and were confirmed by transmission electron microscopy (TEM) and Western blotting detection (Fig. 6A-C). The expression of lncRNA RP11-465B22.8 was decreased in exosomes derived from KYSE-150/si-RP11-465B22.8 cells (si-RP11-465B22.8/exos) compared with exosomes from the control cells (NC/exos) (Fig. 6D). Immunohistochemistry (IHC) analysis showed that the expression of exosome marker CD9 in esophageal tumor tissues was much higher than that in normal tissues (Fig. 6E), confirming the existence of exosomes in EC tissue. Human THP-1 cells were differentiated into macrophages by the treatment with phorbol 12-myristate 13-acetate (PMA). After exposure to PMA, increased CD68 expression was observed in THP-1 cells (Fig. 6F), indicating a successful induction of macrophages. We then examined whether cancer-derived exosomes could be taken up by macrophages. Exosomes derived from KYSE-150 transfected with si-RP11-465B22.8 (si-RP11-465B22.8/exos) or control siRNA (NC/exos) were stained with PKH67 and incubated with PMA-induced macrophages. A number of PKH67 green spots were visualized in macrophages (Fig. 6G). Moreover, treatment with si-RP11-465B22.8/ exos resulted in decreased expressions in M2 markers (CD206 and CD163) and Arginase (Fig. 6H, I), indicating an impairment in M2 polarization of macrophages. In addition, compared with normal tissues, the expression of CD163 was significantly increased in EC tissues (Fig. 6J), indicating a greater population of M2 macrophages in esophageal tumors. To further explore the function of M2 macrophages induced by exosomal lncRNA RP11-465B22.8, an in vitro coculture system with macrophages and EC was utilized (Fig. 7A). The coculture/migration assay showed that M2 macrophages treated with si-RP11-465B22.8/exos remarkably decreased the migration of TE-1 and KYSE-150 cells (Fig. 7B). Similarly, the invasion ability of TE-1 and KYSE-150 cells was significantly inhibited by si-RP11-465B22.8/exos-exposed M2 macrophages (Fig. 7C). Collectively, our results show that exosomal lncRNA RP11-465B22.8 promotes M2 polarization of macrophages, which contributes to EC cell migration.

DISCUSSION
Although the treatment of EC has been improved, the therapeutic efficacy is unsatisfactory and metastasis remains a major threat. The progression of cancer metastasis is complicated, which involves a number of cellular molecules, including lncRNAs. The significance of lncRNAs in cancer development and progression has been documented; however, the function and mechanism of a majority of lncRNAs are not fully understood in EC. In this study, we investigated the biological activity of an uncharacterized lncRNA RP11-465B22.8 in EC. Our data revealed that lncRNA RP11-465B22.8 was upregulated in EC tissues and its high expression predicted poor survival in EC patients. Functional studies showed that lncRNA RP11-465B22.8 promotes the proliferation, migration, and invasion of EC cells in vitro. Moreover, lncRNA RP11-465B22.8 enhanced EC tumorigenesis and metastasis in vivo. Therefore, our results suggest that lncRNA RP11-465B22.8 acts as an oncogene in EC.
Serving as a ceRNA is one of the important mechanisms by which lncRNAs exert their function. Through bioinformatic analysis with online platforms, miR-765 was identified as a potential target of lncRNA RP11-465B22.8. miRNAs are a group of small, wellconserved, noncoding RNAs that regulate the expression of mRNAs [17]. miRNAs are key regulators in many biological and pathological processes, such as cell differentiation, apoptosis, proliferation, migration, and metabolism [17]. Therefore, their translational application as diagnostic markers or potential therapeutic targets for disease treatment has been extensively studied [18]. miR-765 has been reported to function as a tumor suppressor in a variety of cancers, such as pancreatic cancer, ovarian cancer, colorectal cancer, gastric cancer, and renal cell carcinoma [19][20][21][22][23]. In this study, we found that miR-765 was a target of lncRNA RP11-465B22.8. Reinforce of miR-675 abolished the promoting effects of lncRNA RP11-465B22.8 on cell proliferation, migration, and tube formation. Our data suggest that lncRNA RP11-465B22.8 can act as a ceRNA for miR-765 in EC.
KLK4 is a serine protease that belongs to kallikrein-related peptidase, which contains 15 members [14]. Increasing studies have suggested that KLK4 plays a tumor-supporting role in cancer. KLK4 has been indicated to be a biomarker of prognosis in triple-   negative breast cancer and ovarian cancer [14,15]. Inhibition of KLK4 suppresses the growth of oral squamous cell carcinoma cells through the Wnt/β-catenin pathway [16]. Downregulation of KLK4 results in decreased migration and invasion in oral squamous cell carcinoma cells [24]. KLK4 is also reported to contribute to nonsmall cell lung cancer progression [25]. However, the function of KLK4 in EC remains unclear. Herein, we found that KLK4 is a direct target of miR-765. Moreover, overexpression of lncRNA RP11-465B22.8 increased the expression of KLK4, which was decreased by transfection of miR-765. Therefore, our data indicated that lncRNA RP11-465B22.8 enhanced EC cell proliferation and migration via miR-765/KLK4 cascade.
Macrophages are crucial components in the tumor environment, which are important regulators in tumorigenesis and metastasis [26]. Macrophages can be classed into two phenotypes, the proinflammatory M1 and anti-inflammatory M2 macrophages [26,27]. M1 macrophages can phagocytose cancer cells, while M2 macrophages such as tumor-associated macrophages promote tumor cell growth, migration, and invasion [27]. Macrophages infiltrate tumor tissues and gain a polarized M2 phenotype driven by tumor-derived or T cell-derived cytokines/molecules [28]. It has been shown that tumor cells can cross-talk with immune cells by secreting exosomes, thereby inducing immune suppression [29]. A number of exosomal lncRNAs have been reported to be involved in cell-cell communication and are highly associated with macrophage polarization [30][31][32]. Consistently, in this study, we found that exosomal lncRNA RP11-465B22.8 was an inducer of M2 macrophage polarization. Treatment with exosomes derived from si-RP11-465B22.8/KYSE-150 cells could reduce M2 macrophageinduced migration.
In conclusion, in the present study, we found the expression of lncRNA RP11-465B22.8 was increased in EC tissues and high lncRNA RP11-465B22.8 expression was linked to poor survival of EC patients. LncRNA RP11-465B22.8 promoted the proliferation, migration, and invasion of EC cells via miR-765/KLK4 axis. Moreover, our data revealed that exosomal lncRNA RP11-465B22.8 could induce M2 macrophage polarization and thus enhance EC cell motility (Fig. 7D). Therefore, our findings provide evidence that lncRNA RP11-465B22.8 is an oncogene in EC, which may serve as a potential target for EC therapy.

Isolation and characterization of exosomes
KYSE-150 cells were transfected with si-RP11-465B22.8. Forty-eight hours after transfection, the culture medium was collected for exosome isolation. To remove residual cells and debris, the culture medium was centrifuged at 300g for 10 min, 2000g for 10 min, and 10,000g for 30 min. Then the supernatant was ultra-centrifuged at 100,000g for 70 min to pellet the exosomes. Exosomes were washed once with phosphate-buffered saline (PBS) and harvested by ultra-centrifugation at 100,000g for 70 min. The morphology of exosomes was detected by TEM. Briefly, exosomes were resuspended in PBS (Seyotin, China) and dropped on a copper grid. Then, exosomes were fixed with glutaraldehyde, stained with uranyl acetate solution, and photographed.

The uptake of exosomes
Isolated exosomes were labeled with PKH67 dye using a PKH67 Green Fluorescent Cell Linker Kit (Sigma, USA) following the manufacturer's protocols. PKH67-labeled exosomes (2 µg) were incubated with THP-1 cells treated with PMA. After incubation for 24 h, PMA-induced macrophages were fixed with paraformaldehyde, stained with DAPI, and visualized under fluorescence microscopy.

Luciferase reporter assays
HEK293T cells were seeded in six-well plates. To determine the interaction between lncRNA RP11-465B22.8 and miR-765, HEK293T cells were cotransfected with wild-type or mutated lncRNA RP11-465B22.8 and miR-765 mimics (or control miRNA) using Lipofectamine 2000 (Invitrogen, CA, USA). To detect the interaction between miR-765 and KLK4, HEK293T cells were co-transfected with wild-type or mutated KLK4 3′-UTR and miR-765 mimics (or control miRNA). Forty-eight hours post-transfection, cells were collected and the firefly and renilla luciferase activities were determined using a Dual-Luciferase Reporter Assay Kit (Promega, USA).

Quantitative real real-time PCR (qRT-PCR)
Total RNA was extracted using Trizol reagent (Invitrogen) and reversed transcribed to cDNA by using the SuperScript III cDNA Synthesis kit (Seyotin, China). For miRNA detection, cDNA was synthesized with the TaqMan ™ Advanced miRNA cDNA Synthesis kit (Thermo Fisher Scientific, USA). Real-time PCR was performed using the Applied Biosystem SYBR (Seyotin, China) according to the manufacturer's introductions. mRNA expression was normalized to the internal control GAPDH while miRNA expression was normalized to the internal control U6. The relative gene expression was calculated using the 2 −ΔΔCT formula. All primers are listed in Supplementary Table 1, Primers for miR-765 and U6 were obtained from Ribo (Guangzhou, China).

Western blot
Samples of cells or exosomes were lysed in radioimmunoprecipitation assay lysis buffer which was supplemented with protease and phosphatase inhibitors (Sigma). The protein sample was separated by SDS-PAGE gels and transferred onto the polyvinylidene fluoride membrane (Millipore). The membrane was blocked with 5% non-fat milk blocking buffer. After incubation with primary antibodies at 4°C for overnight, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 60 min. The protein bands were detected by using the chemiluminescent Western Blotting Substrate (Seyotin, China).

The migration and invasion assays
Twenty-four-well plates with 8 μm Transwell chambers (Corning, MA, USA) were used for the migration and invasion assays. A total of 1 × 10 5 cells in 100 μl serum-free medium were seeded in the inserts and 600 μl medium with 10% FBS was added into the lower chamber. For coculture assay, macrophages (1 × 10 4 ) in 600 μl medium containing 10% FBS were placed in the lower chamber. After 24 h post-incubation, cells on the top membrane of inserts were scraped by a cotton swab and cells on the bottom of the membrane were stained with 0.5% crystal violet. The number of migrated cells was counted under a light microscope. For the invasion assay, the inserts were coated with Matrigel (BD Bioscience, CA, USA) prior to the assay.

Immunohistochemistry
Tissue samples were fixed and embedded in paraffin. After dewaxing and dehydration, tissue sections were antigen-retrieved and blocked. Then tissues were incubated with primary antibodies against CD9 or CD163. After washing with PBS, the samples were incubated with a biotinylated secondary antibody, followed by incubation with the horseradish peroxidase-streptavidin reagent (Santa Cruz Biotechnology). Tissue sections were detected with diaminobenzidine solution with hematoxylin counterstaining.

Animal experiments
BALB/c nude mice (male, 4-6-weeks-old) were obtained from the Institute of Zoology, Chinese Academy of Sciences, Beijing, China. The mice were randomly divided into two groups and luciferase-labeled TE-1/RP11-465B22.8 or luciferase-labeled control cells were injected into the oxter of nude mice (n = 5). After 4 weeks, luciferase intensity was monitored and mice were sacrificed. Tumors were isolated and photographed. For in vivo metastasis assay, TE-1/RP11-465B22.8 or control cells were injected into mice via tail vein (n = 3). Five weeks later, mice were sacrificed and lung tissues were extracted. The metastatic node in lung tissues was counted and the histological changes of lung tissues were accessed by hematoxylin-eosin staining. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Xinhua Hospital Affiliated with Shanghai Jiaotong University School of Medicine.

Statistical analysis
The sample size in each experiment was chosen to ensure adequate power to detect a pre-specified effect. Studies were not blinded to investigators. Data were expressed as mean ± SD from three independent experiments. Differences between different groups were addressed by the Student's t test or one-way ANOVA analysis. The variance was similar between the groups that were statistically compared. Survival analysis was performed using the Kaplan-Meier method with the log-rank test. A two-tailed value of P < 0.05 was considered statistically significant. All statistical analyses were performed with the SPSS 13.0 statistical software (SPSS Inc., Chicago, IL, USA).