miR-451a was selectively sorted into exosomes and promoted the progression of esophageal squamous cell carcinoma through CAB39

Exosomes are emerging mediators of cell-cell communication, which are secreted from cells and may be conveyed to recipient cells for cell biological processes. Here, we examined exosome miRNA expression in esophageal squamous cell carcinoma (ESCC) cells. We examine different miRNA expressions in cells and exosomes. To study the mechanisms of miRNA packaging into exosomes, we combined exosome proteomic data which is miR-451a interacts with YWHAE. Esophageal squamous tissues and matched tissues were compared from 155 patients, and CAB39 is related to TGF-β 1. We found that miR-451a was encapsulated in the exosomes. Overexpression of YWHAE leads to miR-451a accumulation in the exosomes instead of donor cells. Furthermore, CAB39 was targeted with miR-451a. We found that CAB39 weakens antitumor immunity through TGF-β 1 in ESCC. In summary, our data demonstrated that YWHAE selectively sorted miR-451a into exosomes and through the target of CAB39 weakened antitumor immunity promotes tumor progression.


Introduction
Esophageal cancer is one of the most lethal cancers worldwide.Despite the technical developments in diagnosis and treatment, the disease has limited therapeutic options and the 5-year survival rate ranges from 15-25% [1,2].Histological types of esophageal carcinoma include two main types: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) [3].ESCC is the major type of esophageal cancer in China [2].
The tumor microenvironment plays an important role in the process of tumor metastasis and has been shown to directly affect the occurrence and development of tumors through secreted proteins.The tumor microenvironment includes broblasts, extracellular matrix proteins, and immune cells [4].Furthermore, tumor cells can remodel the tumor microenvironment and maintain their own survival and development conditions by exosomes, which promote the growth and development of tumors [5].
Exosomes miRNA, a class of small noncoding regulatory RNA, can affect cell biological functions by inhibiting or promoting transcription and translation of target genes [7][8][9].Recent studies have shown that exosomes miRNA play an important role in cell-cell signaling, cell growth, differentiation, and transformation.We are considered novel molecules that regulate biological processes [10].miR-451a is a recently identi ed miRNA that has crucial roles in cancer progression including breast cancer, renal cell carcinoma, lung cancer, and gastric cancer [11].For example, miR-451a regulated cell migration and invasion through targeting ATF2 [12].Importantly, it was found that miR-451a was downregulated in gastric cancer tissues and cell lines [13].However, it was not determined whether miR-451a has a role in ESCC.Exosomes play an important role in the initiation and progression of cancer, and that will be important to explore what role exosome miRNA plays in cancer.

Cell culture
All ESCC cell lines were cultured in RPMI 1640 (Hyclone, Logan, UT, USA) media supplementary with 10% fetal bovine serum, 50 U mL − 1 penicillin, and 50 U mL − 1 streptomycin at 37℃ in a 5% CO 2 incubator.All cell lines were obtained from the Key Laboratory of Cellular Physiology, Ministry of Education, Shanxi Medical University, and were identi ed by STR and no mycoplasma contamination.

Isolation and Characterization of ESCC Exosomes
The cell cultures reached 70% density and were incubated with a freshly prepared complete medium to remove the FBS for 48h.Cell culture supernatants were collected and centrifuged at 300g for 10 min and then at 2,000g for 20 min at 4℃ to remove cells, followed centrifuged at 10000g for 30 min to remove cell debris.The exosome pellets were washed with PBS by centrifuged at 100,000g for 70 minutes.Final exosomes were checked using transmission electron microscopic (TEM), nanoparticle tracking analysis, and marker proteins CD81, and Calnexin by Western Blot.

Dual-luciferase reporter assay
The wild-type (WT) CAB39 3'UTR was ampli ed and cloned into the Psicheck2 vector.The mutant (Mut) plasmid was mutated of miR-451a in the 3'UTR of CAB39 and was cloned into the Psicheck2 vector.
293T cells were cultured in 24 well plates co-transfected with miR-451a mimic or NC mimic and CAB39 -WT or CAB39 -Mut using Lipofectamine 2000 (Thermo Fisher Scienti c, Inc).After 48h, Luciferase activity was measured using a Dual Luciferase Reporter Assay kit (Transgene, Beijing, China).All transfection assays are according to the manufacturer's protocol.
Subsequently, the membranes were incubated with secondary antibodies for 1 h at 37˚C.Protein bands were detected using the Odyssey system (Li-Cor) and quanti ed using ImageJ software.

Biotinylated miRNA pull-down
To further con rm the interaction of YWHAE and miR-451a, miRNA of labeled with biotin at 3' end and a scrambled control miRNA were commercially synthesized from Wuhan Kejin Biotechnology.Transfect the cells with control miRNA and 3' biotin-labeled miRNA at a nal concentration of 50 nM by using as a transfection reagent RNAimax (Thermo Fisher Scienti c; Cat#13778150).After Scraping cells add 550 µL lysis buffer (20 mM Tris-HCl pH 7.5, 100 mM KCl, 5 mM MgCl 2 , 0.3% IGEPAL CA-630) supplemented with protease inhibitor (Thermo Fisher Scienti c; Cat#78430) and RNase inhibitor (Thermo Fisher Scienti c; Cat#EO0381) on ice Incubate for 1h.Simultaneously, prepare 50uLStreptavidin beads (Thermo Fisher Scienti c; Cat#11205D) were activated according to the manufacturer's protocol.The beads were incubated with 500 µ L lysis buffer and 10µL yeast tRNA (Thermo Fisher Scienti c; Cat#AM7119) on a rotator at 4°C for 2 h.After washing the beads with lysis buffer, mix 500 µL lysates with the beads and incubate at 4°C on the rotator overnight followed by pulled-down.

Immunoprecipitation-qPCR
We collected 5 − 20×10 6 mammalian cells, washing them three times with 5 mL of ice-cold phosphatebuffered saline (PBS).Pellet by centrifugation 10,000 g for 10 min at 4°C.Resuspend pellet with an approximate volume of lysis buffer supplemented with RNase inhibitors and protease inhibitors (100 mM KCL, 5 mM MgCL 2, 10 mM HEPES, 0.5% NP40,1 mM DTT, 40U/ µL RNase inhibitors and Protease inhibitor cocktail, PMSF).At 4°C, pre-swell protein-A Sepharose beads in NT2(50 mM Tris HCl,150 mM NaCl, 1 mM MgCL 2, 0.05% NP40) supplemented with 5% BSA for at least 1 h before use.Add YWHAE antibody or anti-IgG control to bead and incubate overnight at 4°C.Washing antibody beads with 1 mL NT2 buffer 4 − 5 times.After the nal wash, resuspend beads in 850 µL of NT2 buffer and add 200 units of RNase inhibitor, 10 µL of 100 mM DTT, and EDTA to 20 Mm.Incubate antibody beads and lysis buffer for 4 h at 4°C tumbling end over end and wash beads 4 − 5 times with 1 mL of NT2 buffer.For qPCR analysis, see methods of RNA extraction, reverse transcription-quantitative, and Real-time quantitative PCR (qPCR).

PBMC-cancer cells co-culture
Human PBMCs were isolated from the blood of healthy donors.The resulting cell pellet was resuspended in a culture medium containing RPM1 1640 supplemented with 1%FBS (Gibco, USA), 0.25mg/mL human insulin, 1.3mM L-Glutamine,100U/mL Pen/Strep, and 20mM HEPES.PBMCs activated with Dyna beads® Human T-Activator CD3/CD28(life technology).Then co-cultured with tumor cells at a 10:1 ratio.MTT and apoptosis assays were assessed.

MTT assay
Brie y, co-cultured PBMC and ESCC cells were digested into each well of a 96-well plate and incubated overnight in a nal volume of 200 µL.After that, cells were treated with 20µL of 5 mg/mL MTT (Invitrogen, USA) and incubated for 4 h at 37°C.Then, the MTT solution was removed, and added 200 µL of DMSO to each well.The absorbance was measured with a Spectrophotometer at 490 nm.
Apoptosis assay ESCC cells were co-cultured with PBMC.The cell apoptosis was assessed using the Annexin V-APC/7-AAD Apoptosis Detection kit (KeyGen Biotech Co., Ltd., Nanjing, China) according to the manufacturer's instructions.Brie y, add 500 µ L Binding Buffer, 5 µL Annexin V-APC, and 5 µL 7-AAD, followed by incubating at room temperature for 10 min in the darkness.Subsequently, the cells were analyzed by ow cytometry within 1h.

Animal models
Animal experiments were obtained following the appropriate animal care committee of Shanxi Medical University.In the ex vivo experiment, we used a pad of 6-week-old female Balb/c or SCID mice (n = 5 for each group).2×10 6 cells were injected into mice.Tumor size was determined every ve days.Mice were executed after four weeks, and the tumors were removed and measured.

Immunohistochemistry (IHC)
IHC was performed to detect the protein expression of the genes.Brie y, the sections were dewaxed with xylene and alcohol, then soaked with 3% H 2 O 2 for 15 min at room temperature to block endogenous peroxidase activity.After sections were incubated with primary antibody at 4°C in the wet box overnight, followed by detection with Kit 5020 (Zhongshan, China), and the DAB plus kit (Mai Xin, Fuzhou, China) was used to form dark-brown precipitation.The slides were counterstained with hematoxylin and the images were captured at 20×by Aperio Scan.The antibodies used in this experiment are shown as follows: CAB39 (Abmart; Cat#PS07771), and TGF-β1 (Proteintech; Cat#21898-1).

Statistical analysis
All statistics were performed using GraphPad Prism 6 (GraphPad Software, San Diego, California, USA).
The results were compared using Student's t-test between two groups and one-way analysis of variance (ANOVA) more than two groups.P < 0.05 was considered statistically signi cant.

miR-451a is selectively packaged into exosomes
miRNAs are non-coding RNA that regulate a wide range of biological processes.In addition to playing a role in cells, recent studies have shown that miRNA was secreted in exosomes [14].Exosomes can be actively released from a variety of cell types including cancer cells.Exosomes miRNA has been implicated as informative biomarkers of progression and early diagnosis for cancer [15,16].To determine whether exosomes can be released from ESCC cells, we isolated the exosomes from the conditioned media collected from KYSE-150 and KYSE-450.Transmission electron microscopic examination showed morphological manifestations of exosomes (Supplementary Data Fig. 1a).Then the size of the exosomes was measured using nanoparticle tracking analysis.The diameter of the exosomes was in the range of 30-180 nm (Supplementary Data Fig. 1b).To further determine the isolated exosomes, we used the exosome-speci c marker Calnexin and CD81.These results con rmed that exosomes can be isolated from ESCC cells (Supplementary Data Fig. 1c).To reveal the physiological roles of extracellular miRNAs, we performed miRNA sequencing in exosomes and cells in KYSE150 and KYSE450 (BGI, Shenzhen, China).All data were analyzed on the Dr. Tom online system provided by BGI.Among these differentially expressed miRNAs,156 of the miRNAs were detected in cells and exosomes (Fig. 1a).A heatmap of the 156 selected miRNAs demonstrated miRNA expression according to the level of miRNAs in the exosomes and cells.Our results indicate that the levels of miR-451a and miR-1246 were higher in exosomes than in ESCC cells (Fig. 1b).Serum exosomes miR-451a might serve as a novel diagnostic marker for pancreatic cancer [17].We selected miR-451a for further analysis, we hypothesized that host factors might play a role in sorting from ESCC cells to their exosomes.To validate our hypothesis, the biotin-labeled miR-451a complex was isolated from lysates using streptavidin beads, and gel electrophoresis is shown in Fig. 1C.
Mass spectrometry (MS) analysis was carried out for the identi cation of proteins that are speci cally present in the Bio-miR-451a complex sample but not in the control bio-miRNA complex.
To investigate the molecular machinery that controls the sorting of speci c miRNAs into exosomes, we screened 23 proteins based on "Unique Peptides", "Abundances of miR-451a", "Abundances of miR-451a mimic/ Abundances of NC mimic", "Coverage", "Molecular Weight" and showed them by heat map and Table.Among them, YWHAE showed a higher expression level in the Bio-miR-451a complex sample than in the control bio-miRNA complex in Fig. 1D, 1E.To further explore the mechanism of Bio-miR-451a complex protein, enrichment analysis based on the Gene Ontology (GO).As a result, this implies that these proteins are closely related to the binding (Fig. 1f).To demonstrate that YWHAE is highly expressed in exosomes, we selected KYSE150 and KYSE450 cells for exosomes collection and exosome proteomic sequencing.We compared with KYSE150-exosomes, KYSE450-exosomes, and miRNA pull-down set.Among these differentially expressed proteins, 22 of the proteins were detected in three sets (Fig. 1g).Each point represents the expression value of the protein in the scatter plot.As a result, our data suggest that YWHAE in exosomes and Bio-miR-451a complex sample is higher expression than others (Fig. 1h, Supplementary Data Fig. 1d).This may be one of the mechanisms miR-451a is selectively packaged into exosomes in ESCC cells.
3.2 YWHAE regulates the loading of miR-451a into exosomes RNA-Binding Protein YWHAE suppressed cell proliferation in gastric cancer [18].YWHAE promoted cancer progression and chemoresistance in breast cancer cells [19].To demonstrate the speci c binding of YWHAE and miR-451a controls the sorting into exosomes in vivo, speci c binding of YWHAE to miR-451a was veri ed by immunoprecipitation of YWHAE from KYSE150 lysates followed by qPCR of miRNAs; miR-451a was ampli ed from YWHAE immune precipitates (Fig. 2a).To investigate the role of YWHAE in the packaging of miR451a into exosomes, we assessed the e ciency of silencing and over-expression on KYSE150 by RT-PCR and Western blot (Fig. 2b, 2c).Next, KYSE150 cells transfected with YWHAE siRNA have low levels of miR-451a detected in exosomes, but the level of miR-451a is increased in the ESCC cells.YWHAE overexpression signi cantly increased the levels of the miR-451a in exosomes but signi cantly decreased the levels of the miR-451a in ESCC cells.To further understand the effects of YWHAE on the cells, we co-transfected with YWHAE siRNA and miR-451a inhibitor have low levels of miR-451a detected in exosomes (Fig. 2d).Collectively, these data suggest that miR-451a sorting into exosomes is YWHAE-dependent.

CAB39 is a target gene of miR-451a
To verify the biological effects of miR-451a in ESCC, we measured miR-451a expression in 9 ESCC cell lines by quantitative real-time PCR (RT-qPCR).We found that in these cell lines, miR-451a was relatively higher in TE5 cells but lower in KYSE150 cells (Fig. 3a).Thus, we mimic the expression of miR-451a in KYSE150.We next sought to elucidate the targets of miR-451a.Three predictive tools including TargetScan (http://www.targetscan.org),PITA (https://genie.weizmann.ac.il/pu) and ENCORI (https://rnasysu.com/encori/)were performed to analyze the potential targets of miR-451a (Fig. 3b).According to the analysis, miR-451a likely bind to the 3′ UTR of CAB39 (Fig. 3c).In Nasopharyngeal Carcinoma, CAB39 over-expression dramatically increased the proliferation and colony formation of CNE-1 cells [20].Thus, CAB39 was selected for further study.The dual-luciferase reporter assays indicated that the luciferase activity of CAB39 Wild Type was signi cantly reduced after miR-451a mimic compared with that of NC mimic, while the luciferase activity of mutant CAB39 did not signi cantly after miR-451a mimic compared with that of NC mimic (Fig. 3d).The e ciency of mimic and inhibitor of the miR-451a were con rmed by RT-qPCR in KYSE150 cells and TE5 (Fig. 3e).After we con rmed that mRNA and protein expression levels of CAB39.It was demonstrated that decreased CAB39 expression with miR-451a mimic in KYSE150, whereas was upregulated CAB39 expression with miR-451a inhibitor in TE5 (Fig. 3f,3g).The aforementioned ndings proved that CAB39 was a target of miR451a, indicating that the expression of CAB39 was regulated by binding to its 3'UTR with miR-451a.

CAB39 is in direct correlation with TGF-β1 genes
A previous study showed that miR-451 regulated the production of cytokines by in uenza-infected dendritic cells [21].Macrophages treated with the miR-451a mimic showed lower expression of M1 markers [22].We analyze the relationship between CAB39 and immune checkpoints.Firstly, to identify the relationships between the inhibitory checkpoint genes and CAB39, the correlations were veri ed with our transcriptomic sequencing of fresh frozen tumor tissues and matched normal specimens from 155 ESCC patients in the cohort from another study [23].The network showed the relationship between CAB39 and the inhibitory checkpoint genes (Fig. 4a).Next, the expression correlation of CAB39 and the inhibitory checkpoint genes was supported by Pearson's correlation analysis.Among them, CAB39 was positively correlated with TGF-β1, LGALS9B, and LGALS9C (Fig. 4b).Moreover, we analyzed CAB39 expression with TGF-β1, LGALS9 in mRNA and protein levels.As a result, we found that the transfection of CAB39 siRNA resulted in a signi cant decrease of TGF-β1 mRNA expression with no effect on mRNA expression for LGALS9.When CAB39 was over-expressed, the expression of TGFβ-1 was changed conversely (Fig. 4c, Supplementary Data Fig. 4a).Furthermore, we further con rmed the trends at the protein level via Western Blot (Fig. 4d).TGF-β1 regulates the generation and effector functions of many immune cell types.It controls adaptive immunity by inhibiting the generation and function of effector T cells and natural killer (NK) cells [24].Eduard et al. have shown that tumor cells can also secrete a large amount of TGF-β1 at the later stage of development, which makes tumor cells escape from the immune system [25,26].Thus, CAB39 was forced expressed in KYSE150 cells.Then, we detected the changes in the cell culture medium of TGF-β1 by ELISA (Fig. 4e).The ndings proved that TGF-β1 is higher expression in overexpression of CAB39 than in control.Together, these results indicate that CAB39 is a potential immune checkpoint blocker by the TGF-β1 gene in ESCC cells.Thus, immunohistochemical analyses of CAB39 and TGF-β1 in esophageal squamous cancer tissues from 155 paired of ESCC cohort [23].The results showed that CAB39 expression is signi cantly higher in tumors with relatively higher expression of TGF-β1, and vice versa (Fig. 4f).Together with the results from in vitro studies, our data suggest that correlation between CAB39 and TGF-β1 in ESCC.

CAB39 weakens the sensitivity of PBMC via TGF-β1
TGF-β1 inhibition of cytotoxic T cells is known to be an important mechanism by which tumors evade immune destruction [27].Tumor cells can secrete large amounts of TGF-β1, which can inhibit the activation and differentiation of lymphocytes and cause immune dysfunction [26].Upregulation of TGF-β1 expression in cancer cells is one of the major causes of evading tumor immunity.Thus, we speculated that the expression of CAB39 weakens the sensitivity of peripheral blood mononuclear cells (PBMC) immunity.To test this hypothesis, PBMC was obtained from the Cancer Hospital of Shanxi province.There were co-cultured cancer cells with PBMC.As expected, the proliferation of the KYSE150 cells was signi cantly inhibited by PBMC, and apoptotic cells were dramatically enhanced (Fig. 5a,5b).Furthermore, the MTT assay demonstrated that compared with the control group, overexpression CAB39 signi cantly increased the proliferation and mixed TGF-β1 inhibitors decreased it in the co-cultured PBMC.Flow cytometry assays showed that apoptotic cells were signi cantly reduced after CAB39 overexpression and signi cantly increased after mixed TGF-β1 inhibitors (Fig. 5c,5d).Taken together, these results demonstrated that CAB39 weakens the sensitivity to PBMC via TGF-β1 in ESCC cells thus promoting tumor immunity.

CAB39 increased tumor growth in vivo mouse model
To further con rm the roles of CAB39 using an immunocompetent murine system, we establish a subcutaneous transplantation tumor model into the syngeneic Balb/c mice as well as the immunede cient SCID mice.After 4 weeks, mice were sacri ced and tumors were stripped.We observed that the tumor growth rate of CAB39-exp was signi cantly rapidly than that of the control group and CAB39 exp + LY3200882 in BALB/c mice.Whereas over-expression of CAB39 was over-expressed no signi cant difference in tumor growth was observed in SCID mice, suggesting that immune competency is required for the anti-tumor effect of CAB39 (Fig. 6a,6b).Furthermore, immunohistochemically staining showed that expression of CAB39 was signi cantly higher than that of the control group in vivo (Fig. 6c).Additionally, we further detected TGF-β1 expression in tumor tissues by IHC analyses.The results showed that TGF-β1 in the CAB39-exp group was signi cantly higher than that in the control group and CAB39 exp + LY3200882 in Balb/c mice and SCID mice (Fig. 6d).These data suggested that CAB39 weakened the sensitivity to PBMC via TGF-β1 in ESCC in vivo.

Discussion
Despite chemotherapy and radiotherapy can improve the disease outcome, ESCC patients still suffer from poor prognosis with 20% of 5-year survival rates in China [3].This illustrates the importance of developing novel therapeutic targets.Recently, exosomes miRNA functional effects various cancer hallmarks, highlighting their potential as no invasive therapeutic targets for cancer therapy [28].In this study, we found that YWHAE-regulated miR-451a sorted into exosomes through CAB39-TGF-β1 promotes esophageal squamous cell carcinoma progression.
In the rst part of the study, we illustrated that YWHAE regulated exosome sorting of miR-451a into exosomes.YWHAE promotes cancer progression and can be a potential therapeutic target in multiple cancer types such as breast cancer [19], endometrial cancer [29], and Prostate cancer [30].In this study, we demonstrated that YWHAE transported miR-451a from tumor cells to exosomes.The complex of YWHAE and miR-451a were packed into exosomes leading to the reduction of ESCC cells miR-451a.The fact that YWHAE knockout causes miR-451a accumulation in cells instead of exosomes.Our study demonstrates that YWHAE regulates the loading of miR-451a into exosomes in ESCC.miR-451a targets 3′ UTR of CAB39 mRNA, leading to inhibition of CAB39 protein.However, the expression of CAB39 was correlated with tumor development.CAB39 is a relatively of which the function in immune checkpoint has not been identi ed.In this study, we demonstrated that CAB39 is inversely correlated with TGF-β1 genes in ESCC tumors.Furthermore, our data suggest that it was correlated to CAB39 and TGF-β1 in ESCC.
Previous work concurs in showing that the TGF-β1 inhibition of cytotoxic T cells is known to be an important mechanism by which tumors evade immune destruction [27].It has been reported that tumor cells can also secrete a large amount of TGF-β1 at the later stage of development.It is well-known that the tumor microenvironment has dramatic effects on the outcome of tumor growth.In this study, we show that TGF-β1 is more highly expressed in overexpression of CAB39 than control in cell culture medium.Whether the CAB39 affects the TGF-β1 has not been fully investigated.Another notable nding of the present study was that CAB39 weakens the sensitivity to PBMC via TGF-β1 in vivo and in vitro.
Hence, the central observations of our study suggest that exosomes miR-451a and CAB39 may be therapeutic targets for the treatment of ESCC.However, the effects of TGF-β1 with de ciency on how to regulate immunity remain unde ned.However, the present ndings are exciting for the development of clinically viable targets for treating ESCC.

Supplementary Files
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