Exosomal lncRNA ZFAS1 regulates esophageal squamous cell carcinoma cell proliferation, invasion, migration and apoptosis via microRNA-124/STAT3 axis

The study was approved by the Ethics Committee of The Second Hospital of Hebei Medical University and informed written consent was obtained from all patients. All animal experiments were in line with the Guide for the Care and Use of Laboratory Animal by International Committees.

A total of 136 ESCC patients (mean age of 66.18 ± 7.14 years) who received resection in The Second Hospital of Hebei Medical University from June 2015 to June 2018 were collected. There were 92 males and 44 females, and 50 cases with lymph node metastasis (LNM), and 86 cases without LNM. Meanwhile, the corresponding adjacent normal tissues were taken as the control group. Those patients who have undergone radiotherapy and chemotherapy, patients with infection phenomena, patients who have history of other malignant tumors except for ESCC or patients who have primary immunodeficiency and acquired immure deficiency syndrome (AIDS), were excluded from this study.

Five ESCC cell lines EC9706, Eca109, TE-13, TE-1 and TTN were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). The five cell lines were cultured in medium and renewed every 24 h for subculture. The cell lines with the highest expression of ZFAS1 were screened out for subsequent experiments by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The cells were seeded in 6-well plates at the density of 3 × 10⁵ cells/well. When the cell confluence reached 80%, lipofectamine 2000 (Invitrogen, Carlsbad, California, USA) kit was used for transfection. Four μg target plasmid and ten μL lipofectamine 2000 was diluted by 250 μL serum-free Opti-MEM (Gibco, Carlsbad, California, USA) medium, respectively. After being placed for 20 min, the mixed solution was added to the culture well, and then cultured with 5% CO₂. After 6 h, it was replaced to complete culture medium, and the cells were collected after culturing for 48 h.

The ESCC cells post transfection were seeded in RPMI 1640 medium containing 10% fetal bovine serum (FBS) without exosomes and cultured in a cell culture box at 37 °C with 5% CO₂. After 3 days, cell supernatants were collected and centrifuged to remove cell fragments. According to the Hieff™ Quick exosome isolation kit (article number: 41201ES50, Shanghai YEASEN Biotechnology Co., Ltd., Shanghai, China. The kit was a rapid extraction kit for cell culture supernatant, which was suitable for the extraction of supernatant of the cell culture medium, the urine, or the ascites), the exosomes were extracted. The centrifuged cell supernatant and exosomes separation reagent were centrifuged in the eppendorf (EP) tube at 10,000 g, 4 °C for 1–2 h. The precipitates were the exosomes. According to the ratio of the volume of initial culture medium and the suspension (10: 1), exosomes were resuspended with phosphate buffered saline (PBS). The resuspended exosomes (30 μL) were added with equal volume of radioimmunoprecipitation assay (RIPA) lysate into EP tubes, mixed and placed on ice. The exosomes was continuously lysed by a microwave for 2 times, 10 s each time. And then, the concentration of protein in exosomes was measured by bicinchoninic acid (BCA) quantitative kit (Beyotime Biotechnology, Nantong, China). Exosomes identification: the exosomes marker (CD63, CD9, CD81) was detected by western blot analysis and the morphology of exosomes was observed by a transmission electron microscope (TEM) (JEM-1010, JEOL, Tokyo, Japan). Finally, the exosomes secreted by ESCC cells were obtained.

The exosomes was precipitated and immediately fixed in 2.5% glutaraldehyde at 4 °C. Then the specimens were dehydrated by gradient alcohol and immersed in epoxy resin. The ultra-thin sections were stained with uranyl acetate and lead citrate and observed under a TEM (JEM-1010, JEOL, Tokyo, Japan).

Exo-Red fluorescent staining kit (SBI System Bioscience, San Francisco, CA) was used to label the exosomes. The operation process was carried out according to the instructions of the kit. The exosomes were re-suspended with PBS and mixed with Exo-Glow Red, and then incubated for 10 min. Exo Quick-TC reagent was added to terminate the labeling reaction. After the mixture was centrifuged, the supernatant was discarded and exosomes were resuspended with PBS for further use. Cells (1 × 10⁵) were seeded into a 35-mm dish, after the cell adhered to the wall, the labeled exosomes with 100–150 μL were added, cultured for 24 h, and observed under a confocal microscope.

ESCC cell line Eca109 with a well growth in the logarithmic phase was selected to observe the effect of lncRNA ZFAS1 on the growth, invasion and migration of ESCC cells. The cells were distributed into sh-negative control (NC) group (transfected with sh-ZFAS1 plasmid NC) and sh-ZFAS1 group (transfected with sh-ZFAS1 plasmid). In order to explore the effect of ZFAS1 on the biological functions of ESCC cells in the exosomes, a co-culture model was established to investigate the effect of the exosomal ZFAS1 on the recipient cells. Cells were assigned into the overexpressed (Oe)-NC-exo group (donor Eca109 cells transfected with Oe-ZFAS1 plasmid NC labeled by Cy3 was co-cultured with recipient Eca109 cells), the Oe-ZFAS1-exo group (donor Eca109 cells transfected with Oe-ZFAS1 plasmid labeled by Cy3 was co-cultured with recipient Eca109 cells), the sh-NC-exo group (donor Eca109 cells transfected with sh-ZFAS1 plasmid NC labeled by Cy3 was co-cultured with recipient Eca109 cells), the sh-ZFAS1-exo group (donor Eca109 cells transfected with sh-ZFAS1 plasmid labeled by Cy3 was co-cultured with recipient Eca109 cells) the inhibitor NC-exo group (donor Eca109 cells transfected with miR-124 inhibitor NC labeled by Cy3 was co-cultured with recipient Eca109 cells) and the miR-124 inhibitor-exo group (donor Eca109 cells transfected with miR-124 inhibitor labeled by Cy3 was co-cultured with recipient Eca109 cells). sh-ZFAS1, sh-NC, Cy3-Oe-ZFAS1, Cy3-Oe-NC, Cy3-sh-ZFAS1, Cy3-sh-NC, Cy3-miR-124 inhibitor and Cy3-inhibitor NC were bought from RiboBio Co., Ltd. (Guangdong, China). The transfection of sh-ZFAS1, sh-NC, Cy3-Oe-ZFAS1, Cy3-Oe-NC, Cy3-sh-ZFAS1, Cy3-sh-NC, Cy3-miR-124 inhibitor and Cy3-inhibitor NC into cells were strictly in accordance with the steps of the kit of Lipofectamine™ RNAiMAX (Invitrogen, Carlsbad, CA, USA).

After 36 h transfection of Cy3-Oe-ZFAS1, Cy3-Oe-NC, Cy3-sh-ZFAS1, Cy3-sh-NC, Cy3-miR-124 inhibitor and Cy3-inhibitor NC, Eca109 cells (as donor cells) were collected and inoculated with 1 × 10⁵ cells/well into the apical chamber of Transwell culture plate. Complete medium was replenished to 300 μL. Eca109 cells (as recipient cell) were seeded into the basolateral chamber of Transwell 1 day in advance. The density of the plate in the cell was 1 × 10⁵ cells/well, and 3 parallel wells were set up in each group. After co-culture of apical and basolateral chambers for 24 h, FSX100 organisms could only observe the entry of Cy3-ZFAS1 into recipient cells under a navigator. Meanwhile, the recipient cells were collected and the total RNA was extracted. The expression of ZFAS1 and miR-124 in recipient cells was detected by RT-qPCR.

Cells (4 × 10³ per well) were seeded in 96-well plates and cultured to 80% confluence. Cell proliferation was detected by EdU kit (RiboBio, Guangzhou, China). After removing the original medium, it was incubated with 100 μL 50 μm EdU medium (Dilution of EdU solution with cell medium according to 1000: 1) for 2 h. Cells were fixed with 4% paraformaldehyde (50 μL) for 30 min and incubated with 50 μL 2 mg/mL glycine for 5 min. Cells were incubated with 100 μL 0.5% Triton X-100 osmotic agent per well for 10 min, and incubated in the dark for 30 min with 100 μL of 1× Apollo® staining reaction at room temperature, then infiltrated and decolorized with methanol. Finally, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and examined by a laser confocal microscope (Leica, Carl Zeiss, Jena, Germany).

For colony formation assay, the transfected cells were seeded into 6-well plates at the density of 400 cells per well. After 7–14 days, the culture was terminated after the colonies could be seen by the naked eye. Then the medium was absorbed and washed twice with PBS. Thirty minutes were used to fix the cells by methanol and stained it with 0.1% crystal purple staining solution, and colony imaging was counted. At last, the rate of cell colony formation was calculated.

After 48 h of transfection, the cells were detached with 0.25% trypsin without ethylene diamine tetraacetic acid (EDTA) (PYG0107, Boster, Wuhan, Hubei, China) and collected in a flow tube. The supernatant was discarded by centrifugation. According to the instructions of Annexin-V-fluorescein isothiocyanate (FITC) cell apoptosis detection kit (K201–100, BioVision, Palo Alto, USA), the Annexin-V-FITC, propidium iodide (PI), hydroxyethyl piperazine ethanesulfonic acid (HEPES) buffer solution was prepared into Annexin-V-FITC/PI staining solution at 1: 2: 50. Cells (1 × 10⁶) were resuspended in every 100 μL staining solution, then incubated for 15 min, and 1 mL HEPES buffer was added for mixing. The fluorescence of FITC and PI was detected by 515 nm and 620 nm band pass filters at 488 nm wavelength, and the apoptosis was detected.

The ESCC cells after transfected for 48 h were seeded into 6-well plates with 5 × 10⁵ cells per well. After the cells were completely adhered to the wall, the cells were scratched with a 2-mm cell scraper in the middle of each well and cultured for 24 h. The cells were photographed at 0 h and 24 h after scratching, and the scratch distance was calculated by Image-Pro plus 6.0.

The transwell chamber coated with matrigel was preheated to 37 °C. After detachment and transfection, the cells were divided into the same groups as above. Serum-free medium was used to wash the cells twice and suspended it. The cell density was adjusted to 1 × 10⁵ cells/mL. RPMI 1640 medium (600 μL) containing 20% FBS was added to the transwell basolateral chamber. Cell suspension with 200 μL was added to the apical chamber of transwell and cultured for 48 h. The transwell chamber was taken out, the cells near the lateral membrane of the apical chamber were wiped off, and the cells were fixed with 4% paraformaldehyde solution for 10 min after washing with PBS, followed by crystal violet staining, and then the cell staining was observed under an optical microscope. Five high power visual fields were randomly selected for cell counting, and 3 parallel wells were set up in each group.

The subcellular localization of lncRNA ZFAS1 in cells was identified by FISH technique. According to the instructions of Ribo™ lncRNA FISH Probe Mix (Red) (RiboBio Co., Ltd., Guangzhou, China), the specific methods were as follows: the cover glass was putted in the 24-well culture plate, and the cells were inoculated according to 6 × 10⁴ cells/well, so that the confluence of the cells was about 80%. The slides were taken out and fixed at room temperature with 1 mL 4% paraformaldehyde after cleaning with PBS. After being treated with protease K, glycine and acetylation reagent, cells were added with 250 μL prehybridization solution and incubated at 42 °C for 1 h. The prehybridization solution was sucked out, after which cells were hybridized with 250 μL lncRNA ZFAS1 (300 ng/mL) hybrid solution containing probe overnight at 42 °C. After phosphate-buffered saline with Tween (PBST) cleaning for 3 times, the nucleus was stained with DAPI solution (ab104139, 1: 100, Abcam, Shanghai, China) diluted by PBST, and then added to the 24-well culture plate and dyed for 5 min. Finally, cells were sealed with anti-fluorescent quenching agent, and a fluorescence microscope (Olympus, Tokyo, Japan) was used to observe and take pictures.

The sequence of wild type (WT) of miR-124 and STAT3 mRNA 3′-untranslated region (3′-UTR) and sequence of mutant type (MUT) after site-directed mutation of WT target site were synthesized. pmiR-RB-REPORT™ plasmid (RiboBio Co., Ltd., Guangzhou, China) was digested by restriction endonuclease. Then the synthetic target gene fragments WT and MUT were inserted into pmiR-RB-REPORT™ vector (RiboBio Co., Ltd., Guangzhou, China). At the same time, the empty plasmid was transfected as the control group, and the correct luciferase reporter plasmids WT and MUT were used for subsequent transfection. The vectors of MUT and WT were co-transferred to 293 T cells with mimic-NC or miR-124 mimic together with oe-NC or oe-ZFAS1, respectively. The cells were collected and lysed after 48-h transfection, and the supernatant was obtained by centrifugation for 3–5 min. Luciferase detection kit (RG005, Beyotime Biotechnology Co., Ltd., Shanghai, China) was applied for determining the relative lights units (RLU). Using firefly luciferase as an internal parameter, the RLU value determined by renilla luciferase was divided by the RLU value measured by firefly luciferase, and the relative fluorescence value was obtained.

The binding of lncRNA ZFAS1 to Ago2 was detected by RIP kit (Millipore, Bedford, MA, USA). The cells were washed with precooled PBS, and the supernatant was discarded. The cells were lysed by equal volume of ribonuclease inhibitor and protease inhibitor phenylmethylsulphonyl fluoride (PMSF) for 30 min. After centrifuging at 14,000 rpm (4 °C, 10 min), the supernatant was taken out. Part of the cell extract was taken out as Input, another incubated with antibody for co-precipitation. After cleaning, the magnetic beads-antibody complex was re-suspended at 900 μL RIP Wash Buffer and incubated at 4 °C with 100 μL cell extract. The sample was placed on the magnetic pedestal to collect the magnetic bead-protein complex. The samples and Input were detached by protease K and then RNA was extracted for subsequent PCR detection. The antibodies used in RIP were rabbit anti-Ago2 (ab186733, 1:50, Abcam, Shanghai, China). Rabbit anti-IgG (ab109489, 1: 100, Abcam, Shanghai, China) was used as the NC.

The cells were transfected with 50 nM biotin labeled WT-bio-miR-124 and MUT-bio-miR-124 (GeneCreate Biological Engineering Co., Ltd. Wuhan, China). Forty-eight hours later, the cells were collected and washed with PBS. The cells were incubated for 10 min in a specific lysis buffer (Ambion, Austin, Texas, USA). The lysate was incubated overnight with M-280 streptavidin beads (S3762, Sigma-Aldrich, St Louis, MO, USA) pre-coated with RNase-free bovine serum albumin (BSA) and yeast tRNA (TRNBAK-RO, Sigma-Aldrich, St Louis, MO, USA) at 4 °C. Cells was washed by precooled pyrolysis buffer twice, low salt buffer for 3 times and once with high salt buffer. The bound RNA was purified by Trizol and then lncRNA ZFAS1 enrichment was detected by RT-qPCR.

After collection and treatment of the cells of each group, the total RNA in cells and tissues were extracted by Trizol (TaKaRa, Dalian, China) method. According to the reverse transcription kit (K1621, Fermentas, Maryland, New York, USA) instructions, cDNA was reversely transcribed by RNA. The ZFAS1, miR-124 and STAT3 primer sequences (Table 1) were designed and synthesized by Shanghai Genechem Co., Ltd. (Shanghai, China). The mRNA expression of each gene was detected according to the requirements of fluorescent quantitative PCR kit (TaKaRa, Dalian, China) by RT-qPCR (ABI 7500, ABI, Foster City, CA, USA). U6 was selected as an internal parameter of miR-124, while glyceraldehyde phosphate dehydrogenase (GAPDH) was selected as the internal parameter of ZFAS1 and STAT3. The 2^-ΔΔCt method was used to calculate the relative expression of each target gene.

Western blot assay was used to detect the protein content of STAT3. One hundred μL RIPA lysate (R0020, Solaibao Technology Co., Ltd., Beijing, China) was added to a centrifugal tube. According to bicinchoninic acid kit (AR0146, Boster Biological Technology co. Ltd., Wuhan, Hubei, China), the protein concentration was determined and the concentration of each sample was adjusted to 3 μg/μL. The extracted protein was added into the sample buffer and boiled at 95 °C for 10 min, with 30 μg per well; and 10% polyacrylamide gel electrophoresis was used to isolate the protein. The protein was transferred to polyvinylidene fluoride membrane (P2438, Sigma-Aldrich, St Louis, MO, USA), and sealed with 5% BSA (10 L16, Zhongsheng Likang Technology Co., Ltd., Beijing, China) for 1 h. Subsequently, the rabbit anti-STAT3 (ab119352, 1: 5000), CD63 (ab59479, 1: 1000), CD9 (ab2215, 1: 1000), CD81 (ab79559, 1: 1000) (all from Abcam, Cambridge, USA) were added and incubated at 4 °C overnight, and then corresponding goat anti-rabbit secondary antibody (ab6721, 1: 2000, Abcam, Cambridge, USA) was incubated at room temperature for 1 h. The membrane was developed through chemiluminescence reagent with the internal reference of GADPH (ab181602, 1: 10,000, Abcam, Cambridge, USA) by using Gel Doc EZ imager (Bio-rad, California, USA). Eventually, the gray value of the target band was analyzed by the Image J software.

A total of 20 female BALB/c nude mice aged 4–6 weeks and weighed 16–22 g were purchased from the Animal research center of Chinese Academy of Sciences (Shanghai, China). All nude mice were randomly grouped (n = 5): sh-NC group, sh-ZFAS1 group or Oe-NC-exo group and Oe-ZFAS1-exo group. Each group of nude mice was injected with ESCC cells, and the single cell suspension (4 × 10⁶ cells/mL) was injected subcutaneously into the back of nude mice with a disposable aseptic syringe. After injection, all the nude mice were raised in the animal experimental center. The length diameter (L), width diameter (W) and tumor weight of tumor blocks were measured with a vernier caliper and an electronic balance on the 7th day after injection, and the measurement was conducted once every 7 d. The tumor volume was reckoned as V = W2 × L × 0.52 [16]. The nude mice were euthanized 35 d later, the transplantation tumor was taken out, and the maximum surface of the tumor tissue of nude mice in each group was cut off avoiding the necrotic tissue. The overmuch parts were immersed in 4% neutral formaldehyde solution for overnight fixation and embedded in paraffin. Each wax block was separated with a interval of 50 μm, and 10 sections with thickness of 5 μm were cut continuously for subsequent experiment [17].

Initially, the expression of ZFAS1 and miR-124 was detected in 136 cases of ESCC tissues and its corresponding adjacent normal tissues. The results of RT-qPCR showed that ZFAS1 expression was higher and miR-124 expression was lower in ESCC tissues than that in adjacent normal tissues (both p <  0.05). The results of the correlation analysis of ZFAS1 expression and miR-124 expression in ESCC tissues reported that ZFAS1 expression and miR-124 expression were negatively correlated in ESCC tissues (r = − 0.749, p <  0.001) (Fig. 1a-c). We then divided ESCC patients into high expression group (n = 69) and low expression group (n = 67) on the basis of the median values of ZFAS1 expression in ESCC tissues to further analyze the relationship between the expression of ZFAS1 and the clinicopathological characteristics of the patients with ESCC, and the expression of the ZFAS1 was found to be independent of the patients’ gender and age, and related to the tumor size, the tumor nodes metastasis stage and the presence or absence of LNM, it meant that patients with tumor size more than 3 cm, at TNM III + IV stage and at the presence of LNM had a higher rate of ZFAS1 overexpression (Table 2). Meanwhile, the expression of ZFAS1 in human normal esophageal epithelial cells HEEC and five kinds of ESCC lines EC9706, Eca109, TE13, TE1 and TTN were detected by RT-qPCR. The results suggested that (Fig. 1d) compared with HEEC cells, the expression of ZFAS1 in five kinds of ESCC cells was increased in varying degrees (all p < 0.05). The expression of ZFAS1 in Eca109 cell line was dramatically higher than those in EC9706, TE-13, TE-1 and TTN cell lines, so Eca109 cell line was selected for subsequent experiment.

In order to observe whether ZFAS1 affects the development of ESCC, we used RT-qPCR to detect the expression of ZFAS1 in Eca109 cells after transfection. The analysis displayed that (Fig. 2a) compared with the sh-NC group, the expression of ZFAS1 in the sh-ZFAS1–1 group, the sh-ZFAS1–2 group and the sh-ZFAS1–3 group were declined, and ZFAS1 expression in the sh-ZFAS1–1 group was the lowest (all p < 0.05). Therefore, the sequence in the sh-ZFAS1–1 group was selected to silence ZFAS1 in the following experiments and named it as sh-ZFAS1. RT-qPCR was also utilized to detect miR-124 expression in Eca109 cells after transfected with sh-ZFAS1, and the results demonstrated that (Fig. 2b) in relation to the sh-NC group, miR-124 expression elevated in the sh-ZFAS1 group (p < 0.05). The findings of EdU assay, colony formation assay (Fig. 2c-d), scratch test and Transwell assay (Fig. 2f-g) showed decreased proliferation, colony formation, migration, and invasion in cells treated with sh-ZFAS1 (all p < 0.05). Besides, flow cytometry results revealed promoted apoptosis in cells treated with sh-ZFAS1 (p < 0.05) (Fig. 2e). Tumor xenografts in nude mice tested the effect of sh-ZFAS1 on the growth of tumor in vivo, the results revealed that compared to the sh-NC group, the tumor growth ability in the sh-ZFAS1 group was declined (p < 0.05) (Fig. 2h). These findings demonstrated that silencing of ZFAS1 could suppress the proliferation, colony formation, invasion, migration of ESCC cells and tumor growth in vivo but facilitate the cell apoptosis.

We further investigated that the mechanism of the effect of ESCC cells on the surrounding cells. White precipitates were isolated from the culture medium of ESCC cell line Eca109 by differential centrifugation. Under a TEM, the white precipitates were microvesicles with bilayer membrane structure, which were round or oval, and the diameter of vesicles was about 30–60 nm, which was in consistent with the morphological characteristics of exosomes (Fig. 3a). Western blot assay found that the white precipitates had the expression of the exosomes marker (CD63, CD9, CD81) (Fig. 3b). Therefore, we confirmed these white precipitates were the exosomes. The results observed by a laser confocal microscope revealed that exosomes was uptake by Eca109 recipient cells and distributed around the nucleus (Fig. 3c). Therefore, ESCC cells transmit ZFAS1 to surrounding cancer cells through exosomes, which affects the development of ESCC. At the same time, RT-qPCR was utilized to detect the expression of ZFAS1 in donor Eca109 cells, exosomes and recipient Eca109 cells after transfected with Oe-ZFAS1 plasmid and sh-ZFAS1 plasmid. The results showed that (Fig. 3d) ZFAS1 expression elevated in Eca109 donor cells, exosomes and recipient cells relative to that in corresponding NC cells after transfected with Oe-ZFAS1 plasmid (all p < 0.05), but declined in Eca109 donor cells, exosomes and recipient cells after transfected with sh-ZFAS1 plasmid (all p < 0.05).

In order to explore the mechanism of ZFAS1, RNA-FISH was used to detect the subcellular localization of ZFAS1. The results demonstrated that ZFAS1 was concentrated in cytoplasm (Fig. 4a), indicating that ZFAS1 may play a role in cytoplasm. In addition, ZFAS1 could bind to miR-124 (Fig. 4b) through the RNA22 website (https://​cm.​jefferson.​edu/​rna22/​Precomputed/​). The results of dual luciferase reporter gene assay showed that the luciferase activity of WT-miR-124 decreased in cells treated with oe-ZFAS1 (p < 0.05). However, there was no significant difference in luciferase activity of MUT-miR-124, which indicating that miR-124 may specifically bind to ZFAS1 (Fig. 4c). The results of RIP assay revealed there was higher specific adsorption level of ZFAS1 on Ago2 (p < 0.05) (Fig. 4d). RNA pull-down assay was employed to further verify whether ZFAS1 could be used as the ceRNA to adsorb miR-124, and the results showed that the enrichment level of ZFAS1 in the Bio-miR-124-WT group was significantly higher than that in the Bio-probe NC group (p < 0.05). There was no distinct difference in the enrichment level of ZFAS1 in the Bio-miR-124-MUT group and the Bio-probe NC group (p > 0.05) (Fig. 4e). The above results suggest that lncRNA ZFAS1 can act as a ceRNA to adsorb miR-124, thereby affecting the expression of miR-124. Additionally, RT-qPCR tested the expression of miR-124 in each group after transfected with Oe-ZFAS1 plasmid and miR-124 mimics. The results presented that in relation to the Oe-NC group, miR-124 expression was degraded in the Oe-ZFAS1 group (p < 0.05). In contrast to the Oe-ZFAS1 + mimics NC group, miR-124 expression was raised in the Oe-ZFAS1 + miR-124 mimics group (p < 0.05) (Fig. 4f). At the same time, we predicted the target gene of miR-124 on the RNA22 website. It was found that there were binding site between miR-124 and STAT3 (Fig. 4g). Meanwhile, we found that miR-124 bound to STAT3, and STAT3 was the target gene of miR-124 by double luciferase reporter gene assay (Fig. 4h). Meanwhile, RT-qPCR tested the expression of STAT3 in each group after transfected with miR-124 mimics and Oe-STAT3 plasmid. The results presented that in contrast with the mimics NC group, STAT3 expression was degraded in the miR-124 mimics group (p < 0.05). In contrast to the miR-124 mimics + Oe-NC group, STAT3 expression was raised in the miR-124 mimics + Oe-STAT3 group (p < 0.05) (Fig. 4i). It was speculated that ZFAS1 could inhibit the expression of miR-124, thus up-regulating the expression of STAT3.

As shown in Fig. 5a-e, the circumstance and effect of exosomal ZFAS1 shuttling into recipient cells was studied by establishing a co-culture model. After co-culture of donor cells and recipient cells with Cy3-labeled Oe-ZFAS1 plasmid, sh-ZFAS1 plasmid or miR-124 inhibitor for 24 h, the effects of exosomal ZFAS1 on proliferation, apoptosis, migration and invasion of ESCC cells were detected. It presented that proliferation, migration and invasion of ESCC cells in the Oe-ZFAS1-exo group increased, while the apoptosis of cells decreased relative to the Oe-NC-exo group (all p < 0.05). In relation to the sh-NC-exo group, proliferation, migration and invasion of cells declined and apoptosis raised in the sh-ZFAS1-exo group (all p < 0.05). RT-qPCR was adopted to measure the expression of miR-124 and STAT3 in exosomes and recipient cells after treatment of Oe-ZFAS1-exo and sh-ZFAS1-exo, which demonstrated that miR-124 down-regulated and STAT3 up-regulated in exosomes and recipient cells after donor cells with overexpressed ZFAS1 and co-cultured with recipient cells, the trend was apposite in the sh-ZFAS1-exo group and the Oe-ZFAS1-exo group. By comparison with the inhibitor NC-exo group, cell proliferation, migration and invasion ability increased while apoptosis decreased in the miR-124 inhibitor-exo group (all p < 0.05). RT-qPCR detected the expression of miR-124 and STAT3 after miR-124 inhibition in exosomes and recipient cells. The results suggested that the donor cells with miR-124 inhibition were co-cultured with the recipient cells, and miR-124 down-regulated and STAT3 up-regulated in exosomes and recipient cells. The obtained data proved that exosomal ZFAS1 may up-regulate STAT3 and down-regulate miR-124 to promote the proliferation, migration and invasion of ESCC cells, inhibit their apoptosis, and then lead to occurrence and development of ESCC tumor.

As shown in Fig. 6a, compared with the Oe-NC-exo group, the tumor growth ability in the Oe-ZFAS1-exo group increased significantly from the 3rd week (p < 0.05). RT-qPCR revealed that the expression of miR-124 in the Oe-ZFAS1-exo group was notably reduced, while the expression of STAT3 was up-regulated (both p < 0.05) relative to the Oe-NC-exo group (Fig. 6b). The results of western blot assay showed that the expression of STAT3 in the Oe-ZFAS1-exo group was significantly higher than that in the Oe-NC-exo group (p < 0.05) (Fig. 6c). Taken together, overexpression of ZFAS1-exo can promote the growth of nude mice in vivo.