Extracellular vesicle derived miR-544 downregulates expression of tumor suppressor promyelocytic leukemia zinc finger resulting in increased peritoneal metastasis in gastric cancer

PLZF expression is decreased in GC tissues and PM lesions

We analyzed the expression of PLZF in GC patients. Compared with control tissues, PLZF mRNA and protein levels were significantly reduced in GC tissues (Figure 1A, 1B). However, no significant differences of PLZF mRNA and protein levels were found in GC tissues between GC patients with PM and without PM (Figure 1A, 1B). Furthermore, we compared the PLZF levels in PM lesions and normal peritoneal tissues. Remarkably, decreased mRNA and protein levels of PLZF were found in PM lesions compared to normal peritoneal tissues (Figure 1C, 1D), suggesting that the changes of PLZF in PM lesions of GC patients may be regulated by other mediators, such as EVs in the peritoneal fluid.

Figure 1. PLZF mRNA and protein levels in GC patients. (A) mRNA and (B) protein expression of PLZF in GC, and control adjacent tissues. (C) mRNA and (D) protein expression of PLZF in PM lesions and control tissues of GC patients. (n=68 for GC patients without PM, n=65 for GC patients with PM, one way ANOVA for A, B, two-tailed unpaired student’s t-tests for C, D).

Peritoneal fluid in GC patients contains EVs

To explore the mechanism by which the PLZF expression is decreased in GC patients with PM, we first examined whether peritoneal fluid of GC patients with and without PM contains EVs. As shown in Figure 2A, many EVs were identified in the peritoneal fluid. Western blot analysis demonstrated that TSG101, CD63 and CD9, two commonly used EV markers, were present in EV fractions isolated from peritoneal fluids (Figure 2B), indicating that the peritoneal fluid contains EVs.

Figure 2. EVs identification in peritoneal fluid. (A) Transmission electron microscopy demonstrating many EVs <200 nm in diameter. (B) Western blotting illustrating the presence of TSG101 and CD63, two common EV markers, in EV fraction isolated from peritoneal fluid.

GC-associated EVs decrease PLZF expression in peritoneal mesothelial cells

We then investigated whether the GC-associated EVs might affect invasion and migration ability of human peritoneal mesothelial HMrSV5 cells. HMrSV5 cells were co-cultured with EVs isolated from peritoneal fluid from GC patients with and without PM. CCK-8 assay showed that EVs from GC patients with PM significantly increased the viability in HMrSV5 cells compared to that of EVs from GC patients without PM (Figure 3A). As shown in Figure 3B, HMrSV5 cells incubated with EVs isolated from peritoneal fluid from GC patients with PM had an increased invasive ability compared with cells incubated with EVs from patients without PM. In addition, HMrSV5 cells exhibited an increased migration when pretreated with EVs from GC patients with PM compared to those without PM (Figure 3C). Analysis of PLZF protein levels by immunofluorescence and western blotting showed that pretreatment of HMrSV5 cells with EVs from patients with PM reduced the PLZF levels compared to pre-incubation with EVs from patients without PM (Figure 3D, 3E). These findings suggest that the PM-derived EVs reduce the PLZF expression in peritoneal mesothelial cells, and promote GC peritoneal metastasis.

Figure 3. PM-derived EVs reduce PLZF expression in HMrSV5 cells and promote their invasive ability. (A) CCK-8 assay indicated that EVs isolated from GC patients with PM significantly increased the viability of HMrSV5 cells compared to that of without PM. (n=3, one way ANOVA) (B) Trans-well assay illustrating invasion of HMrSV5 cells incubated with EVs isolated from peritoneal fluid of GC patients with and without PM. (C) For migration assays, more adhesive HMrSV5 cells were found in those pretreated by EVs isolated from peritoneal fluid in GC patients with PM compared those without PM. (D) Immunofluorescence and (E) western blotting showing that pretreatment with EVs isolated from patients with PM reduces the PLZF expression in HMrSV5 cells. (n=3, Student’s t-test for B, C, D, E).

miR-544 suppresses PLZF in GC-derived EVs of PM patients

Having found that the GC-derived EVs decrease the PLZF expression in peritoneal mesothelial HMrSV5 cells, we next analyzed the EV PLZF levels in GC patients with and without PM. However, no significant differences were found in PLZF mRNA and protein levels in the EVs isolated from GC patients with and without PM (Figure 4A, 4B). Interestingly, microarray analysis of exosomal miRNAs of GC patients identified five miRNAs that may target PLZF, including miR-342, miR-223-3p, miR-19a-5p, miR-21-5p, and miR-544-5p; these miRNAs were upregulated in patients with PM. RT-PCR confirmed the increased expression of miR-223-3p, miR-21-5p, and miR-544-5p in EVs isolated from GC patients with PM compared to patients without PM (Figure 4C).

Figure 4. miR-544 from GC-derived EVs of PM patients reduces PLZF expression in HMrSV5 cells. (A) mRNA and (B) protein expression of PLZF in EVs isolated from GC patients with and without PM. (n=3, Student’s t-test for A, B) (C) RT-PCR demonstrating increased levels of miR-223-3p, miR-21-5p, and miR-544-5p in EVs isolated from GC patients with PM. (D) RT-PCR of miR-544 in HMrSV5 cells transfected with miR-544 mimics or miR-544 inhibitors. (E) Western blotting of PLZF in HMrSV5 cells transfected with miR-544 mimics or miR-544 inhibitors. (n=3, one way ANOVA for C, D, E).

Since a previous report has indicated that PLZF is a target gene of miR-544 [18], we analyzed the PLZF regulation by miR-544 in HMrSV5 cells. RT-PCR analysis showed that transfection with miR-544 mimics significantly increased the level of miR-544, but transfection with miR-544 inhibitors significantly decreased the level of miR-544 (Figure 4D). Importantly, overexpression of miR-544 significantly suppressed the PLZF protein levels, while miR-544 inhibition increased the PLZF expression in HMrSV5 cells (Figure 4E). Together, these data indicate that PLZF is a target gene of miR-544 in peritoneal mesothelial cells, and in GC-derived EVs of PM patients.

miR-544 is transferred from GS-derived EVs to peritoneal cells

Since the GC-derived EVs could reduce the PLZF expression in HMrSV5 cells, we investigated whether the GC-derived EVs could transfer miR-544 into peritoneum. We first evaluated the miR-544 expression in GC tissues and EVs from GS patients. Compared with adjacent normal tissues, the miR-544 levels were significantly increased in GC tissues (Figure 5A). Moreover, the miR-544 levels were significantly increased in the EVs isolated from GC patients with PM compared to patients without PM (Figure 5B).

Figure 5. miR-544 is transferred from GS-derived EVs to peritoneal cells. (A) RT-PCR of miR-544 in GC tissues. (B) RT-PCR of miR-544 in EVs isolated from GC patients with and without PM. (n=68 for GC patients without PM, n=65 for GC patients with PM, two-tailed unpaired student’s t-tests) (C) RT-PCR of miR-544 in GC cell lines MGC803, BGC823, MKN45, HGC27, and SGC7901. (D) RT-PCR of miR-544 in EVs isolated from GC and GES-1 cells. (E) PKH67-labeled EVs in HMrSV5 cells (bar represents 10 μm). (F) PLZF expression in HMrSV5 cells co-cultured with MGC-803-and GES-1-derived EVs. (n=3, one way ANOVA for C, D, E and F).

Based on the above findings, we further explored the expression of miR-544 in GC cell lines. Compared to normal human gastric epithelial cell line GES-1, the miR-544 expression was significantly increased in gastric cancer cell lines, including MGC803, BGC823, MKN45, HGC27, and SGC7901 (Figure 5C). In addition, EVs isolated from the GC cell lines MGC803, BGC823, MKN45, HGC27, and SGC7901, demonstrated an increased miR-544 expression compared to GES-1 cells (Figure 5D). Since the EVs from MGC803EV cells exhibited the highest miR-544 levels (Figure 5D), MGC803 cells were used for further experiments.

EVs isolated from the gastric cancer MGC-803 cells and control gastric epithelial GES-1 cells were incubated with HMrSV5 cell culture supernatants. As shown in Figure 5E, PKH67 (green)-labeled EVs could be observed in HMrSV5 cells. Importantly, RT-PCR analysis demonstrated that the level of miR-544 increased in HMrSV5 cells co-cultured with MGC-803-derived EVs compared with GES-1-derived EVs. In addition, the PLZF levels were reduced in HMrSV5 cells co-cultured with MGC-803-derived EVs compared with GES-1-derived EVs (Figure 5F). These results indicated that miR-544 might be transferred from the GC-derived EVs to peritoneal cells, where it suppresses the PLZF expression.

Inhibition of miR-544 in MGC803 cells increases PLZF expression in HMrSV5 cells

To examine the possibility that the upregulation of miR-544 in HMrSV5 cells was directly due to the miR-544 transfer from MGC803 cells, we transfected MGC803 cells with a lentivirus construct that inhibits the miR-544 expression. RT-PCR analysis confirmed that the level of miR-544 was significantly decreased in MGC803 cells transfected with the Len-miR-544-inhibitor compared to the control vector Len-miR-544-NC (Figure 6A). Importantly, the PLZF expression was significantly increased in MGC803 cells transfected with Len-miR-544-inhibitor compared to Len-miR-544-NC (Figure 6B). In addition, the exosomal miR-544 derived from MGC803 cells transfected with Len-miR-544-inhibitor was significantly decreased compared to cells transfected with Len-miR-544-NC (Figure 6C). However, no changes in PLZF mRNA were found between the EVs derived from MGC803 cells transfected with Len-miR-544-inhibitor and Len-miR-544-NC (Figure 6D).

Figure 6. miR-544 inhibition increases PLZF expression in HMrSV5 cells. (A) RT-PCR of miR-544 in EVs isolated from MGC803 cells transfected with Len-miR-544-inhibitor or control Len-miR-544-NC. (B) Western blotting of PLZF in MGC803 cells transfected with Len-miR-544-inhibitor or Len-miR-544-NC. (C) RT-PCR of exosomal miR-544 derived from MGC803 cells transfected with Len-miR-544-inhibitor or Len-miR-544-NC. (D) PLZF mRNA in MGC803 cells transfected with Len-miR-544-inhibitor or Len-miR-544-NC. (E) RT-PCR of miR-544 in Len-miR-544-inhibitor group and Len-miR-544-NC group in HMrSV5 cells co-cultured with EVs derived from MGC803 Len-miR-544-NC or Len-miR-544-inhibitor. (F) PLZF expression in EVs isolated from MGC803 cells transfected with Len-miR-544-inhibitor or Len-miR-544-NC. (n=3, Student’s t-test)

Next, HMrSV5 cells were co-cultured with EVs derived from MGC803 cells transfected with Len-miR-544-NC or Len-miR-544-inhibitor. As shown in Figure 6E, the miR-544 levels in the Len-miR-544-inhibitor group were distinctly decreased compared to the Len-miR-544-NC group. Moreover, the PLZF expression was increased in EVs isolated from MGC803 cells transfected with Len-miR-544-inhibitor compared to cells transfected with Len-miR-544-NC (Figure 6F). These results demonstrated that the exosomal miR-544 could be transferred from MGC803 cells to HMrSV5 cells.

miR-544 induces malignant phenotype of peritoneum cells by downregulating PLZF

The above findings demonstrated that miR-544 could be transferred from GC cells to peritoneum cells, but whether the transfer resulted in a malignant phenotype of HMrSV5 peritoneum cells has not been revealed. To address this, we introduced a lentivirus vector overexpressing PLZF into HMrSV5 cells (Figure 7A). As shown in Figure 7B, PLZF overexpression significantly reduced migration and invasion of HMrSV5 cells, indicating that PLZF functions as a tumor suppressor in peritoneum cells. In contrast, PLZF suppression by PLZF specific siRNA (Figure 7C), significantly increased HMrSV5 cell migration and invasion (Figure 7D). Moreover, when PLZF was silenced, even transfection with Len-miR-544-inhibitor could not effectively reverse the increased HMrSV5 cell migration and invasion (Figure 7C, 7D). These data demonstrate that miR-544 induces the malignant phenotype of peritoneum cells via targeting PLZF.

Figure 7. miR-544 induces malignant phenotype in HMrSV5 cells via targeting PLZF. (A) Western blotting of PLZF in Len-PLZF-transfected HMrSV5 cells overexpressing PLZF. (n=3, Student’s t-test) (B) Migration and invasion of Len-PLZF-transfected HMrSV5 cells. (C) Western blotting of PLZF in PLZF siRNA-transfected HMrSV5 cells. (n=3, one way ANOVA) (D) Migration and invasion of PLZF siRNA-transfected HMrSV5 cells.

Patient samples

60 paired GC and adjacent non-cancer tissues from patients who underwent surgery at Affiliated Hangzhou First People Hospital were obtained between January 2018 and December 2018. At the same time, peritoneal lavage fluid was obtained. Laparoscopic observation or positive cytology of peritoneal lavage fluids was used for the diagnosis of peritoneal metastasis. Surgically resected samples, including primary tumors, paired adjacent non-cancerous tissues, suspicious peritoneal metastatic lesions, and normal peritoneal tissues adjacent to the corresponding possible peritoneal metastasis sites were analyzed (Table 1). Exclusion criteria: (1) Patients who ever received chemotherapy or radiation therapy; (2) Patients with distant metastasis; (3) Patients with gastric stump carcinoma. Inclusion criteria: (1) Patients over 18 years old; (2) Patients with histologically proven adenocarcinoma of the stomach or esophagogastric junction (Siewert type 2 or 3); (3) Patients who received CT or PET-CT scan before surgery.

Cell culture

Human GC cell lines MGC803, BGC823, MKN45, HGC27, and SGC7901, normal human gastric epithelial cell line GES-1, and human peritoneal mesothelial cell line HMrSV5 were purchased from the Cell Center of Shanghai Institutes for Biological Sciences (Shanghai, China). All cell lines were cultured in RPMI-1640 (GE Healthcare Life Sciences, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, USA), streptomycin (100 mg/ml), and penicillin (100 U/ml) at 37° C in a humidified atmosphere containing 5% CO₂.

Isolation of EVs

EVs were isolated from peritoneal fluid samples by centrifuging at 2,000 g for 10 minutes. The supernatants were then filtered through 800 nm filter (Millipore, USA) to remove cell debris, and ultra-centrifuged at 150,000 g for 70 minutes at 4^oC. In addition, EVs were isolated from GC cells cultured in RPMI-1640 supplemented with 10% exosome-free FBS (ExoPerfectTM Exo-free FBS, SUER250QY, Hangzhou Qiannuo Biotechnology Co., LTD, Hangzhou City, China) for 48 h, using the GET™ Exosome Isolation Kit (GET301-10, Genexosome Technologies, USA). In brief, 5ml of exosome concentration solution (ECS reagent) was added to 5ml of serum and was then diluted with 5ml of 1 × PBS. After adding 2.5 ml ECS reagent, the mixture was turned upside down, and then placed at 4^oC for overnight. The mixture was centrifuged for 60 min at 10,000 g. The supernatant was discarded and EVs was rich in the precipitate. Then, 400 μ L of 1 × PBS was used to suspend the precipitate and EVs were collected for the subsequent experiments. The presence of isolated EVs was validated using an HT-7700 transmission electron microscope (Hitachi High-Technologies, Tokyo, Japan) (bar = 50 nm). Purified EVs were labeled with the PKH67 green fluorescent linker Mini Kit (Sigma, USA) according to the instructions. Briefly, EVs were suspended in 180μl of PBS with 20μl of 1:50 diluted PKH67. After 3 min of incubation at room temperature, 3.8 ml of exosome-free medium was added to terminate the labeling reaction, and EVs were harvested and washed twice with PBS by centrifugation at 100,000 g for 1 h. After 48 h of co-culture, GC cells were washed twice with PBS, fixed with 4% paraformaldehyde for 10 min, washed twice or thrice with PBS for 10 min and then observed under a laser confocal microscope (Zeiss, Germany).

RNA isolation and qRT-PCR

RNA was extracted from EVs and cells using the miRNeasy Mini Kit (Qiagen, Hilden, Germany). Concentration and purity of RNA was determined by measuring the optical density at 260 and 280 nm. RNA reverse transcription was performed using the TaqMan™ MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific USA). The reagent concentrations were 3.0 μh RNA, 3.0 μl RT primer, 0.3μl dNTP, 3.0 μl MultiScribe Reverse Transcriptase, 1.5μl 10e RT buffer, 1.01μl Nuclease-free water. The temperature protocol used for RT was as follows 72° C for 10 min; 42° C for 60 min, 72° C for 5 min and 95° C for 2 min. SYBR Green Super mix (Biorad, USA) was used for real-time quantitative PCR. The PCR amplifications were performed in a 10 μl reaction system containing 5 μl SYBR Green Supermix, 0.4 μl forward primer, 0.4 μl reverse primer, 2.2 μl double distilled H₂O and 2 μl template cDNA. PCR reaction cycles were as follows: 95^oC for 30 seconds, 45 cycles of 5 seconds at 95^oC and 30 seconds at 60 ^oC. Relative mRNA expression was normalized to U6 using the 2^{-Δ ΔCq} method [29]. The primers used in the present study were listed in Table 2. Cell migration assays were performed using Boyden chambers (8-μm pore filter; Corning Inc, USA). Cells (1 × 10⁵/well) were plated into the top chamber and 10% FBS containing medium was placed into the bottom chamber. After incubation at 37° C in 5% CO₂ for 12 h, the cells remaining at the upper surface of the membrane were removed with a cotton swab. The cells that migrated through the 8 μm sized pores and adhered to the lower surface of the membrane were fixed with 4% paraformaldehyde, stained with crystal violet, and photographed.

Cell transfections

The lentivirus vector overexpressing PLZF (Len-PLZF), control vector (Len-NC), inhibitory miR-544 (Len-miR-544-inhibitor), and control miR (Len-miR-544-NC) were purchased from Genechem (Shanghai, China).

siRNA transfections, MGC-803 cells were seeded at the density of 10⁵ cells/well in a 6-well plate, and transfected with siRNA targeting PLZF or a non-specific siRNA at a final concentration at 20 nM, using the HiPerfect transfection reagent (Qiagen GmbH, Hilden, Germany).

Cell viability analysis

To examine cell viability, HMrSV5 cells were seeded in 96-well plates at a density of 1.0x10⁴ cells/per well. Exosomes from GC patients without PM or with PM were co-cultured with MGC-803 cells for 24, 48, 72 h. Then, Cell numbers were calculated using a hemocytometer and cell proliferation was analyzed using a Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Inc., Kumamoto, Japan), according to the manufacturer’s protocol.

Western blotting

Proteins were extracted using RIPA buffer (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China). Equal amounts of protein (15 μg/lane) were separated by 10% SDS-PAGE, and transferred onto a PVDF membrane. The membranes were incubated with primary antibodies (RT, 2 h), washed in TBST, and incubated (RT, 2 h) with HRP-conjugated goat anti-rabbit IgG (1:5,000; ZB-2306, Zhongshan Gold Bridge Biological Technology Co., Beijing, China). After washing, the signal was detected using enhanced chemiluminescence (Merck KGaA, Darmstadt, Germany). ImageJ 1.8.0 (National Institutes of Health, Bethesda, MD, USA) was used to quantify the relative protein levels. GAPDH was used as an internal control.

Statistical analysis

Data were expressed as the mean ± standard error. Each experiment was carried out in triplicates. The two-tailed unpaired student’s t-tests were used for comparisons of two groups. Multiple comparisons were performed using one-way one way ANOVA followed by Tukey’s multiple comparison test; P<0.05 was considered significant. The data were analyzed using SPSS software, version 20.0 (SPSS, Inc., Chicago, IL, USA).