Down‐regulated lncRNA SBF2‐AS1 in M2 macrophage‐derived exosomes elevates miR‐122‐5p to restrict XIAP, thereby limiting pancreatic cancer development

Abstract Evidence has indicated that M2 macrophages promote the progression of cancers, but few focus on the ability of M2 macrophage‐derived exosomes in pancreatic cancer (PC). This study aims to explore how M2 macrophages affect malignant phenotypes of PC through regulating long non‐coding RNA SET‐binding factor 2 antisense RNA 1 (lncRNA SBF2‐AS1)/microRNA‐122‐5p (miR‐122‐5p)/X‐linked inhibitor of apoptosis protein (XIAP) axis. THP‐1 cells were transformed into M1 macrophages by lipopolysaccharide and interferon‐γ treatment, and into M2 macrophages after interleukin‐4 treatment. The PANC‐1 PC cell line with the largest lncRNA SBF2‐AS1 expression was selected, and M2 macrophage‐derived exosomes were isolated and identified. A number of assays were applied for the examination of lncRNA SBF2‐AS1 expression, PC cell biological functions and subcellular localization of lncRNA SBF2‐AS1. XIAP expression was detected, along with the interaction among lncRNA SBF2‐AS1, miR‐122‐5p and XIAP. M2 macrophage exosomal lncRNA SBF2‐AS1 expression's effects on the tumorigenic ability of PANC‐1 cells in nude mice were also investigated. M2 macrophage‐derived exosomes promoted progression of PC cells. Overexpressed lncRNA SBF2‐AS1 promoted progression of PC cells. LncRNA SBF2‐AS1 was found to act as a competing endogenous RNA to repress miR‐122‐5p and up‐regulate XIAP. Constrained lncRNA SBF2‐AS1 in M2 macrophage‐derived exosomes contributed to restraining tumorigenic ability of PC cells. Collectively, our study reveals that constrained lncRNA SBF2‐AS1 in M2 macrophage‐derived exosomes increases miR‐122‐5p expression to restrain XIAP expression, which further inhibits PC progression.


| INTRODUC TI ON
Pancreatic cancer (PC) is a major cause accounting for the cancer mortality in developed countries, and it is mainly comprised of adenocarcinoma (85%) and pancreatic endocrine tumours (under 5%).1 There are a multitude of risk factors in PC, including smoking, obesity, alcoholism, age, genetic factors and pre-existing chronic pancreatitis.2 PC is chiefly manifested by anorexia, asthenia abdominal pain and weight loss, but there is often deferred diagnosis as a result of no particular symptoms.3 Moreover, little improvement has been made in the prevention and treatment of advanced PC patients. 4 As PC causes great threats to human lives, this study aimed to make new explorations of therapies for PC treatment on the basis of previous studies. and some tumours' development. 8 As a newly discovered lncRNA, SET-binding factor 2 antisense RNA 1 (SBF2-AS1) has been found to be involved in non-small-cell lung cancer and results in unfavourable prognosis in patients. 9 By functioning as a competitive endogenous RNA, lncRNA SBF2-AS1 is found to elevate twinfilin-1 (TWF1) to sponge miR-142-3p and participate in gemcitabine resistance in PC. 10 Long et al11 have demonstrated that SBF2 up-regulation can substantially depress cell proliferation and promote apoptosis in PC possibly through restriction of transforming growth factor β/SMAD signalling pathway. There is a study showing that in cervical cancer, lncRNA SBF2-AS1 regulates microRNA (miRNA)-361-5p/forkhead box M1 (FOXM1) axis to contribute to tumour progression.12 MiRNAs have been proposed to correlate with cancer development. 13 There is evidence indicating that miR-122 is down-regulated in PC.14 Besides that, lower miR-122-5p expression is demonstrated in PC tissues. 15 A study has showed that X-linked inhibitor of apoptosis protein (XIAP) is a direct target of miR-130, 16 and it is a cytosolic suppressor of caspases 3, 7 and 9.17 Young Kim et al18 have revealed that XIAP silencing is able to increase PC cell apoptosis stimulated by tumour necrosis factor-related apoptosis-inducing ligand.
Nevertheless, few studies have paid attention to the role of M2 macrophage-derived exosomes in PC. Hence, this study is meant to discuss how M2 macrophages affect malignant phenotypes of PC through ln-cRNA SBF2-AS1/miR-122-5p/XIAP axis.

| Exosome isolation and identification
Exosomes were isolated by ultracentrifugation. The cell culture supernatant of M2 macrophages was centrifuged at 500 g for 10 minutes to remove the precipitates, and the supernatant was centrifuged at 2000 g for 10 minutes at 4°C to remove cell debris. The obtained supernatant was filtered through a 0.22-μm filter and centrifuged at 100 000 g for 4 hours in an ultracentrifuge tube. The precipitates were resuspended in phosphate-buffered saline (PBS) and centrifuged at 100 000 g for 70 min. The obtained precipitates were exosomes.24 Exosome morphology was detected by a transmission electron microscope (TEM). The obtained exosomes (50 μL) were dropped on a 200-mesh copper mesh and incubated at room temperature for 5 minutes. Then, the exosomes were stained with 1% phosphotungstic acid for 1 minute and rinsed 1 time or 2 times with distilled water.
Exosome surface markers were identified by Western blot analysis, and the exosome protein content was measured by bicinchoninic acid (BCA) kit (23227, Thermo Fisher Scientific) after the concentration of the exosome suspension. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was prepared, followed by protein denaturation and electrophoresis. Then, the exosomes were transferred to the membrane, and the expression of exosome-specific marker protein TSG101, CD63, CD81 and GRP94 (1:1000, Abcam Inc.) was examined.

| Exosome uptake
The exosomes dissolved in PBS were supplemented to 0.3 mL Diluent C for dilution and then stained with 4 μL PKH67 staining solution (Sigma) and incubated at 37°C for 5 minutes. Next, the exosomes were added with 2 mL PBS containing 0.1% bovine serum albumin to stop staining and then centrifuged at 110 000 g for 1 hour at 4°C. The supernatant was removed, and then, the exosomes were supplemented with appropriate medium for suspension. The nuclei of PANC-1 cells were stained with 4',6-diamidino-2-phenylindole (DAPI). The labelled exosomes were added to cultured PANC-1 cells to a final concentration of 10 mg/L. Four hours later, a fluorescence microscope was used to observe the uptake of fluorescence-labelled exosomes by PANC-1 cells.

| Cell treatment, grouping and transfection
In order to observe the effect of M2 macrophage-derived exosomes on PANC-1 cells, PANC-1 cells were grouped as the control group

| 5-Ethynyl-2′-deoxyuridine (EdU) assay
The cells were inoculated in a 96-well plate at 4 × 10 3 cells per well and cultured to 80% confluence, followed by cell proliferation detection by an EdU kit (RiboBio). The cells were incubated in a new EdU medium (100 μL, 50 μm, diluted with a cell culture medium at 1000:1) for 2 hours before two PBS washes (5 minutes once) with the medium removed. Subsequently, the cells were fixed by 50 μL 4% paraformaldehyde, incubated with 50 μL 2 mg/mL glycine and supplemented with 100 μL 0.5% Triton X-100 penetrant in each well for 10-min incubation before 5-min PBS washes. Then, the cells were cultivated with 100 μL 1 × Apollo® staining reaction solution avoiding light for 30 minutes, infiltrated, de-colorized with methanol, stained with DAPI and captured by a laser confocal scanning microscope (Leica).

| Flow cytometry
The cells were detached with 0.25% trypsin after 48-hours trans-

| Scratch test
After 48 hours of transfection, the PC cells were inoculated on 6-well plates at 5 × 10 5 cells/well. When complete cell adherence occurred, a 2-mm cell scraper was used to scratch in the middle of each well, followed by 24-h culture. Photographs were taken at the 0 and 24th hours after cell scratching, and the scratch distance was calculated with Image-Pro plus 6.0.

| Transwell assay
The Matrigel-coated Transwell chamber was preheated to 37°C to detach the transfected cells. The cells were grouped as the above way. The transfected cells were resuspended in serum-free medium and altered to 1 × 10 5 cells/mL after counting. RPMI 1640 medium (600 μL) containing 20% FBS was appended to the lower chamber, Three duplicates were set in each group.

| RNA-fluorescence in situ hybridization (FISH) assay
FISH assay was conducted to identify the subcellular localization of lncRNA SBF2-AS1 in cells. This assay was performed following the requirements of Ribo™ lncRNA FISH Probe Mix (Red, RiboBio Co., Ltd.). To begin with, the cells were seeded on a coverslip on the 24-well culture plate at 6 × 10 4 cells/well to achieve about 80% confluence and fastened by 1 mL 4% paraformaldehyde fixation.
After proteinase K, glycine and acetamidine reagent treatment, 250 μL prehybridization solution was added for incubation, which was then replaced with lncRNA SBF2-AS1 hybridization solution containing probe overnight at 42°C. Diluted DAPI (ab104139, 1:100, Abcam) was added for 5-min cell staining. Finally, the plate was sealed with an anti-fluorescence quencher, followed by fluorescence microscopy observation (Olympus) and photographing.

| Dual-luciferase reporter gene assay
The wild-type (WT) and mutant type (MUT) sequences of lncRNA SBF2-AS1 and XIAP mRNA 3′-untranslated regions (3′-UTR) were inserted into the pmiR-RB-REPORT™ vector (RiboBio). The correctly sequenced WT and MUT were transfected with mimic-NC, miR-122-5p mimic into 293T cells. After 48-h transfection, the cells were lysed before 3-to 5-min centrifugation. With the firefly luciferase as a loading control, the relative light unit value of Renilla luciferase divided by that of firefly luciferase was the relative fluorescence value.

| RT-qPCR
TRIzol method (Takara) was applied for extracting total RNA from both cells and tissues. On the basis of the reverse transcription kits (K1621, Fermentas), cDNA was obtained by RNA reverse transcription. LncRNA SBF2-AS1, miR-122-5p, XIAP and other genes' primer sequences were entrusted by Shanghai GeneChem Co., Ltd.
( Table 1). Fluorescence quantitative PCR kit (Takara,) was adopted to lncRNA, mRNA and miRNA detection by employing RT-qPCR (ABI 7500, ABI). U6 was a loading control for miR-122-5p and glyceraldehyde phosphate dehydrogenase (GAPDH) for lncRNA SBF2-AS1, XIAP and other genes. 2 -ΔΔCt method was utilized for the calculation of the relative expression of target genes.
Then, membranes were mixed with the primary antibodies XIAP

| Tumour xenografts in nude mice
Thirty-two BALB/c female nude mice were obtained from the Experimental Animal Center of the Chinese Academy of Science

| Statistical analysis
All data were analysed with the SPSS 21.0 software package (IBM Corp.) and consistent with the normal distribution and the homogeneity variance test. The measurement data were depicted as mean ± standard deviation. Comparisons between two groups were made by independent sample t test while those among multiple groups by one-way analysis of variance (ANOVA), after which Tukey's post hoc test was performed. The difference was statistically significant at P < .05. and non-exosome marker protein GRP94. TSG101, CD63 and CD81

| Isolation and identification of M2 macrophagederived exosomes
were expressed, but GRP94 was not expressed by M2 macrophagederived exosomes, which further confirmed the success of exosome isolation ( Figure 1E).

PANC-1 cells for 48 h, and the uptake of exosomes by PANC-1 cells was observed under an inverted fluorescence microscope. The results
showed that PKH67-labelled exosomes with green fluorescent appeared in the co-culture group of PANC-1 and exosomes ( Figure 1F).

| LncRNA SBF2-AS1 represses miR-122-5p to up-regulate XIAP
RNA-FISH results showed that lncRNA SBF2-AS1 was mainly functioned in the cytoplasm ( Figure 4A). Through the RNA22 website (https://cm.jeffe rson.edu/rna22 /Preco mpute d/), it was predicted that lncRNA SBF2-AS1 could bind to miR-122-5p ( Figure 4B vs the control group; all the above results were measurement data expressed by mean ± standard deviation, and comparison between two groups was analysed by independent sample t test; the cell experiment was repeated three times assay with the corresponding findings revealing that the luciferase activity of cells cotransfected with WT-SBF2-AS1 and miR-122-5p mimic descended greatly (P < .05), while no palpable difference was seen in the luciferase activity of cells cotransfected with MUT-SBF2-AS1 and miR-122-5p mimic (P > .05), indicating that miR-122-5p may specifically bind to lncRNA SBF2-AS1 ( Figure 4C). RIP assay showed that the lncRNA SBF2-AS1's specific sponge of Ago2 ascended markedly vs the IgG group (P < .05; Figure 4D). RNA pull-down assay verified that lncRNA SBF2-AS1 enrichment in the bio-miR-122-5p-WT group increased clearly (P < .05), while no marked difference was seen in the same parameter in the bio-miR-122-5p-MUT group vs the bio-probe NC group (P > .05; Figure 4E), indicating lncRNA SBF2-AS1 can function as a sponge to miR-122-5p, thereby affecting miR-122-5p expression. At the same time, we also predicted the target gene of miR-122-5p in the RNA22 website and found that a targeted binding site existed between miR-122-5p and XIAP ( Figure 4F).
Dual-luciferase reporter gene assay also revealed that ( Figure 4G) miR-122-5p specifically bound to XIAP, and XIAP was the target gene of miR-122-5p. To further verify our inference, we adopted RT-qPCR and Western blot assay to determine miR-122-5p and XIAP expression in PANC-1 cells, and the results illustrated that  All in all, our study revealed that knock-down of lncRNA SBF2-AS1 in M2 macrophage exosomes promoted miR-122-5p expression and then declined XIAP expression, thereby constraining PC development ( Figure S1). This study provides new clues for the function of lncRNA SBF2-AS1/miR-122-5p/XIAP axis in PC progression and more significantly, a new direction for PC treatment, which is of great importance to human beings.

| D ISCUSS I ON
Nevertheless, more studies are necessary to further elaborate the relevant mechanisms of M2 macrophage-derived exosomes on PC development.

ACK N OWLED G EM ENT
We would like to acknowledge the reviewers for their helpful comments on this paper.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.
Consent for publication: Not applicable.
Availability of data and material: Not applicable. Zhang.

E TH I C A L S TATEM ENT
Animals were treated humanely, and the protocol was approved by