Exosome-derived lnc-HOXB8-1:2 induces tumor-associated macrophage infiltration to promote neuroendocrine differentiated colorectal cancer progression by sponging hsa-miR-6825-5p

Patients

From April 2015 to July 2016, at the Department of Gastrointestinal Surgery of Sun Yat-sen Memorial Hospital, Sun Yat-sen University, we selected 105 patients with CRC who underwent surgery for retrospective analysis. The inclusion criteria were as follows: (1) pathological diagnosis of colorectal adenocarcinoma; (2) the markers of NED, including CgA and Syn, were detected by immunohistochemistry; (3) available histologic samples (i.e., surgically resected specimens); and (4) valid clinical data, including surgery, treatment and follow-up data, which were collected through case reviews and telephone follow-up. The following follow-up data were recorded: current physical condition, date of local recurrence and/or metastasis, date and cause of death, etc. Overall survival (OS) was defined as the interval from surgery to the date of mortality or the final follow-up. Progression-free survival (PFS) was defined as the interval from surgery to the date of recurrence, metastasis, or mortality. The end point was set as July 31, 2021. Patients who died perioperatively (within 3 months after surgery) or received preoperative chemotherapy and/or radiotherapy were excluded.

Cell culture

The LoVo colon cancer cell line, 293 T cells and THP-1 cells were purchased from the Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences. The LoVo colon cancer cell line was selected to transform into stable CgA neuroendocrine-like cells (LoVo-CgA) and control cells (LoVo-NC) according to the method described previously [5], the CgA vector and NC vector were labeled with green fluorescent protein (GFP) gene. The PCR fragment of lnc-HOXB8-1:2 was inserted into the pcDNA3.1( +) vector, which was used to infect LoVo-CgA cells, in addition to a separate control vector experiment. Lipofectamine 2000 was used for transfection according to the manufacturer’s instruction. Following infection, selective media containing G418 were used until the stable clone cell line (LoVo-CgA-OE) and control cell line (LoVo-CgA-NC) were obtained after 2 weeks. THP-1 cells were treated with 320 nM PMA for 6 h and then 20 ng/mL IL-4 was added for another 18 h to obtain tumor-associated macrophages [5]. TAMs were transfected with hsa-miR-6825-5p mimics as well as the corresponding control oligonucleotides using reagent Lipofectamine RNAimax based on the manufacturer’s instructions. Stable clone LoVo colon cancer cells and 293 T cells were incubated in DMEM-12 supplement with 10% FBS, and the other cells were incubated in RPMI-1640 supplement with 10% FBS at 37 °C with 5% CO2. All specific overexpression plasmids were designed and synthesized by General Biosystems Co., Ltd (Anhui, China) and hsa-miR-6825-5p mimics were purchased from GenePharma (Suzhou, China). The details of main reagents were listed in Table S1.

Coculture of tumor-associated macrophages with exosomes or colon cancer cells

Approximately 1 × 106 tumor-associated macrophages were inoculated in the upper chamber of 6-well plates, and cocultured with LoVo-CgA-OE or LoVo-CgA-NC cells at a ratio of 1:1 or with different exosomes (20 µg/mL) [22] in the lower transwell chamber for 48 h, separately. The tumor-associated macrophage cells were washed for subsequent experiments after coculture.

Immunohistochemistry (IHC) assay

An IHC assay was used to detect CgA, Syn, CD68 and CXCR3 proteins. The staining procedure was conducted using the Envision two-step method as previously reported [5]. PBS replaced the primary antibody as a negative control. Primary antibodies against CgA (1:500), Syn (1:500), CD68 (1:500) and CXCR3 (1:600) were used. Horseradish peroxidase (HRP)-linked polyclonal rabbit anti-mouse IgG was used as the secondary antibody (1:1000). The details of main antibodies were listed in Table S2.

Extraction and identification of exosomes

The original culture media of cells was aspirated, and then the cells were washed twice with PBS, and replenished with serum-free culture media for 36 h. The culture media was then harvested. The cell media was centrifuged at 2000 × g for 30 min to remove cells and debris, and the supernatant containing the cell-free culture media was transferred into a new tube without disturbing the pellet. Total Exosome Isolation Reagent (from cell culture media) was used to extract exosomes from different cell culture media according to the manufacturer's instructions. After exosomes were isolated, the morphology of exosomes was observed by transmission electron microscopy (TEM), and the particle size was detected by Zeta View (Particle Metrix). Total RNA and protein of exosomes were purified for subsequent RNA sequencing and western blot assays, respectively, using the Total Exosome RNA and Protein Isolation Kit.

RNA isolation and quantitative reverse transcription PCR (qRT–PCR) assay

Total RNA from cell lysates was isolated using TRIzol and reverse transcription was conducted using M-MLV Reverse Transcriptase. GoTaq® RT–PCR Master Mix was applied to perform qRT–PCR according to the manufacturer’s protocol to examine the level of mRNAs, miRNAs and lncRNAs. The specific sequences of the qRT–PCR primers were listed in Supplementary Table S3.

Western blot assay

Cells were collected and lysed in RIPA buffer on ice. The Bradford assay was performed to detect the protein concentration. Equivalent amounts of protein were separated on 10% and 12% SDS–PAGE gels and transferred onto PVDF membranes. Five percent nonfat milk was used to seal the membranes. After blocking, the membranes were incubated at 4 °C overnight with primary antibodies against CgA (1:1000), CD68 (1:1000), CXCR3 (1:1000), CD63 (1:1000), HSPA8 (1:1000), Alix (1:1000) and TSG101(1:1000) and then incubated with HRP-linked polyclonal rabbit anti-mouse IgG (1:2000) for 1 h at room temperature after washing. An enhanced chemiluminescence (ECL) kit was used to visualize the labeled proteins. GAPDH served as an internal reference. The details of main antibodies were listed in Table S2.

Cell migration and invasion assay

For cell migration assays, single-cell suspensions were diluted to 1 × 106 cells in serum-free RPMI-1640 medium and 100 µL was seeded into the upper transwell chamber, followed by the addition of 600 µL RPMI-1640 medium containing 10% FBS in the lower chamber. The cells were allowed to incubated at 37 °C with 5% CO2 in a humidified atmosphere. After 24 h, nonmigrating cells were removed and the other cells were fixed with 4% paraformaldehyde for 10 min, stained with 0.1% crystal violet for 10 min, washed with PBS, and counted in under a microscope. Cell migration assays were also conducted using the same transwell inserts with 10% Matrigel spread onto the upper chamber. The invasive cells were incubated for 48 h. Three random fields were selected for cell counting.

Next-generation sequencing (NGS)

Total RNA was extracted from exosomes secreted by LoVo-CgA cells (CgA Exo) and LoVo-NC cells (NC Exo) for next-generation sequencing (NGS). NGS and gene differential expression analysis were conducted by Forevergen (Guangzhou, China) to select lncRNAs for subsequent study. The inclusion criteria of lncRNAs were as follows: (1) upregulated expression in CgA Exo; (2) number of miRNA potential binding sites in common with CXCR3 mRNA greater than or equal to three; (3) only one transcript, with length between 200 and 2500 base pairs; (4) multiple top 20 of differentially expressed lncRNAs between CgA Exo and NC Exo; and (5) available to search in the LNCipedia database (https://lncipedia.org/).

Bioinformatics analysis

The association between the gene expression level of CgA or CXCR3 and the infiltration of macrophages, especially M2 macrophages, in different cancers was determined based on the Timer 2.0 database (http://timer.cistrome.org/). According to competitive endogenous RNA (ceRNA) theory [19], miRanda software (Memorial Sloan-Kettering Cancer Center, USA) was used to predict the miRNAs with the most binding sites that could simultaneously bind to lnc-HOXB8-1:2 and CXCR3 mRNA. RNA22 database (https://cm.jefferson.edu/rna22) and Target Scan database (https://www.targetscan.org) were used to validate this prediction.

Fluorescence in situ hybridization (FISH) assay

Specific fluorescently labeled lnc-HOXB8-1:2 FISH probes were designed and synthesized by GenePharma Co. (Shanghai, China). The specific sequence of the FISH probe was listed in Supplementary Table S4. Paraffin sections were placed at room temperature for 60 min, soaked in xylene for dewaxing and immersed in graded alcohol for rehydration. Subsequently, the slides were heated in 0.01 M sodium citrate buffer solution (pH 6.0) at approximately 95 °C for 15 min, and treated with proteinase K (20 µg/mL) at 37 °C for 30 min. The slides were then treated with lnc-HOXB8-1:2 probe hybridization solution (2 µM) at 37 °C overnight. Subsequently, the slide was washed with 50% formamide at 42 °C, thoroughly rinsed using 2 × saline sodium citrate buffer (SSC), and blocked in 5% normal goat serum at room temperature for 30 min. The slides were used for subsequent immunofluorescence (IF) assays to detect the expression level and site of CD68 protein in CRC tissue specimens.

Immunofluorescence (IF) assay

A PKH67 Green Fluorescent Cell Linker Mini Kit was used to detect whether labeled exosomes were absorbed by macrophages [23] according to the manufacturer's protocol.

To determine the proportion of M2 macrophages, treated TAMs were grown to 40%-60% confluence in glass bottom dishes, washed with PBS three times and fixed in 4% paraformaldehyde. The cells were incubated with primary antibodies against CD68 (1:600) and CD206 (1:200) at 4 °C overnight. After washing with PBS, the cells were incubated with Alexa Fluor 488-donkey anti-rabbit IgG secondary antibody (1:500) and Alexa Fluor 546-goat anti-mouse IgG secondary antibody (1:500) at room temperature away from light for 1 h and stained with DAPI for 10 min.

To detect the expression level and site of CD68 proteins in CRC tissue specimens, a primary antibody against CD68 (1:400), and Alexa Fluor 594-goat anti-rabbit IgG secondary antibody (1:500) were used in the IF assay procedure mentioned above. The details of main antibodies were listed in Supplementary Table S2.

The fluorescent cells of the IF assay and FISH assay were observed using a Nikon A1Si laser scanning confocal microscope (Nikon Instruments Inc., Japan). The proportion of fluorescent cells was quantified for data analyses.

Dual-luciferase reporter assay

Dual-luciferase reporter assay was applied to confirm the direct binding between lnc-HOXB8-1:2 and hsa-miR-6825-5p and to explore whether CXCR3 was the direct target of hsa-miR-6825-5p. The sequence of lnc-HOXB8-1:2 and the 3’UTR sequence of CXCR3 containing the wild-type or mutant binding sites with hsa-miR-6825-5p (Supplementary Table S5) were cloned into the psiCHECK-2 vector including Firefly luciferase gene (Fluc) and Renilla luciferase gene (Rluc), respectively. lnc-HOXB8-1:2 vectors and CXCR3 vectors were cotransfected with NC or has-miR-6825-5p mimics into 293 T cells. The relative values of Fluc and Rluc were measured by Centro LB960 XS3 (Berthold, German) using Dual-Luciferase Reporter Assay Systems.

Statistical analysis

Statistical analysis and graph generation were performed with GraphPad Prism 8.0 (GraphPad Software, USA) and SPSS 20.0 (IBM Corp, USA). Qualitative data were assessed by Pearson’s chi-square test or the Mann–Whitney U rank-sum test. Student’s t test or Welch’s t test was used to perform statistical analysis for quantitative data between two groups. The Kaplan–Meier method and log-rank test were performed to analyze overall survival and progression-free survival. The associations between risk factors and OS or PFS were quantified by hazard ratios (HRs) and 95% confidence intervals (CIs) using a Cox proportional hazards model. A 2-tailed P value < 0.05 was considered statistically significant.

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