Exosomal miR-628-5p from M1 polarized macrophages hinders m6A modification of circFUT8 to suppress hepatocellular carcinoma progression

Cell culture

HCC cell lines (Huh7, HCCLM3, Hep3B, MHCC97H), immortalized human liver epithelial THLE-3 cell line, and macrophage THP-1 were all obtained from ATCC (Manassas, VA, USA). The THLE-3 cell line and four HCC cell lines were cultured in RPMI-1640 medium (A4192301, Gibco, Grand Island, New York, USA) with 10% fetal bovine serum (FBS; no. 12483020, Gibco, Grand Island, New York, USA), and 1% penicillin–streptomycin (no. 15070063, Gibco, Grand Island, New York, USA). The THP-1 cell line was cultivated in RPMI-1640 medium containing 10% heat-inactivated FBS, 10 mM Hepes (no. 15630-056, Gibco, Grand Island, New York, USA), 1 mM pyruvate (#11360-039, Gibco, Grand Island, New York, USA), 2.5 g/L D-glucose, and 50 pM β-mercaptoethanol (no. 31350-010, Gibco, Grand Island, New York, USA). Cells were cultured in a humid incubator with 5% CO2 at 37 °C.

Macrophage polarization model

The macrophage polarization model was constructed as previously described [11]. THP-1 cells were differentiated into M0 macrophages by 24 h incubation with 150 nM phorbol 12-myristate 13-acetate (PMA; P8139, Sigma-Aldrich, St. Louis, Missouri, USA) followed by 24 h incubation in RPMI medium. M0 macrophages were polarized in M1 macrophages by incubation with 20 ng/ml of IFN-γ (no. 285-IF, R&D system, Minneapolis, Minnesota, USA) and 10 pg/ml of lipopolysaccharide (LPS; no. 8630, Sigma-Aldrich, St. Louis, Missouri, USA).

Cell transfection

For the overexpression of circFUT8, METTL14, and CHMP4B, the whole sequences were separately synthesized and subcloned into pcDNA3.1 vectors with a pcDNA3.1 empty vector (V79520, Invitrogen, Carlsbad, CA, USA) as a negative control (NC), while for the overexpression of miR-552-3p and miR-628-5p, related miRNA mimics and NCs were used. For the knockdown of circFUT8, METTL14, and CHMP4B, specific short hairpin RNAs (shRNAs) were respectively designed and established with nontargeting shRNA (sh-NC), while miR-552-3p/miR-628-5p inhibitors were used to silence miR-552-3p/miR-628-5p expression. In line with the supplier’s protocols, transfections were conducted with Lipofectamine 2000 (Invitrogen).

Quantitative real-time RT–PCR (RT–qPCR)

Based on the user’ guidance for Trizol reagent (15,596,018, Invitrogen, Carlsbad, CA, USA), total RNA was extracted from HCC cells and then reverse transcribed into cDNA using SuperScript III First-Strand Synthesis SuperMix (11,752,050, Invitrogen, Carlsbad, CA, USA). The RT–qPCR reaction was achieved with the SYBR Green PCR Master Mix (4,364,346, Applied Biosystems, Foster City, CA, USA) and gene expression was calculated by the 2−ΔΔCt method. In relevant assays, U6 and GAPDH served as the endogenous controls.

Colony formation assay

Huh7 and MHCC97H cells were seeded into 6-well plates for 2 weeks of incubation. The culture medium was discarded and cells were washed with phosphate-buffered saline (PBS) twice. Methanol solution was applied for cell fixation for 15 min and crystal violet was utilized for cell staining for 10 min at room temperature. Finally, colonies with no less than 50 cells were manually counted.

5-Ethynyl-20-deoxyuridine (EdU) incorporation assay

Using BeyoClick EdU-488 kit (C0071S, Beyotime, Shanghai, China), HCC cells at the logarithmic growth stage were taken and seeded in 96-well plates to the normal growth stage. An appropriate amount of 50 μM EdU culture medium was prepared by dilution of EdU solution by 1:1000. Each well was added with 100 μL 50 μM EdU assay kit for cultivation for 2 h, and then the culture medium was discarded. PBS was used to wash the cells for 5 min, and 4′,6-diamidino-2-phenylindole (DAPI) was added to stain the nucleus for 5 min at room temperature. Finally, cell proliferation was monitored under fluorescence microscopy.

Western blot

Total protein extracted from HCC cell lines was isolated by Radio Immunoprecipitation Assay (RIPA) buffer, and after being separated through Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins were transferred to polyvinylidene difluoride (PVDF) membranes and blocked in 5% skim milk. The membranes were cultivated with primary antibodies against CD63 (ab134045, Abcam, Cambridge, MA, USA) and CD81 (ab109201, Abcam, Cambridge, MA, USA) overnight at 4 °C, followed by cultivation with secondary antibody for 1 h. After washing in TBS with Tween-20 (TBST), the secondary antibodies were added, finally assayed by enhanced chemiluminescence (ECL) Substrate. In related assays, β-actin (ab8226, Abcam, Cambridge, MA, USA) was used as the internal control.

Transwell assays

HCC cells were planted on the top of 24-well Matrigel-coated transwell chambers at a density of 2 × 104 cells per well. The lower chambers were loaded with complete medium. Twenty-four hours later, cells in the upper layer were removed and then fixed in methanol solution for 15 min. Crystal violet was adopted to stain the membranes for 10 min, and the invaded cells were observed and counted under a microscope (10 × 10).

RNA immunoprecipitation (RIP) assay

With the Imprint RNA Immunoprecipitation Kit, RIP in Huh7 or MHCC97H cells was achieved with specific antibodies and normal control anti-IgG antibody (no. 3420, cell signaling, Boston, Massachusetts, USA). Lysates were obtained from HCC cell lines using RIP lysis buffer. The lysis was incubated with the magnetic beads conjugated with the AGO2 antibody (no. 2897, cell signaling, Boston, Massachusetts, USA) or IgG antibody (negative control). The precipitated RNAs went through RT–qPCR analysis.

Dual luciferase reporter assay

For gene promoter analysis, CHMP4B promoter was subcloned into the pGL3-basic vector (E1751, Promega, Madison, WI, USA) and co-transfected with M1-Exo into Huh7 and MHCC97H cells. In addition, circFUT8-WT/Mut, CHMP4B-WT/Mut, and METTL14-WT/Mut vectors were respectively subcloned into the pmirGLO luciferase reporter vector (E1330, Promega, Madison, WI, USA) and then transfected with different plasmids in HCC cells. The Dual Luciferase Reporter Assay System (E1910, Promega, Madison, WI, USA) was used for the analysis.

Flow cytometry

After 48 h transfection, HCC cells were collected and washed with PBS. They were double stained in a darkroom for 15 min, and then subjected to staining using the eBioscience Annexin V-FITC Apoptosis Detection Kit (85-BMS500FI-300, Invitrogen, Carlsbad, CA, USA). Finally, the apoptosis rate was analyzed through flow cytometry (BD Biosciences, Franklin Lake, New Jersey, USA).

Subcellular fractionation detection

The PARIS kit (AM1921, Invitrogen, Carlsbad, CA, USA) was used for this assay, following the manufacturer’s instructions.The expression pattern of circFUT8, U3 (nucleus control), or ACT8 (cytoplasmic control) in two fractions was assessed by RT–qPCR. In related assays treated with M1-Exo, the percent of circFUT8 in the control or Exo group was determined by RT–qPCR.

RNA pull down assay

The biotin-labeled circFUT8 probe with Bio-NC was obtained from RiboBio (Guangzhou, Guangdong, China) for the RNA pull down assay. Cells were lysed with lysis buffer, and then the lysates were incubated with specific biotin-labeled probes for 2 h. Then, the mixtures were incubated with the streptavidin beads to pull down the biotin-labeled RNA complex for another 4 h. After washing, the RNA complex was extracted with TRIzol and RNA enrichment was achieved through RT–qPCR.

In vivo tumorigenesis experiment

Related in vivo studies were performed as previously described, with the approval of the Animal Care and Use Committee guidelines of our hospital (Date of the Ethic Committee decision: 25 January 2022) [20]. Male BALb/c nude mice (three mice per group) were bought from Guangdong Medical Laboratory Animal Center, and indicated HCC cells were subcutaneously injected into the flanks of nude mice at 2 × 106 cells per site. Four weeks after injection, the mice were sacrificed, and tumors were removed to be weighed and measured.

Statistical analysis

Each experiment was performed in triplicate. Data was exhibited as the mean ± standard deviation (SD). Statistical analyses were made in the form of Student’s t test (comparison for two groups) and one-way/two-way ANOVA (comparison for more than two groups). **P-values less than 0.01 (P < 0.01), representing the data that exhibited a significant statistical difference in the experimental group in comparison with the control.

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