Modulation of miR-146b by N6-methyladenosine modification remodels tumor-associated macrophages and enhances anti-PD-1 therapy in colorectal cancer

Purpose MicroRNA-146b (miR-146b) alleviates experimental colitis in mice by mediating macrophage polarization and the release of inflammatory factors. Our goals were to evaluate the antitumor efficacy of miR-146b in colorectal cancer (CRC) and to investigate the underlying mechanisms. Methods We used murine models of CRC to evaluate whether miR-146b influenced the progression of tumors independent of tumor-associated macrophages (TAMs). RNA immunoprecipitation, N6-methyladenosine (m6A) RNA immunoprecipitation and in vitro pri-miRNA processing assays were conducted to examine whether m6A mediates the maturation of pri-miR-146b/miR-146b. In a series of in vitro and in vivo experiments, we further defined the molecular mechanisms of methyltransferase-like 3 (METTL3)/miR-146b-mediated antitumor immunity and its efficacy in combination with anti-PD-1 immunotherapy. Results We found that miR-146b deletion supported tumor progression by increasing the number of alternatively activated (M2) TAMs. Mechanistically, the m6A-related “writer” protein METTL3 and “reader” protein HNRNPA2B1 controlled miR-146b maturation by regulating the m6A modification region of pri-miR-146b. Furthermore, miR-146b deletion promoted the polarization of M2-TAMs by enhancing phosphoinositide 3-kinase (PI3K)/AKT signaling, and this effect was mediated by the class IA PI3K catalytic subunit p110β, which reduced T cell infiltration, aggravated immunosuppression and ultimately promoted tumor progression. METTL3 knockdown or miR-146b deletion induced programmed death ligand 1 (PD-L1) production via the p110β/PI3K/AKT pathway in TAMs and consequently augmented the antitumor activity of anti-PD-1 immunotherapy. Conclusions The maturation of pri-miR-146b is m6A-dependent, and miR-146b deletion-mediated TAM differentiation promotes the development of CRC by activating the PI3K/AKT pathway, which induces upregulation of PD-L1 expression, inhibits T cell infiltration into the TME and enhances the antitumor activity of anti-PD-1 immunotherapy. The findings reveal that targeting miR-146b can serve as an adjuvant to anti-PD-1 immunotherapy. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s13402-023-00839-0.


Background
Colorectal cancer (CRC) is one of the most lethal human malignancies in the world [1].In recent years, immunotherapy has rapidly developed and benefits some cancer patients by affecting immune checkpoints, including the cytotoxic T lymphocyte associated protein 4 (CTLA-4) or the programmed cell death 1 (PD-1) signaling pathway [2,3], which is a promising approach to activate antitumor immunity and can improve T cell function and reduce tumor burden by strengthening cytotoxic CD8 + T cell (CTL) infiltration to slow tumor progression and prolong patient survival.However, the majority of CRC patients with mismatch-repair-proficiency and microsatellite instability-low (pMMR-MSI-L) tumors are unresponsive to treatment [4,5].Thus, the mechanism of immunotherapy resistance must be elucidated, and novel markers to predict immunotherapy resistance need to be identified.
Tumor-associated macrophages (TAMs) are critical drivers of the immunosuppressive microenvironment and are recruited to tumors by colony stimulating factor (CSF), transforming growth factor (TGF-β1) and chemokines, such as CCL2 (MCP-1) [6], and these cells mostly exhibit an alternatively activated macrophage (M2)-like phenotype [7].M2-TAMs can directly stimulate tumor cell proliferation and invasion and suppress T cell recruitment and activation through the production of cytokines, such as interleukin (IL)-10 or epidermal growth factor (EGF) [8].Hence, modifying the properties and function of TAMs in tumors can enhance antitumor immune responses.In addition, resistance to immunotherapies can be mediated by abundant infiltration of tumor-associated myeloid cells, including macrophages [9].However, accumulating evidence suggests that programmed death ligand 1 (PD-L1) + TAMs in tumors could better predict a patient's response to immunotherapy than PD-L1 − TAMs in non-small-cell lung cancer patients [10,11].These data suggested that PD-L1 + TAMs were related to anti-PD-1/PD-L1 therapy resistance in the tumor microenvironment (TME).A better understanding of the mechanism by which PD-L1 regulates immune checkpoints in TAMs is necessary [12,13].
MicroRNAs (miRNAs) widely participate in macrophage polarization in the TME, and their aberrant expression could affect the balance of macrophage polarization [14].Evidence from our laboratory has demonstrated that microRNA-146b (miR-146b) inhibits tumor progression in a colitis-associated cancer (CAC) mouse model by targeting TAMs with miR-146b mimic-containing nanoparticles [13], but the mechanism remains unclear.In the current study, we demonstrated that N6-methyladenosine (m 6 A) is a key regulator that modifies the transcription of pri-miR-146b to mature miR-146b in macrophages.MiR-146b deficiency increased the number of M2-TAMs accompanied by the upregulation of PD-L1 expression through PI3K/AKT pathway activation, which inhibited T cell infiltration in the TME and resulted in immune suppression and tumor development.Importantly, the combination of miR-146b depletion and a PD-1/PD-L1 checkpoint inhibitor was shown to enhance the antitumor activity of effector immune cells.In summary, our findings revealed the mechanism by which miR-146b functions in colorectal cancer to promote PD-1 blockade, thereby providing potential new biomarkers and a therapeutic avenue for tumors refractory to ICI treatment.

Mouse husbandry
WT C57BL/6 mice (6-8 weeks old) were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China), and miR-146b deficient (miR-146b −/− ) mice on a C57BL/6 background were housed in the Guangzhou Medical University Animal Experiment Center (Guangzhou, China).All animal protocols were approved by the Animal Ethics Committee of Guangzhou Medical University.The animals were maintained under specific pathogen-free conditions in the Guangzhou Medical University Animal Experiment Center (Guangzhou, China).

Tumor studies
Six-to eight-week-old female or male WT and miR-146b −/− mice were implanted with 5 × 10 5 MC38 cells in 100 μl of phosphate-buffered saline (PBS) by subcutaneous injection in the right flank, and tumor growth was monitored for up to 21 days.Tumor volumes were determined by measuring the length (l) and width (w) and were calculated as follows: tumor volume = (l 2 x w)/2.All in vivo monoclonal antibodies (mAbs) were from BioX-Cell (New Hampshire, USA).Animals were euthanized before the maximum IACUC allowable tumor burden of 2 cm 3 /mouse was exceeded [12].Tumor dimensions were measured 2 times per week beginning on day 7.
For miR-146b agomir administration, 6-to 8-weekold miR-146b −/− mice were implanted with 5 × 10 5 MC38 cells, and after 1 week, the mice were intraperitoneally administered 10 mg/kg miR-146b agomir twice per week, while the control group was administered the same dose of NC for 2 weeks.
In vivo macrophage adoptive transfer experiments were performed.Primary bone marrow-derived macrophages (BMDMs) were polarized to the M2 phenotype as described in the Supplemental Material.M2 cells were mixed 1:1 with MC38 tumor cells, and a total of 1 × 10 6 cells were injected subcutaneously.Primary BMDMs were polarized to the M2 phenotype, and macrophage-conditioned medium (CM) was harvested, centrifuged at 800 × g for 10 min and filtered through a 0.22-μm syringe filter (Millipore, Billerica, MA, USA).Then, CM mixed with 5 × 10 5 MC38 tumor cells was injected subcutaneously, and inoculated mice were further treated by intradermal injection with conditioned medium at 3 and 6 days post inoculation.
For anti-PD-1 mAb treatment, 5 × 10 5 MC38 cells were subcutaneously injected into the flanks of the mice.Tumors were allowed to grow for seven days, and then the mice were treated i.p. with 200 μg of anti-PD-1 mAb (Clone J110) or rat IgG2a isotype control every 3 days for 2 weeks.In the colitis-associated cancer model, mice were intraperitoneally injected with 10 mg/kg azoxymethane (AOM, Sigma-Aldrich Corp, St Louis, MO, USA).One week later, the mice were administered 2% dextran sodium sulphate (DSS, MW 40000-50,000) (MP Biomedicals, Solon, Ohio, USA) in distilled water for 5 days, followed by 14 days of normal drinking water [13].This cycle was repeated three times.After 5 weeks, the mice were administered 200 μg of anti-PD-1 mAbs or Rat IgG2a isotype control every 3 days for the indicated time.The mice were sacrificed after the third cycle ended.

Deletion of miR-146b in macrophages impairs T cell infiltration and promotes the immunosuppressive TME
To understand how the TME was changed by miR-146b depletion, we performed RNA-seq and compared WT and miR-146b −/− tumor-bearing mice.Transcriptome analyses of whole tumor tissue mRNA expression showed 111 upregulated and 138 downregulated genes in miR-146b −/− mice compared with WT mice (Supplementary Fig. 2).As expected, Gene Ontology (GO) enrichment analysis indicated that a portion of the genes were associated with T cell activation and stimulation (Fig. 3A).The downregulated genes were enriched for molecules involved in "Innate immunity, T cell activation and stimulation and antigen presentation", and the upregulated genes were enriched for molecules involved in "tumor progression" (Fig. 3B).These results were validated by qPCR on a second set of samples, including Stat4, Icos, Gzmk, Il18r, Cd3e, Cxcr3 and Card11 (Fig. 3C).Using flow cytometry (FACS), we further confirmed that the depletion of miR-146b decreased the infiltration of CD3 + T, CD4 + T and CD8 + T cells in tumors (Fig. 3D and Supplementary Fig. 1E), although Treg and CTLs did not significantly change (Fig. 3E-F).
Since miR-146b depletion inhibited proinflammatory responses in macrophages, we next examined whether macrophage miR-146b depletion slowed adaptive immunity and promoted tumor progression.BMDMs from WT and miR-146b −/− mice were stimulated with the classic M2-polarizing cytokine IL-4 plus IL-10, mixed with MC38 cells and adoptively transferred into new WT recipient mice.Tumor growth was significantly promoted in the presence of M2 macrophages, and miR-146b −/− M2 macrophages were significantly more likely to promote tumor progression than WT M2 macrophages (Fig. 3G).T cells were significantly decreased in tumors treated with miR-146b −/− M2 cells compared with tumors treated with WT M2 macrophages (Supplementary Fig. 3A), confirming that miR-146b depletion in macrophages inhibited T cell recruitment to tumors.Moreover, the levels of proinflammatory factors in tumors treated with miR-146b −/− M2 cells were significantly decreased, and anti-inflammatory factors were increased (Supplementary Fig. 3B).To determine whether macrophage-derived cytokines control tumor growth, we implanted MC38 cells mixed with CM derived from in vitro cultured macrophages into new WT mice.Tumor growth was enhanced by IL-4 plus IL-10-stimulated macrophage CM, and tumor growth was more obvious in cells mixed with CM derived from miR-146b −/− macrophages (Fig. 3H).These results indicated that miR-146b deletion in macrophages impaired T cell infiltration in CRC tissues.

miR-146b targets p110β and inhibits the PI3K/ AKT signaling pathway in the regulation of M2 macrophage polarization
We used two publicly available algorithms to identify potential miR-146b target genes, and bioinformatics analysis showed that murine p110β was one of the predicted targets of miR-146b (data not shown), which is an isoform of PI3K that cooperates with p110γ to induce PI3K/AKT signaling activation [15].We thus constructed reporter genes containing the p110β 3′ untranslated region (UTR) (Supplementary Fig. 4).We found that the miR-146b mimic significantly decreased the luciferase activity in 293 T cells transfected with the WT p110β plasmid; however, the miR-146b mimic had no effect on the mutant p110β plasmid (Fig. 4A).Further experiments showed that p110β protein levels were markedly increased in miR-146b −/− M2 macrophages compared with WT M2 macrophages but dramatically decreased in cells with the miR-146b mimic (Fig. 4B).To confirm the effect of miR-146b on p110β during M2 polarization, miR-146b −/− BMDMs were transfected with si-p110β or treated with a p110β inhibitor (TGX221) [16].Knocking down or inhibiting p110β reduced Arg-1 and Ym-1 mRNA levels and cytokine secretion in miR-146b −/− macrophages (Fig. 4C-D and Supplementary Fig. 5A).These results indicate that p110β is involved in the M2 differentiation of miR-146b −/− macrophages.AKT signaling is regulated by p110β [15], and the PI3K/ AKT signaling pathway can control the switch between immune stimulation and suppression in cancer by the C/ CATT enhancer binding protein (C/EBPβ) [8].We examined whether miR-146b affects PI3K/AKT signaling and C/ EBPβ phosphorylation and found that miR-146b deletion increased AKT and C/EBPβ phosphorylation (Fig. 4E).This result was confirmed by C/EBPβ transcription factor assay kits, the data showed that miR-146b ablation enhanced the DNA binding activity of C/EBPβ (Fig. 4F).C/EBPβ induces immune suppression by controlling Arg-1 expression [12,17].We found that Arg-1 activity was enhanced in miR-146b −/− M2 macrophages (Fig. 4G).These results suggest that miR-146b is an upstream inhibitor of AKT signaling and C/EBPβ activation.We further found that a PI3K inhibitor (LY294002) and an AKT inhibitor (GSK2141795) could substantially abolish the effects of miR-146b deletion on the anti-inflammatory effects of M2 macrophages (Fig. 4H-I).In addition, miR-146b −/− M2 macrophages treated with si-Cebpβ substantially abrogated the miR-146b deletion induced anti-inflammatory effect (Fig. 4J-K and Supplementary Fig. 5B).
To confirm that miR-146b controls tumor growth through the p110β-dependent PI3K/AKT pathway, we treated miR-146b −/− macrophages ex vivo with inhibitors of p110β, PI3K and AKT prior to mixing the macrophages with tumor cells and implanting them in mice.Blockade of the p110β/PI3K/AKT pathway in miR-146b −/− macrophages suppressed tumor growth (Fig. 4L).p110β/PI3K/ AKT inhibition stimulated T cell recruitment (Supplementary Fig. 6A), enhanced the expression of proinflammatory factors and inhibited the expression of immunosuppressive factors (Supplementary Fig. 6B).These results indicate that miR-146b inhibits tumor growth through p110β/PI3K/ AKT-mediated immune activation and that p110β/PI3K/ AKT inhibition reverses these effects by polarizing macrophages toward a proinflammatory phenotype.

METTL3-dependent m 6 A methylation regulates the maturation of pri-miR-146b in macrophages
Recent studies have reported that m 6 A modification is involved in the life cycle of RNA and in primary micro-RNA (pri-miRNA) processing [20].However, the potential roles of m 6 A modification in miR-146b transcription and maturation remain elusive.To study the effects of m 6 A modification on the processing of pri-miRNA-146b to mature miR-146b in the context of macrophage differentiation, we first examined whether m 6 A levels varied with macrophage differentiation under polarized conditions.The m 6 A RNA methylation level was analyzed in BMDMs under resting macrophage (M0), M1 and M2 type macrophage conditions.The results showed that M2-type stimulation inhibited the m 6 A modification compared to that in the M0 and M1 states (Fig. 6A).The m 6 A-related gene Mettl3 was notably decreased under M2 type stimulation, but levels of the methyltransferase genes Mettl14 and Wtap as well as the demethylase genes Alkbh5 and Fto did not change significantly (Fig. 6B).To investigate the biological relevance of the m 6 A modification in macrophage differentiation, we used MO-Mettl3 [21], which decreased METTL3 protein levels (Supplementary Fig. 7A).We found that pri-miR-146b expression was significantly increased, but mature miR-146b expression was downregulated by MO-Mettl3 compared to that in the control group (Fig. 6C).The m 6 A "reader" protein HNRNPA2B1 binds to m 6 A in pri-miRNA transcripts to promote primary miRNA processing by interacting with the microRNA microprocessor complex protein DGCR8 [22].HNRN-PA2B1 knockdown caused pri-miR-146b upregulation and a decline in mature miR-146b (Supplementary Fig. 7B Fig. 2  and Fig. 6D).Using a coimmunoprecipitation assay, we also confirmed that the METTL3 interaction with DGCR8 was mediated by RNA [23], given that RNase treatment disrupted this association (Fig. 6E).In addition, HNRN-PA2B1 interacted with DGCR8 through protein-protein interactions (Fig. 6F) [22].
To demonstrate the direct role of the m 6 A modification in pri-miR-146b in mature miR-146b processing, m 6 A-specific RNA immunoprecipitation (RIP) coupled with qRT-PCR was used, and the results showed that pri-miR-146b was significantly enriched by the anti-m 6 A antibody (Fig. 6G).Subsequent RIP-qPCR analysis showed that the METTL3, HNRNPA2B1 and DGCR8 proteins interacted with pri-miR-146b (Fig. 6H).Furthermore, we found that the deceased level of pri-miR-146b was immunoprecipitated by anti-m 6 A in METTL3-knockdown cells and was immunoprecipitated by anti-DGCR8 and anti-METTL3 antibodies (Fig. 6I-J).In addition to METTL3 silencing, HNRNPA2B1 knockdown substantially reduced the interaction of HNRNPA2B1 and DGCR8 with pri-miR-146b (Fig. 6K).These data suggested that METTL3, DGCR8 and HNRNPA2B1 could disturb the interaction between m 6 A and pri-miR-146b.To directly confirm that the processing of pri-miR-146b to mature miR-146b was mediated by m 6 A modification, we performed an in vitro pri-miRNA processing assay to examine this relationship.We observed that [m 6 A] pri-miR-146b was more rapidly and efficiently processed to pre-miR-146b followed by mature miR-146b than its unmethylated counterpart (Fig. 6L, Supplementary Fig. 8).However, when the potential m 6 A motif in pri-miR-146b was mutated, the processing of pri-miR-146b to mature miR-146b was inhibited (Fig. 6M).Taken together, these results suggest that METTL3 and HNRNPA2B1 promote the processing of pri-miR-146b to mature miR-146b in an m 6 A-dependent manner.

METTL3 modulates TAMs via the miR-146b/ p110β/PI3K/AKT axis
m 6 A deposition influences CRC growth [24,25], and the protumoral function of TAMs is promoted by reduced m 6 A levels [26].To determine METTL3 function in M2 macrophage differentiation, we assessed macrophage development after METTL3 knockdown.As expected, the M2-TAMs markers Arg-1 and Ym-1 were significantly increased in MO-Mettl3-treated macrophages compared to MO-nc cells (Fig. 7A).ELISA analysis showed that the secretion of IL-10 and TGF-β1 was significantly increased in MO-Mettl3-treated macrophages (Fig. 7B).To further confirm that macrophage METTL3 controls tumor growth, BMDMs derived from WT mice were transfected with MO-nc or MO-mettl3, mixed with MC38 cells, and then adoptively transferred into new WT recipient mice.Knocking down Mettl3 in macrophages promoted MC38 tumor growth (Fig. 7C-D).Consistently, impaired T cell infiltration into MC38 cell-derived tumor tissues occurred when the cells were mixed with MO-Mettl3 macrophages compared with cells mixed with MO-nc (Supplementary Fig. 9A).Accordingly, TGF-β1, IL-10 and CCL2 protein levels were significantly increased, and TNFα, IL-6, IL-1β and IL-12p40 protein levels were significantly decreased in tumor tissues in mice with MO-Mettl3 macrophages compared with mice with MO-nc macrophages (Supplementary Fig. 9B).

Loss of METTL3 sensitizes CRC to anti-PD-1 treatment
We next examined the role of METTL3 in PD-L1 expression in M2 macrophages.In addition to the effect of miR-146b, PD-L1 protein expression was significantly elevated in the METTL3-knockdown M2 macrophages (Fig. 7K) but reduced in the METTL3-knockdown M2 macrophages upon treatment with miR-146b mimic (Fig. 7L).In addition, PD-L1 protein levels were obviously decreased in the METTL3-knockdown M2 macrophages upon treatment with GSK2141795, LY294002 or TGX221 (Fig. 7M).

Discussion
In this study, we demonstrate that the METTL3/miR-146b axis reprograms macrophages to exert protumor effects.METTL3 knockdown or miR-146b ablation alone significantly increases the TAM population, reduces T cell infiltration, and promotes tumor progression.However, METTL3 or miR-146b deficiency increases PD-L1 expression in TAMs, and the therapeutic effect of anti-PD1 mAb is markedly improved and leads to the regression of even well-established CRC tumors.Mechanistically, we demonstrated that miR-146b deficiency promoted the polarization of TAMs by inducing oncogenic PI3K-AKT signaling; specifically, miR-146b expression is decreased in TAMs, which significantly increases p110β expression and promotes PI3K/ AKT signaling activation.Moreover, we found that the m 6 A modification was the main factor associated with miR-146b expression and determined that knockdown of the m6A "writer" protein METTL3 and the "reader" protein HNRN-PA2B1 significantly decreased miR-146b expression and promoted M2 macrophage polarization.These data suggest that the METTL3/miR-146b axis may represent an effective therapeutic target to reprogram TAMs, improve the immunosuppressive microenvironment of CRC tumors, and increase the efficacy of immunotherapy.
Many innate and adaptive immune cells are recruited to the TME, and macrophages are the most abundant immune cells [27].Macrophages are the first line of defense against pathogenic insults, suggesting that these cells can be tumoricidal after being activated; however, cancer-associated macrophages are usually polarized to a protumoral M2 phenotype at the time of tumor initiation, and these cells are named TAMs [28].Therefore, the polarization of TAMs can indirectly stimulate CTL activation and synergize with checkpoint inhibitors, suggesting improved cancer outcomes [8].MiRNAs have also been identified as important determinants of the protumoral activity of TAMs.In our study, we observed that miR-146b participated in the initiation and progression of CAC, which significantly reduced tumor numbers and tumor areas [13].Accordingly, Arg-1 expressing macrophages were sharply reduced [13].miR-146b −/− mice had significantly increased M2-TAM numbers, reduced T cell recruitment, and promoted immune suppression and tumor progression.Our in vitro study further confirmed that the miR-146b mimic could inhibit M2 macrophage polarization under M2 conditions.p110γ is the most highly expressed PI3K isoform in myeloid cells, and PI3K/AKT is involved in the macrophage phenotype transitions in the TME [8,12].We found that miR-146b ablation promoted PI3K/AKT signaling pathway activation, exacerbated the upregulation of the PI3K/AKT signaling pathwayassociated target C/EBPβ in M2 macrophages and modulated Arg-1 expression by binding to the response element of Arg-1.These results suggest that miR-146b repressed the polarization of TAMs by targeting the PI3K/AKT signaling pathway.In our study, miR-146b deficient BMDMs promoted p110β expression but not p110γ expression.Intriguingly, both p110β and p110γ contribute to AKT activation [15].Taken together, these results suggest that miR-146b can regulate the polarization of TAMs through PI3K/AKT signaling and that these activities may be highly dependent on p110β expression.
TAMs can suppress T cell recruitment and activation, thereby exacerbating immunosuppression [8].We found that miR-146b deficiency reduced the T cells recruited to tumors.In addition, PD-L1 expression can be mediated by PI3K/AKT signaling [18].These effects indicate that miR-146b may affect T cell-based immunotherapy.David L. Rimm et al. recently showed that macrophages expressing PD-L1 can enhance anti-PD-1 mAb therapy [10].We assessed the percentage of PD-L1 in TAMs and showed that PD-L1 expression in TAMs was significantly increased in macrophage bearing tumors.Furthermore, we found that high expression of PD-L1 in miR-146b −/− macrophages Fig. 6 m 6 A modification mediates pri-miR-146b maturation in M2 macrophages.(A-B) BMDMs from WT mice were stimulated with IL-4 plus IL-10 (M2) or LPS plus IFNγ (M1), and the m 6 A levels of mRNAs were assessed using the EpiQuik m6A RNA Methylation Quantification Kit.Mettl3, Mettl14, Wtap, Alkbh5 and Fto mRNA levels were determined by qPCR.(C-D) BMDMs from WT mice were transfected with MO-Mettl3 or si-hnrnpa2b1 for 24 h and then stimulated with IL-4 plus IL-10 for 4 h.The mRNA levels of pri-miR-146b and miR-146b were determined by qPCR.(E) RAW264.7 cells were treated with IL-4 (20 ng/ml) plus IL-10 (10 ng/ml) for 24 h to generate M2 macrophages.Coimmunoprecipitation of the METTL3-interacting protein DGCR8 was performed.After immunoprecipitation, the samples were washed and incubated with RNase as indicated.Western blotting was used to detect METTL3 and DGCR8, and IgG served as a control for immunoprecipitation. (F) RAW264.7 cells were treated with IL-4 plus IL-10 for 24 h.Coimmunoprecipitation of the HNRNPA2B1-interacting protein DGCR8 was performed.After immunoprecipitation, the samples were washed and incubated with RNase as indicated.Western blotting was used to detect HNRNPA2B1 and DGCR8.(G) RAW264.7 cells were treated with IL-4 plus IL-10 for 24 h.Coimmunoprecipitation was performed with an anti-m 6 A antibody.After immunoprecipitation, the samples were washed, and pri-miR146b expression was determined by qRT-PCR.(H) RAW264.7 cells were treated with IL-4 plus IL-10 for 24 h.Western blot analysis was used to detect METTL3, HNRNPA2B1 and DGCR8 expression in the input or immunoprecipitation group, and RIP assays showed significant enrichment of pri-miR146b in RNA binding to METTL3, HNRNPA2B1 and DGCR8.(I) RAW264.7 cells were transfected with MO-Mettl3 for 24 h and then stimulated with IL-4 plus IL-10 for 4 h.Immunoprecipitation of m 6 A-modified RNA in control or METTL3-knockdown cells was followed by qRT-PCR to assess pri-miR146b m 6 A modification levels.(J) Western blot analysis was used to detect METTL3 expression in the input and immunoprecipitation group, and RIP assays showed pri-miR146b binding to METTL3 or DGCR8.(K) RAW264.7 cells were transfected with si-hnrnpa2b1 for 24 h and then stimulated with IL-4 plus IL-10 for 4 h.Western blot analysis was used to detect HNRN-PA2B1 expression in the input and immunoprecipitation group, and RIP assays showed pri-miR146b binding to HNRNPA2B1 or DGCR8.(L) In vitro reaction system containing the starting materials of pri-miR-146 or [m 6 A]pri-miR-146 and the whole cellular lysates of 293 T cells transfected with plasmids carrying DROSHA and DGCR8.pri-miR-146b, pre-miR-146b and miR-146b mRNA levels were determined by qPCR.Pri-miR-1-1, which has no methylation site, was included as a control.(M) In vitro reaction system containing the starting materials of [m 6 A]pri-miR-146 WT or [m 6 A] pri-miR-146 Mut.pri-miR-146b, pre-miR-146b and miR-146b mRNA levels were determined by qPCR.All experiments were repeated three times independently.The data represent the mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant ◂ indicated a better immunotherapeutic effect.Given that these outcomes occurred in miR-146b −/− mice, we hypothesize that miR-146b deficiency-mediated polarization of TAMs promotes tumor progression, whereas increased PD-L1 expression potentially drives a response to anti-PD1 immunotherapy.
RNA m 6 A modification is a common epigenetic regulation that is involved in a variety of cellular processes [29,30].The decrease in the m 6 A "reader" METTL14 in TAMs determines T cell phenotypes in tumors [26].In our study, m 6 A modification was decreased in M2 macrophages by the m 6 A methyltransferase METTL3, and we found that m 6 A methylation mediated the maturation process of pri-miR-146b.Knockdown of both METTL3 and the m 6 A "reader" HNRN-PA2B1 impaired the maturation process of pri-miR-146b and led to a decrease in mature miR-146b expression.Furthermore, knockdown of both METTL3 and HNRNPA2B1 induced M2 macrophage polarization.When miR-146b mimics were transfected into METTL3-knockdown cells, the miR-146b mimic rescued the M2 macrophage polarization induced by METTL3.An in vivo study further confirmed that knockdown of METTL3 could decrease miR-146b expression, leading to an increase in TAMs and ultimately promoting tumor progression.More interestingly, we showed that METTL3 knockdown increased PD-L1 expression via miR-146b in M2 macrophages, thereby activating the p110β/PI3K/AKT pathway and inducing an immunosuppressive status.More interestingly, METTL3 knockdown led to sensitization of macrophages to anti-PD-1 therapy.These results suggested that METTL3 promotes the maturation of pri-miR-146b in CRC TAMs in an m 6 A-dependent manner.Targeting the enzymatic activity of METTL3 may be an anti-CRC strategy.For example, STM2457, the first bioavailable inhibitor of METTL3, may be useful, though future research is needed.In addition, we only quantified the m 6 A level in M2 macrophages using a colorimetric method, which was not quite accurate.Further might quantify m 6 A levels by mass spectrometry, and these results need to be confirmed in TAMs from human CRC tissues.
In conclusion, we have identified the role of the m 6 A-miR-146b/p110β/PI3K/AKT axis in the polarization of TAMs in CRC tissue, which might be associated with CRC development and progression.Our results also suggest that m 6 A modification may play an important role in CRC and other types of cancer linked with miR-146b decrease.
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Fig. 3
Fig. 3 miR-146b deletion promotes T-cell exhaustion.(A) GO analysis of differentially expressed genes between tumors from WT and miR-146b −/− mice.(B) Heatmap of differentially expressed immune response-related genes between tumors from WT and miR-146b −/− mice.(C) Relative mRNA expression of immune response-related genes between tumors from WT and miR-146b −/− mice.(D) Flow cytometric analysis and quantification of T-cell populations in tumors from WT and miR-146b −/− mice.(E-F) Flow cytometric analysis