Therapeutic targeting miR130b counteracts diffuse large B-cell lymphoma progression via OX40/OX40L-mediated interaction with Th17 cells

Abstract

MicroRNAs (miRNAs) are involved in lymphoma progression by regulating the tumor microenvironment. Serum miR130b is overexpressed in diffuse large B-cell lymphoma (DLBCL), inducing Th17 cell alterations. To further illustrate its biological significance and therapeutic rationale, miR130b was detected by quantitative real-time PCR in the serum samples of 532 newly diagnosed DLBCL patients. The mechanism of miR130b on lymphoma progression and the tumor microenvironment was investigated both in vitro and in vivo. Therapeutic targeting miR130b was also evaluated, including OX40 agonistic antibody and lipid nanoparticles (LNPs)-miR130b antagomir. The results showed that serum miR130b significantly correlated with tumor miR130b and serum interleukin-17, indicating lymphoma relapse and inferior survival of DLBCL patients. MiR130b overexpression altered tumor microenvironment signaling pathways and increased Th17 cell activity. As mechanism of action, miR130b downregulated tumor OX40L expression by directly targeting IFNAR1/p-STAT1 axis, recruiting Th17 cells via OX40/OX40L interaction, thereby promoting immunosuppressive function of Th17 cells. In co-culture systems of B-lymphoma cells with immune cells, miR130b inhibited lymphoma cell autophagy, which could be counteracted by OX40 agonistic antibody and LNPs-miR130b antagomir. In murine xenograft model established with subcutaneous injection of A20 cells, both OX40 agonistic antibody and LNPs-miR130b antagomir remarkably inhibited Th17 cells and retarded miR130b-overexpressing tumor growth. In conclusion, as an oncogenic biomarker of DLBCL, miR130b was related to lymphoma progression through modulating OX40/OX40L-mediated lymphoma cell interaction with Th17 cells, attributing to B-cell lymphoma sensitivity towards OX40 agonistic antibody. Targeting miR130b using LNPs-miR130b antagomir could also be a potential immunotherapeutic strategy in treating OX40-altered lymphoid malignancies.

Fig. 1

Elevated serum miR130b was significantly associated with lymphoma progression in DLBCL. a Real-time PCR analysis of miR130b expression in relapse patients (n = 148) and non-relapse patients (n = 384). b, c Progression-free survival (PFS) (b) and overall survival (OS) curves (c) in DLBCL patients according to miR130b expression. d Correlation between serum miR130b and tumor miR130b expression (n = 124) calculated by Pearson correlation coefficient analysis

Fig. 2

MiR130b affected signaling pathways involved in the tumor microenvironment of DLBCL. a Differentially expressed gene ontology (GO) terms according to miR130b expression. −Log (P value) of dysregulated pathways was indicated by color of points using RNA-sequencing. The size of points indicates the number of genes included in each gene set. b Distribution of immune subpopulations in miR130b high group (n = 64) and miR130b low group (n = 60) analyzed by transcriptomics data using TIP analysis. c Serum IL17 level of DLBCL patients measured by ELISA in miR130b high group (n = 64) and miR130b low group (n = 60). Data are summarized as mean ± SD. d Correlation between serum IL17 and tumor Th17 cell (n = 124) calculated by Pearson correlation coefficient analysis. e Differentially expressed immune checkpoints identified by RNA-sequencing between miR130b high group (n = 64) and miR130b low group (n = 60). f The immune activity scores of Th17 cells of DLBCL patients in OX40L low group (n = 62) and OX40L high group (n = 62). g Immunohistochemical study of Th17 cells in tumor samples of DLBCL patients in OX40L low group (n = 62) and OX40L high group (n = 62). h Pathway enrichment analysis in DLBCL patients according to OX40L expression using gene ontology database. i Heatmap diagram of the differentially expressed gene in type I interferon pathway between OX40L low group (n = 62) and OX40L high group (n = 62). Mean expression values are shown. Red, increased expression; blue, decreased expression; and white, mean value. j RNA-sequencing was used to determine gene–gene interaction network of type I interferon pathway. Genes that were more likely to be functionally related were presented as larger nodes. k Correlation between miR130b and IFNAR1 expression (n = 124) calculated by Pearson correlation coefficient analysis

Fig. 3

MiR130b modulated OX40/OX40L-mediated B-lymphoma cell interaction with Th17 cells via IFNAR1/p-STAT1 axis. a Potential binding sites of miR130b with the 3′-UTR of IFNAR1 (left panel) were predicted by bioinformatics analysis and the relative luciferase activities in HEK-293T cells transfected with IFNAR1^WT or IFNAR1^MUT were assessed. Data are summarized as mean ± SD (n = 3). b Flow cytometry analysis of OX40L expression on lymphoma cells in the miR130b-overexpressing DB co-culture system and miR130b-knockdown OCI-ly10 co-culture system. c Immunofluorescence assay of OX40 and OX40L in the IFNAR1-knockdown DB co-culture system and IFNAR1-overexpressing OCI-ly10 co-culture with Th17 cells. Representative immunofluorescent visions of OX40L [green]/OX40 [red] and nucleus counterstained with DAPI [blue]). The fluorescence intensity of OX40L, OX40, and OX40/OX40L interaction were quantified using the Image J software from five visions selected at random and subjected for statistical analysis. Data are summarized as mean ± SD (n = 5). d Flow cytometry analysis of OX40L expression on lymphoma cells and OX40 expression on Th17 cells in the IFNAR1-knockdown DB co-culture system and IFNAR1-overexpressing OCI-ly10 co-culture system. e Western blot analysis of p-STAT1 and STAT1 expression in IFNAR1-knockdown DB cells and IFNAR1-overexpressing OCI-ly10 cells. f Western blot analysis of p-STAT1 and STAT1 expression in IFNAR1^Δ486-511 DB cells and IFNAR1^Δ486-511 OCI-ly10 cells. g Flow cytometry analysis of OX40L expression on lymphoma cells and OX40 expression on Th17 cells in the STAT1-knockdown DB co-culture system and STAT1-overexpressing OCI-ly10 co-culture system. Data are summarized as mean ± SD (n = 3). h Flow cytometry analysis of OX40L expression on lymphoma cells in DB cells and OCI-ly10 cells upon treatment with p-STAT1 inhibitor. Data are summarized as mean ± SD (n = 3). i Potential binding sites of p-STAT1 with OX40L promotor region (−1130 bp to −1140 bp) predicted by bioinformatics analysis and the relative luciferase activities in HEK-293T cells transfected with OX40L^WT or OX40L^MUT. Data are summarized as mean ± SD (n = 3). j Flow cytometry analysis of OX40L expression in STAT1^Y701F DB cells and STAT1^Y701F OCI-ly10 cells. Data are summarized as mean ± SD (n = 3). k Th17 cell percentage and IL17 level were assessed in the OX40L-knockdown DB co-culture system and OX40L-overexpressing OCI-ly10 co-culture system. Th17 cell percentage (CD4+ RORγt+) was assessed by flow cytometry. IL17 level was measured by ELISA. Data are summarized as mean ± SD (n = 3)

Fig. 4

MiR130b induced Th17 cell accumulation and IL17 secretion. a Th17 cell percentage and IL17 level were assessed in the control mimics-transfected DB co-culture system and miR130b mimics-transfected DB co-culture system upon treatment with or without OX40 agonistic antibody. Th17 cell percentage (CD4+ RORγt+) was assessed by flow cytometry. IL17 level was measured by ELISA. Data are summarized as mean ± SD (n = 3). b Th17 cell percentage and IL17 secretion were assessed in the control inhibitor-transfected OCI-ly10 co-culture and miR130b inhibitor-transfected OCI-ly10 co-culture upon treatment with or without OX40 agonistic antibody. Th17 cell percentage (CD4+ RORγt+) was assessed by flow cytometry. IL17 level was measured by ELISA. Data are summarized as mean ± SD (n = 3). c Th17 cell percentage and IL17 level were assessed in the control mimics-transfected DB co-culture system and miR130b mimics-transfected DB co-culture system upon treatment with or without LNPs-miR130b antagomir. Th17 cell percentage (CD4+ RORγt+) was assessed by flow cytometry. IL17 level was measured by ELISA. Data are summarized as mean ± SD (n = 3). d Th17 cell percentage and IL17 level were assessed in the control inhibitor-transfected OCI-ly10 co-culture and miR130b inhibitor-transfected OCI-ly10 co-culture upon treatment with or without LNPs-miR130b antagomir. Th17 cell percentage (CD4+ RORγt+) was assessed by flow cytometry. IL17 level was measured by ELISA. Data are summarized as mean ± SD (n = 3)

Fig. 5

OX40 agonistic antibody modulated B-lymphoma cell autophagy through OX40/OX40L-mediated lymphoma cell interaction with Th17 cells both in vitro and in vivo. a Cell growth in the control mimics-transfected DB co-culture system and miR130b mimics-transfected DB co-culture system upon treatment with or without OX40 agonistic antibody. MTT assay was adopted to measure cell viability. Data are summarized as mean ± SD (n = 3). b Cell growth in the control inhibitor-transfected OCI-ly10 co-culture and miR130b inhibitor-transfected OCI-ly10 co-culture upon treatment with or without OX40 agonistic antibody. MTT assay was adopted to measure cell viability. Data are summarized as mean ± SD (n = 3). c Flow cytometry analysis of LC3B in the control mimics-transfected DB co-culture system and miR130b mimics-transfected DB co-culture system upon treatment with or without OX40 agonistic antibody. Data are summarized as mean ± SD (n = 3). d Western blot of LC3B and p62 in the control mimics-transfected DB co-culture system and miR130b mimics-transfected DB co-culture system upon treatment with or without OX40 agonistic antibody. e Flow cytometry analysis of LC3B in the control inhibitor-transfected OCI-ly10 co-culture and miR130b inhibitor-transfected OCI-ly10 co-culture upon treatment with or without OX40 agonistic antibody. Data are summarized as mean ± SD (n = 3). f Western blot of LC3B and p62 in the control inhibitor-transfected OCI-ly10 co-culture and miR130b inhibitor-transfected OCI-ly10 co-culture upon treatment with or without OX40 agonistic antibody. g Transmission electron microscope showed typical autophagosomes in the miR130b mimics-transfected DB co-culture system and miR130b inhibitor-transfected OCI-ly10 co-culture upon treatment with or without OX40 agonistic antibody. The cells were counted from five visions selected at random and subjected for statistical analysis. Data are summarized as mean ± SD (n = 5). h pGMLV-vector and pGMLV-miR130b cells were injected into nude mice subcutaneously with or without OX40 agonistic antibody treatment, and tumor volume was determined. Data are summarized as mean ± SD (n = 4). i Standardized uptake value intensity in pGMLV-vector group and pGMLV-miR130b group upon treatment with or without OX40 agonistic antibody were detected by micro PET-CT. j IL17 level was measured by ELISA in pGMLV-vector group and pGMLV-miR130b group upon treatment with or without OX40 agonistic antibody. Data are summarized as mean ± SD (n = 4). k Transmission electron microscope showed typical autophagosomes in pGMLV-vector group and pGMLV-miR130b group upon treatment with or without OX40 agonistic antibody. The cells were counted from five visions selected at random and subjected for statistical analysis. Data are summarized as mean ± SD (n = 5)

Fig. 6

LNPs-miR130b antagomir exhibited in vitro and in vivo activity on OX40-impaired B-cell lymphoma. a Cell growth in the control mimics-transfected DB co-culture system and miR130b mimics-transfected DB co-culture system upon treatment with LNPs-miR130b control or with LNPs-miR130b antagomir. MTT assay was adopted to measure cell viability. Data are summarized as mean ± SD (n = 3). b Cell growth in the control inhibitor-transfected OCI-ly10 co-culture and miR130b inhibitor-transfected OCI-ly10 co-culture upon treatment with LNPs-miR130b control or with LNPs-miR130b antagomir. Cell viability was measured by MTT. Data are summarized as mean ± SD (n = 3). c Transmission electron microscope showed typical autophagosomes in the miR130b mimics-transfected DB co-culture system and miR130b inhibitor-transfected OCI-ly10 co-culture upon treatment with LNPs-miR130b control or LNPs-miR130b antagomir. The cells were counted from five visions selected at random and subjected for statistical analysis. Data are summarized as mean ± SD (n = 5). d Standardized uptake value intensity of Cy5.5/Fam-labeled LNPs-miR130b antagomir after intravenous injection (left panel) were measured by micro PET-CT. The column indicated ex vivo fluorescence images of the tumor and major organs, including brain, heart, lung, liver, spleen, and kidney, from mice at 24 h after intravenous injection (right panel). e pGMLV-vector and pGMLV-miR130b cells were injected into nude mice subcutaneously with LNPs-miR130b control or LNPs-miR130b antagomir treatment, and tumor volume was determined. Data are summarized as mean ± SD (n = 4). f Micro PET-CT showed standardized uptake value intensity in pGMLV-vector group and pGMLV-miR130b group upon treatment with LNPs-miR130b control or LNPs-miR130b antagomir. g Serum ALT, AST, and Serum BUN, CREA were measured using a clinical chemistry analyzer in pGMLV-vector group and pGMLV-miR130b group between untreated group and treatment with LNPs-miR130b control group or LNPs-miR130b antagomir group. Data are summarized as mean ± SD (n = 4). h Western blot analysis of IFNAR1, p-STAT, STAT1 in pGMLV-vector group and pGMLV-miR130b group upon treatment with LNPs-miR130b control or LNPs-miR130b antagomir. i IL17 level was measured by ELISA in pGMLV-vector group and pGMLV-miR130b group upon treatment with LNPs-miR130b control or LNPs-miR130b antagomir. Data are summarized as mean ± SD (n = 4). j Transmission electron microscope showed typical autophagosomes in pGMLV-vector group and pGMLV-miR130b group upon treatment with LNPs-miR130b control and LNPs-miR130b antagomir. The cells were counted from five visions at random and subjected for statistical analysis. Data are summarized as mean ± SD (n = 5). k Graphic working model illustrated that miR130b counteracts diffuse large B-cell lymphoma progression via OX40/OX40L-mediated interaction with Th17 cells