Mechanism of LINC01018/miR-182-5p/Rab27B in the immune escape through PD-L1-mediated CD8+ T cell suppression in glioma

Abstract

Background: Glioma is a malignant tumor associated with poorer prognosis. This study aims to elucidate the mechanism of LINC01018/miR-182-5p/Rab27B axis in PD-L1-mediated CD8⁺ T cell suppression in the progression of gliomas.

Methods: LINC01018, miR-182-5p, and Rab27B expression levels in glioblastoma tissues were measured. The proportion of infiltrating macrophages and monocytes and CD8⁺ T cell function were assessed. The relationship between miR-182-5p and Rab27B was analyzed. Glioma cell activity, invasion, and migration were measured. The expression of E-cadherin, N-cadherin, Vimentin, PD-L1, iNOS, and CD206 was determined. Glioma cell-derived EVs were isolated, and the co-localization of Rab27B and PD-L1 and the binding of Rab27B to PD-L1 were analyzed. The endocytosis of EVs by microglia was assayed. The impact of LINC01018/miR-182-5p/Rab27B on glioma growth was observed. The function of macrophages and CD8⁺ T cells in tumors was analyzed.

Results: Rab27B was downregulated, and infiltrating macrophages and monocytes were increased in glioblastoma. miR-182-5p inhibited Rab27B expression. Rab27B knockdown reverses the inhibitory effect of LINC01018 overexpression on glioma cell growth. Glioma cells-derived EVs with low Rab27B expression carried more PD-L1 to increase PD-L1 expression and M2 polarization in microglia. LINC01018 overexpression reduced macrophages in orthotopic tumors. CD8⁺ T cell numbers showed no significant difference, but TIM-3 increased and IFN-γ decreased. miR-182-5p inhibition enhanced the therapeutic effect of anti-PD-L1, which was reversed after glioma cell-derived EVs.

Conclusion: LINC01018 promotes PD-L1-mediated CD8⁺ T cell suppression via the miR-182-5p/Rab27B axis in glioma cell-derived EVs, thereby contributing to immune escape in gliomas.

Keywords: EVs; Glioma; Immune escape; LINC01018; Rab27B; miR-182-5p.

Fig. 1

Rab27B expression is reduced in glioma tissues. A: Rab27B expression in GBM (Glioblastoma multiforme) and LGG (Brain Lower Grade Glioma) was predicted online using the GEPIA2 database (http://gepia2.cancer-pku.cn/#index) [34]. B: qRT-PCR was performed to detect the expression of LINC01018, miR-182-5p, and Rab27B in tumor tissues and adjacent normal tissues from 78 glioblastoma patients. C: Western blot analysis was conducted to assess the expression of Rab27B in tumor tissues and adjacent normal tissues from 78 glioblastoma patients, with representative band images shown. D: Pearson correlation analysis was performed to evaluate the correlation between the expressions of LINC01018, miR-182-5p, and Rab27B. E: qRT-PCR was used to measure the expression of LINC01018, miR-182-5p, and Rab27B in NHA and human glioma cell lines (U251, T98G, LN229, A172). with cell experiment repeated three times. F: Western blot analysis was employed to determine the expression of Rab27B in NHA and human glioma cell lines (U251, T98G, LN229, A172), with the experiment repeated three times. In panels B and C, ** p < 0.01; In panels E and F, * versus NHA, p < 0.05, ** versus NHA, p < 0.01. Data comparison between two groups in panels B and C was analyzed using t-test, and multiple group comparison in panels E and F was analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons test

Fig. 2

LINC01018/miR-182-5p targets Rab27B expression. A: Dual-luciferase reporter assays were conducted to investigate the binding relationship between miR-182-5p and Rab27B. B-C: The expression of Rab27B in U251 and T98G cells under different treatments was assessed using qRT-PCR and Western blot. The cell experiments were repeated three times. ** p < 0.01. Comparison of data was analyzed using two-way ANOVA, followed by Tukey’s multiple comparisons test

Fig. 3

Rab27B inhibition reverses the inhibitory effects of overexpressed LINC01018 on proliferation and metastasis of glioma cells. sh-Rab27B was transfected into U251 and T98G cells, with sh-NC as a control, followed by combined experiments with LINC01018 overexpression. A: qRT-PCR was performed to detect the expression of Rab27B in the cells. B: Cell viability was assessed using CCK-8 assay. C: Transwell assay was used to evaluate cell invasion and migration. D: Western blot analysis was conducted to measure the expression of Rab27B, E-cadherin, N-cadherin, and Vimentin in the cells. The cell experiments were repeated three times. ** p < 0.01. Comparison of data was analyzed using two-way ANOVA, followed by Tukey’s multiple comparisons test

Fig. 4

Overexpression of LINC01018 suppresses glioma growth via the miR-182-5p/Rab27B axis. Glioma cells (U251, T98G) with stable overexpressed LINC01018 (Lv-LINC01018) was generated by lentiviral infection, with Lv-NC as the control. These stably expressing cells were subcutaneously injected into nude mice. A: Tumor volume was measured every 7 days, and the tumors were harvested after 28 days when the mice were euthanized. N = 12. B: Tumor weight was recorded. N = 12. C: IHC was performed to assess the expression of ki67 in the tumors. N = 6. D: qRT-PCR was used to detect the expression of LINC01018, miR-182-5p, and Rab27B in the tumors. N = 6. E: Western blot analysis was conducted to measure the expression of Rab27B in the tumors. N = 6. ** p < 0.01. Comparison of data in panels A, B, C, and E was analyzed using two-way ANOVA, followed by Tukey’s multiple comparisons test; data in panel D were analyzed using t-test

Fig. 5

Rab27B is involved in immune evasion of glioma cells. Glioblastoma patients were divided into high expression group (n = 39) and low expression group (n = 39) based on the expression of Rab27B. A-C: The proportions of monocytes (CD45⁺CD14⁺), macrophages (CD45⁺CD14⁺CD68⁺), and CD8⁺ T cells (CD45⁺CD3⁺CD8⁺) in each glioma tissue were analyzed by flow cytometry. D-E: The functional status of CD8⁺ T cells was analyzed by flow cytometry. F: Western blot was performed to detect the expression of PD-L1 in the tissues. ns p > 0.05, * p < 0.05, ** p < 0.01. Data were analyzed using t-test, with representative band images shown

Fig. 6

The LINC01018/miR-182-5p/Rab27B axis is involved in the transport of PD-L1 in glioma cell-derived EVs. A: Immunoprecipitation was performed to detect the binding of Rab27B to PD-L1 in cells and EVs. B: Western blot was conducted to measure the expression of PD-L1 in cells and EVs. C: Western blot was used to detect the expression of Rab27B, CD9, and CD63 in EVs. The cell experiments were repeated three times. **p < 0.01. Data in panels B and C were analyzed using two-way ANOVA, followed by Tukey’s multiple comparisons test

Fig. 7

Glioma cell-derived EVs delivery PD-L1 and increase PD-L1 expression in microglial cells. A: Glioma cells (U251, T98G) with or without GW4869 were co-cultured with human microglial cells HMC3, and the expression of PD-L1 in HMC3 cells after co-culture was detected by Western blot. B: The CreLoxP system was used to evaluate the delivery of EV cargo from glioma cells to microglial cells. C: Western blot was used to detect the expression of PD-L1, iNOS and CD206 in HMC3 cells treated with different EVs. The cell experiments were repeated three times. ** p < 0.01. Data were analyzed by two-way ANOVA, followed by Tukey's multiple comparisons test.

Fig. 8

Rab27B mediates glioma cell-derived EVs to regulate PD-L1 transfer and promotes immune escape in vivo. The GL261 cells with stable low expression of miR-182-5p (Lv-antagomiR) were injected into mice, with Lv-NC serving as the control. Anti-PD-L1 treatment was administered every 3 days along with intravenous injection of glioma cell-derived EVs. A: qRT-PCR was conducted to detect the expression of miR-182-5p and Rab27B in the tumor, n = 6; B: Western blot was performed to assess Rab27B expression in the tumor, n = 6; C: Survival status of mice in different treatment groups was recorded, n = 12; D: Flow cytometry was used to determine the proportions of CD8⁺ T cells (CD45⁺CD8⁺), macrophages (CD45⁺F4/80⁺CD11b⁺), and TAM (F4/80⁺ CD11b⁺ CD206⁺) in the tissues, n = 6; E: The functional status of CD8⁺ T cells was analyzed by flow cytometry, n = 6; F: Western blot was utilized to measure PD-L1 expression in the tumor, n = 6; ns p > 0.05, * p < 0.05, ** p < 0.01. Data in panels A and B were analyzed by t-test; data in panels D, E, and F were analyzed by one-way ANOVA analysis, followed by Tukey’s multiple comparisons test

Fig. 9

LINC01018 is downregulated in glioma cells. LINC01018 promotes the transport of PD-L1-carrying EVs into microglia and increases PD-L1 expression through the miR-182-5p/Rab27B axis, inhibiting CD8⁺ T cell function and promoting immune evasion in glioma cells