PGM5-AS1 Promotes Progression of Diffuse Large B-Cell Lymphoma and Immune Escape by Regulating miR-503-5p

Abstract

Purpose: Diffuse large B-cell lymphoma (DLBCL) is a prevalent malignant condition with a dismal prognosis. LncRNA PGM5 antisense RNA 1 (PGM5-AS1) appears to be intricately involved in the progression of DLBCL, yet the modulatory mechanism remains unclear. The purpose of this study was to explore the expression of lncRNA PGM5-AS1 in DLBCL and its effect on the disease progression of DLBCL, as well as to explore its mechanisms.

Patients and methods: A total of 35 patients were included in the study. The expression levels of PGM5-AS1 and miR-503-5p in DLBCL tumor tissues and cell lines were detected by RT-qPCR. Cell proliferation was assessed using CCK8. Apoptosis rate was determined by flow cytometry. Cell invasion was examined by transwell assays. The specific interaction between PGM5-AS1 and miR-503-5p was verified through dual luciferase reporter gene assays. The immune related factors were detected by ELASA kits. The CD8⁺ T cells cytotoxicity was evaluated by LDH cytotoxicity kit.

Results: In DLBCL tumor tissues and cells, upregulated PGM5-AS1 expression, downregulated miR-503-5p expression, and elevated PD-L1 expression were observed. PGM5-AS1 functioned as a regulator in controlling DLBCL cell proliferation, apoptosis, and invasion by downregulating miR-503-5p expression. When CD8⁺ T cells were co-cultured with cells transfected with si-PGM5-AS1, the secretion of immunoregulatory factors increased, and the cytotoxicity of CD8⁺ T cells increased. These effects were mitigated by miR-503-5p inhibitors.

Conclusion: PGM5-AS1 accelerated DLBCL development and facilitated tumor immune escape through the miR-503-5p. Our discoveries offered an insight into lncRNA PGM5-AS1 serving as a prospective therapeutic target for DLBCL.

Keywords: DLBCL; PGM5-AS1; immune escape; miR-503-5p.

Figure 1

PGM5-AS1 and miR-503-5p were differently expressed in DLBCL tissues and cells. (A and B) The levels of PGM5-AS1 and miR-503-5p in DLBCL tumor tissues (n=35) were detected by RT-qPCR (C) Pearson correlation analysis between PGM5-AS1 and miR-503-5p. (D and E) The levels of PGM5-AS1 and miR-503-5p in DLBCL cell lines were detected by RT-qPCR.

Figure 2

Downregulating PGM5-AS1 resulted in variations in cell proliferation, apoptosis, and invasion capabilities of DLBCL cells. (A) The levels of PGM5-AS1 in DLBCL cells were verified by RT-qPCR. (B and C) Viability of DLBCL cells was assessed by CCK-8. (D and E) Apoptosis in DLBCL cells was quantified by flow cytometry. (F and G) The invasion capability of DLBCL cells was assessed by the transwell assay.

Figure 3

Inhibition of miR-503-5p resulted in variations in cell proliferation, apoptosis, and invasion capabilities of DLBCL cell lines. (A) The levels of miR-503-5p in OCI-LY-7 and OCI-LY-10 cells were verified by RT-qPCR. (B and C) Viability of DLBCL cells was assessed by CCK-8. (D and E) Apoptosis in DLBCL cells was quantified by flow cytometry. (F and G) The invasion capability of DLBCL cells was assessed by the transwell assay. *P<0.05, **P<0.01, ***P<0.001.

Figure 4

PGM5-AS1 functions as a miR-503-5p sponge. (A) The binding site sequence between PGM5-AS1 and miR-503-5p was forecasted utilizing the lncRNASNP2 database. (B and C) The specific interaction between PGM5-AS1 and miR-503-5p was investigated using a dual luciferase reporter gene assay.

Figure 5

MiR-503-5p inhibitor reversed the effects on proliferation, apoptosis and invasion induced by si-PGM5-AS1. (A) The levels of miR-503-5p were assessed by RT-qPCR after si-PGM5-AS1 and miR-503-5p inhibitor treatment. (B and C) Cell proliferation was assessed using the CCK8 assay at intervals of 0, 24, 48, and 72 hours following si-PGM5-AS1 and miR-503-5p inhibitor administration. (D and E) Apoptosis in DLBCL cells was quantified by the Annexin V/PI assay. (F and G) The invasion capability of DLBCL cells was assessed by the transwell assay.

Figure 6

PGM5-AS1 exerts a regulatory influence on PD-L1 expression, IFN-γ and TNF-α secretion, and CD8⁺ T cell cytotoxicity by targeting miR-503-5p. CD8⁺ T cells were co-cultured with OCI-Ly7 and OCI-Ly10 cells with si-PGM5-AS1 and miR-503-5p inhibitor treatment. (A and B) The PD-L1 levels in DLBCL tumor tissues (n=35) and cell lines were detected by RT-qPCR. (C and D) Pearson correlation analysis between PGM5-AS1 and PD-L1, miR-503-5p and PD-L1. (E) PD-L1 levels were assessed by RT-qPCR after si-PGM5-AS1 and miR-503-5p inhibitor treatment. (F and G) Concentrations of TNF-α and IFN-γ were determined via ELISA kit. (H) The CD8⁺ T cells cytotoxicity was assessed using the LDH cytotoxicity assay kit.