The Role of Lncrna-AF117829.1 in the Immunopathogenesis of CD8+ T Cells in Patients with Severe Aplastic Anemia

Background: Severe aplastic anemia (SAA) is a kind of autoimmune disease, which is mainly caused by activated T-lymphocytes and immune abnormality. With the development of RNA sequencing technology, LncRNAs play an important role in gene transcription, cell differentiation, cell proliferation and cell cycle in a series of cell processes.

Methods: In this study, RNA SEQ technology was used to detect the expressions of messager RNA (mRNA) and long non coding RNA (LncRNA) in CD8⁺ T cells from peripheral blood of newly diagnosed SAA patients and healthy controls. The differential expressions of mRNAs and LncRNAs were obtained by bioinformatics analysis. After sceening and verification, LncRNA-AF117829.1 was selected for future study. LncRNA-AF117829.1 was overexpressed in CD8+ T cells from SAA patients by lentivirus vector. Then the expression levels of perforin and granzyme B were detected by flow cytometry (FCM), its possibly regulated target gene mRNA RIPK2 was detected by western blot and PCR. Finally, CD8⁺ T cells of SAA patients were intervened with RIP2 kinase inhibitor (GSK583) in vitro.

Results: A total of 194 differential expressed LncRNAs were screened, including 107 LncRNAs were up-regulated, 87 LncRNAs were down regulated. The differentially expressed LncRNAs were also related to MAPK and NOD signaling pathways (Fig A, B). The expression of lncRNA AF117829.1 was verified to be downregulated in SAA patients by PCR. Bioinformatics predicted that RIP2 was the target gene of lncRNA AF117829.1, which was proved to be opposite trend to that of lncRNA AF117829.1 (Fig C, D). After overexpression of lncRNA AF117829.1 in CD8⁺ T cells of SAA patients, mRNA-RIP2 expression decreased, the expression of perforin and granzyme B decreased and RIP2 signaling pathway related proteins decreased (Fig E-G). After CD8⁺ T cells from SAA patients were intervened by GSK583 in vitro, RIP2 signaling pathway related protein decreased, the expression of perforin and granzyme B decreased in CD8⁺ T cells (Fig H, I).

Conclusions: A total of 194 differential expressed LncRNAs were screened in CD8⁺ T cells of SAA patients. LncRNA-AF117829.1 was selected and verified in vitro, which can promote CD8⁺ T cell by upregulating the expression of target gene RIP2. These results maybe helpful to further explore the molecular mechanism of immune pathogenesis of SAA and provided potential targets for diagnosis and treatment of SAA.