Shishan Xiao, Hongqian Zhu*
Department of Hematology, Guizhou Provincial People's Hospital, Guiyang 550002, Guizhou Province, China
Objective: B lymphocyte miR-150-targeting c-Myb is involved in the occurrence and development of autoimmune haemolytic anaemia (AIHA).
Methods: Our sample included 20 patients with AIHA haemolytic attacks treated in the Haematology Department of our hospital between January 2020 and January 2021, defined as the attack group, as well as 10 healthy controls. Peripheral blood mononuclear cells were extracted from the two groups. CD19+B lymphocytes in the peripheral blood of the two groups were sorted and purified using magnetic attraction. Total RNA was extracted using the TRIzol method. The number of CD5+B cells before transfection of peripheral blood CD19+B lymphocytes was detected using flow cytometry. The hsa-miR-150 high expression plasmid was transfected into cells using the Lipofectamine™️ 2000 liposome transfection reagent. The expression of IL-10 on the cell surface was measured using an enzyme-linked immunosorbent assay. Real-time fluorescent PCR was used to detect the expression of miR-1 and 50c-Myb. A luciferase kit was used to detect relative luciferase activity and analyse the transcription activity of the target gene.
Results: The expression of miR-150 in the attack group before transfection was significantly lower than in the control group (p<0.05). There was no significant difference in the proportion of peripheral blood CD5+CD19+B cells in the attack group compared with before transfection (p>0.05). There was a significant difference between the attack group and the control group (P<0.05). The proportion of peripheral blood CD5+CD19+B cells in IL-10 cells in the attack group was significantly lower than before transfection (p<0.05); There was a significant difference between the attack group and the control group (p<0.05). After transfection, the expression level of miR-150 in peripheral blood CD19+B lymphocytes was significantly lower than in the control group (p<0.05). The expression level of the c-Myb gene was significantly higher than in the control group (p<0.05). The double luciferase experiment showed that the has-miR-150 high expression plasmid in B lymphocytes can significantly inhibit the luciferase activity of c-Myb (i.e. the transcriptional activity of c-Myb), proving that the c-Myb group in B lymphocytes is the target of miR-150.
Conclusion: The up-regulated expression of miR-150 in B lymphocytes of AIHA patients can inhibit the expression of the c-Myb gene, which may be involved in the occurrence and development of the disease.
B lymphocytes, autoimmune haemolytic anaemia, transfection, dual luciferase test.