Abnormal Function of Circulating Follicular Helper T Cells Leads to Different Manifestations of B Cell Maturation and Differentiation in Patients with Osteosarcoma.

Objective: The objective of this study is to investigate the effect of dysfunctional circulating follicular helper T cells (Tfh) on B cell maturation and differentiation in patients with osteosarcoma (OS). Method: Data from 30 OS patients who underwent diagnosis and treatment in our hospital, as well as those of 30 healthy subjects (HC), were collected at the same time. Flow cytometry was employed to identify proportions of CD4+CXCR5+Tfh cells and Tfh cell subtypes Tfh17, Tfh1, and Tfh2 in the patient's peripheral blood. CD40 L and IFNγ levels were detected after stimulating Tfh cells with an influenza antigen; the positive rates of CD27 and CD38 in B cells were detected before and after coculture with Tfh cells. qRT-PCR was carried out for Blimp-1 expression in B cells, and ELISA was employed to identify the levels of IgM, IgG, and IgA in B cells and IL-2, IL-10, and IL-4 in Tfh cells before and after coculture. Results: The percentage of CD4+CXCR5+Tfh cells in OS patients' peripheral blood increased significantly. The Tfh cell ratio increased along with the TNM stage, and the Tfh cell ratio in OS metastasis patients is greater than that in nonmetastatic patients. In addition, Tfh2 and Tfh17 cells increased drastically in OS patients, and no meaningful change was seen in Tfh1 cells. CD40 L levels of Tfh cells in OS patients were less than those of the HC group, and IFNγ was substantially increased. After coculturing the OS group's B cells with Tfh cells, the CD27+ and CD38+ cells of B cells were drastically greater, and Blimp-1 expression was also significantly increased. In addition, the levels of IL-21, IL-4, and IL-10 of Tfh cells in the OS group and the levels of IgA, IgG, and IgM in B cells were significantly reduced after coculture. Conclusion: Dysfunctional Tfh in OS patients can severely inhibit B cell development, maturation, and differentiation.

The Effect of miR-138 on the Function of Follicular Helper T Cells and the Differentiation of B Cells in Osteosarcoma.

OBJECTIVE: To explore the effect of miR-138 on the function of follicular helper T (Tfh) cells and the differentiation of B cells in osteosarcoma. METHODS: Clinically collect peripheral blood from osteosarcoma (OS) patients and healthy volunteers (HC), as well as OS tumor tissues (OS tumor) and adjacent tissues with normal histology (normal group). The CD4+CXCR5+Tfh cells of OS patients were screened and isolated by flow cytometry, and the expression of Tfh cell membrane surface antigens PD-1 and CTLA-4 was detected. In addition, qRT-PCR was used to detect the expression of miR-138 in tissues and Tfh cells, and the correlation relationship between miR-138 and PD-1 and CTLA-4 was analyzed. After interference or overexpression of miR-138 in Tfh cells, coculture with untreated B cells was done, and the levels of IL-10, IL-12, IL-21, and INF-γ in Tfh cell culture medium and the levels of IgM, IgG, and IgA in B cell culture medium after coculture were measured by ELISA. Flow cytometry was used to detect the expression of B cell membrane surface antigens CD27 and CD38 after coculture. RESULTS: The rate of PD-1- and CTLA-4 positive cells in the peripheral blood and tissues of the OS group was significantly increased, the expression of miR-138 was significantly reduced, and the expression of miR-138 was negatively correlated with the expression of PD-1 and CTLA-4. In addition, upregulation of miR-138 can lead to a significant increase in the level of IL-10 in the supernatant of Tfh cells and a significant decrease in the levels of IL-12, IL-21, and INF-γ, which in turn leads to increased levels of IgM, IgG, and IgA released by B cells. At the same time, it significantly increases the rate of CD27- and CD38-positive cells and promotes the maturation of B cells. Downregulating miR-138 has the opposite effect. CONCLUSION: Downregulating the expression of miR-138 in osteosarcoma can improve the dysfunction of CD4+CXCR5+Tfh cells and promote the differentiation of B cells.

MicroRNA-138 functions as a tumor suppressor in osteosarcoma by targeting differentiated embryonic chondrocyte gene 2.

BACKGROUND: MicroRNA-138 (miR-138) has been proven to be a tumor suppressor gene in various types of tumors. However, the expression and the role of miR-138 in human osteosarcoma are still poorly understood. We investigated the function and the underlying mechanism of miR-138 in osteosarcoma. METHODS: The expression of miR-138 in human osteosarcoma tissues and cell lines was detected by real-time PCR analysis. The gain-of-function and loss-of-function experiments were performed on osteosarcoma cell lines to investigate the effects of miR-138 on osteosarcoma progression, and to determine whether differentiated embryonic chondrocyte gene 2 (DEC2) mediates these effects. Cell proliferation, apoptosis and invasion were assessed by MTT, flow cytometry and transwell-matrigel assays. Dual-luciferase reporter assay was used to identify whether DEC2 is a direct target of miR-138. RESULTS: MiR-138 was significantly downregulated in human osteosarcoma tissues and cell lines. Moreover, miR-138 expression was significantly lower in metastatic osteosarcoma tissues than that in non-metastatic tissues. The in vitro gain-of-function and loss-of-function experiments demonstrated that miR-138 inhibited cell proliferation and invasion, and promoted cell apoptosis of human osteosarcoma cells. DEC2 was verified as a direct target of miR-138, and DEC2 could reverse the inhibitory effect of miR-138 on osteosarcoma progression. CONCLUSIONS: These findings suggested that miR-138 acts as a tumor suppressor in osteosarcoma.miR-138 inhibited cell proliferation and invasion, as well as promoted cell apoptosis of human osteosarcoma cells, at least partially, by inhibiting the expression of DEC2. MiR-138/DEC2 may be a novel therapeutic target in osteosarcoma.