Background:The progression of osteosarcoma is closely related to the immune microenvironment. Related studies have found that the RNA-binding motif protein, X-linked (RBMX), plays a regulatory role in modulating the biological characteristics of the tumor microenvironment (TME). However, its regulatory mechanism in osteosarcoma remains unclear.Methods:In this study, the expression of RBMX in osteosarcoma was analyzed using the results of bulk and single-cell transcriptome sequencing of human osteosarcoma. The RBMX knockout cell line was constructed via lentivirus transfection. The mouse subcutaneous implantable tumor model and single-cell transcriptome sequencing analysis revealed the effects of RBMX on the osteosarcoma microenvironment, as verified via multiplex immunofluorescence, flow cytometry, and PCR experiments.Results:Using the TARGET database and multiplex immunofluorescence, we found that RBMX is highly expressed in human osteosarcoma and is associated with poor prognosis. The high expression of RBMX may mediate the immunosuppressive microenvironment of human osteosarcoma. In vitro cell experiments showed that knockout of RBMX significantly inhibited the proliferation of mouse osteosarcoma cells. Through single-cell transcriptome sequencing analysis of subcutaneous implantable tumors in mice, we determined that RBMX deletion substantially elevated the recruitment of cytotoxic CD8+T cells within the mouse TME, which was further verified through flow cytometry analysis. Cell coculture assay confirmed that knockout of RBMX significantly enhanced the cytotoxic activity of CD8+T cells. Finally, cell communication and in vitro experimental verification revealed that knocking out RBMX might enhance the infiltration of CD8+T cells by upregulating histocompatibility 2, K1, and K region (H2-K1) and downregulating thrombospondin 1 (THBS1).Conclusions:This study may provide potential targets for reshaping the immune microenvironment of osteosarcoma and improving its therapeutic efficacy.
背景：骨肉瘤的进展与免疫微环境密切相关。相关研究发现，RNA结合基序蛋白X连锁（RBMX）在调控肿瘤微环境（TME）生物学特性中发挥调节作用，但其在骨肉瘤中的调控机制尚不明确。 方法：本研究利用人骨肉瘤的批量及单细胞转录组测序结果分析RBMX在骨肉瘤中的表达情况。通过慢病毒转染构建RBMX敲除细胞系，结合小鼠皮下移植瘤模型及单细胞转录组测序分析揭示RBMX对骨肉瘤微环境的影响，并通过多重免疫荧光、流式细胞术及PCR实验进行验证。 结果：通过TARGET数据库及多重免疫荧光检测发现，RBMX在人骨肉瘤中高表达且与不良预后相关，其高表达可能介导人骨肉瘤的免疫抑制微环境。体外细胞实验表明，敲除RBMX显著抑制小鼠骨肉瘤细胞增殖。通过对小鼠皮下移植瘤的单细胞转录组测序分析发现，RBMX缺失可显著提升小鼠TME内细胞毒性CD8+T细胞的募集水平，该结果经流式细胞术分析得到进一步验证。细胞共培养实验证实，敲除RBMX能显著增强CD8+T细胞的细胞毒性活性。最后通过细胞通讯分析及体外实验验证发现，敲除RBMX可能通过上调组织相容性2-K1-K区（H2-K1）和下调血小板反应蛋白1（THBS1）来增强CD8+T细胞浸润。 结论：本研究可能为重塑骨肉瘤免疫微环境、提升其治疗效果提供潜在靶点。
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