骨肉瘤转化生长因子β1/B7-H3正反馈调控机制研究

B7-H3 is highly expressed in osteosarcoma tissue, which was deeply involved in tumor invasion and metastasis, and correlated with clinical staging and prognosis in patients. The role of overexpressed B7-H3 in osteosarcoma, however, remains unknown. Our previous studies have shown that the expression level of B7-H3 was positively correlated with the secretion of TGF-β1, and TGF-β1 could induce the expression of B7-H3 in osteosarcoma cells in vitro, while treatment of OS cells with B7-H3 siRNA could induce the inhibition of secretion of TGF-β1 on the contrary. Therefore, it is hypothesized that there might exist a positive feedback loop between B7-H3 expression and TGF-β1 secretion, which could result in the overexpression of B7-H3 and TGF-β1 secretion in osteosarcoma. To be specific, the autocrine or paracrine TGF-β1 could act through activation of specific signaling pathways to regulate the expression of B7-H3 at the transcriptional level or/and post transcriptional level in osteosarcoma cells. Furthermore, the high expression of B7-H3 could probably promote the secretion of TGF-β1 in osteosarcoma cells or tumor infiltrating lymphocytes. Therefore, the aim of present study is to examine the relationship between B7-H3 expression and TGF-β1 secretion in clinical osteosarcoma tissues, and further explore the mutual positive feedback and regulation principle together with the corresponding intracellular signal transduction and gene expression regulation mechanism examined by cellular functional experiments in vitro and animal experiments in vivo. It will be of great help to provide promising and effective for immunotherapy target in osteosarcoma.

B7-H3高表达于骨肉瘤组织，深度参与了肿瘤的侵袭转移，且与患者预后密切相关，然高表达B7-H3的原因尚不清楚。预实验发现，骨肉瘤组织中TGF-β1与B7-H3的表达呈正相关，TGF-β1可诱导骨肉瘤细胞B7-H3表达升高，且沉默B7-H3基因却导致TGF-β1分泌水平下降。因此我们推测，骨肉瘤组织存在B7-H3表达与TGF-β1分泌的正反馈机制，即自分泌或旁分泌的TGF-β1通过活化骨肉瘤细胞特定信号通路，从转录或/和转录后水平调控B7-H3表达，而高表达的B7-H3会促进骨肉瘤细胞本身或浸润淋巴细胞分泌TGF-β1，可能是骨肉瘤细胞发生免疫逃逸的重要原因。本研究通过检测骨肉瘤标本B7-H3和TGF-β1表达的相关性，进行体内外实验验证TGF-β1/TGF-βR/B7-H3正反馈机制及调控规律，并探明相应的胞内信号转导通路和基因表达调控机制，为骨肉瘤的免疫治疗提供理论基础和有效靶点。

骨肉瘤肿瘤微环境复杂，肿瘤细胞异质性强导致极易发生免疫逃逸。本研究发现骨肉瘤细胞通过自分泌或旁分泌TGF-β1，作用于细胞本身TGF-β1受体，活化特定信号通路，从转录水平或/和转录后水平上调B7-H3表达，而骨肉瘤细胞高表达B7-H3分子通过特定机制进一步促进了肿瘤细胞TGF-β1的表达和分泌。B7-H3和TGF-β1形成的正反馈环，是骨肉瘤高度恶性、进展迅速、预后差的重要原因之一。主要研究内容及结果如下：1.在骨肉瘤标本中TGF-β1和B7-H3的表达存在一定的相关性，即TGF-β1染色水平越高，相应组织B7-H3的表达也越高。患者血清中TGF-β1与sB7-H3的含量显著高于骨纤维发育不良患者及健康对照，并具有显著正相关。骨肉瘤患者血清sB7‑H3的ROC 曲线下面积为 0.863，最佳临界值为60.94 ng/mL，灵敏度为75.7%，特异性为83.8%，对骨肉瘤早期预测具有一定价值；2.低浓度组TGF-β1可上调骨肉瘤细胞的B7-H3基因表达。沉默TGF-β1基因后，可特异性下调骨肉瘤细胞中B7-H3基因的表达。MG-63B7-H3-/-骨肉瘤细胞中显著表达差异的circRNA有652条，402条circRNA表达上调；250条circRNA表达下调，其中hsa_circ0021347被证实是表达改变最显著的一条circRNA，其表达与B7-H3 的表达呈负相关。在骨肉瘤组织及多种细胞系中均得到了进一步的验证，发现hsa_circ0021347表达显著降低，并可能是通过miR-646/NOB1轴发挥重要调控作用；3. 给与适宜浓度TGF-β1作用后，MG-63B7-H3-/-细胞与对照细胞基因表达差异显著，其中上调基因394个，下调基因733个。经验证，Siglec15基因在该过程中变化显著，且可能成为替代B7-H3发挥免疫逃逸作用的重要分子；4. 进一步研究发现，Siglec-15分子在人骨肉瘤组织中高度表达，其表达与患者肿瘤分期、肺转移呈正相关，高Siglec-15组患者的总生存率显著低于低Siglec-15组；5. 沉默Siglec-15基因24小时后，骨肉瘤细胞生长速度开始减慢，沉默48和72小时后，骨肉瘤细胞增殖受到显著抑制。细胞克隆实验进一步证明了上述结果。转染Siglec-15 siRNA后，骨肉瘤细胞的体外侵袭能力和平面运动能力均明显减弱。