组蛋白H3.3 G34W通过p62蛋白调控骨巨细胞瘤地舒单抗治疗后单核基质细胞表型转化的机制研究

我国骨巨细胞瘤（giant cell tumor of bone, GCTB）的发病率约为美国和日本的总和，手术后易复发。作为唯一的靶向药物，地舒单抗疗效确切，但近年来出现用药后易误诊、促恶变等新问题，均与用药后GCTB单核基质细胞表型转化有关。组蛋白H3.3 G34W是单核基质细胞标志蛋白，可调控细胞表型转化，但机制不明。前期研究发现p62蛋白表达与单核基质细胞表型相关，并且，地舒单抗治疗后，组蛋白H3.3 G34W与p62蛋白表达均下调，组蛋白H3.3 G34W可能通过p62蛋白调控单核基质细胞表型转化。本研究比较地舒单抗治疗前后单核基质细胞表型变化，在H3F3A G34突变型及野生型单核基质细胞中，研究组蛋白H3.3 G34W与p62蛋白相互作用机制；对地舒单抗治疗后耐药的单核基质细胞，CRISPR编辑关键基因，观察细胞表型变化及耐药能否逆转，为临床制定新的靶向治疗策略提供理论依据。

Giant cell tumor of bone (GCTB) is a common intermediate bone tumor that is locally aggressive and rarely metastasis. In 2017, GCTB new cases of China were about the sums of America and Japan. Recurrence rate was high, ranging from 16.7% to 39% after surgery alone. As the only totally humanized monoclonal antibody against RANKL, denosumab achieved a good response in GCTB. However, some new problems arose after denosumab treatment in recent years including misdiagnosis in pathology, higher recurrence rate after curettage surgery and sarcoma changing. Through extensive pathology literatures summary, we analyzed that GCTB monocyte stromal (MS) cells phenotype transformation was an important reason of these new problems..Histone 3.3 G34W is a new biomarker for GCTB. Our previous research showed histone 3.3 G34W locates in MS cells’ nucleus. It regulated MS cells phenotype transformation after denosumab treatment. But the regulation mechanism was not clear. Our previous research also showed p62 protein was overexpressed in GCTB tumor tissues, which promoted MS cells proliferation and invasion by regulating apoptosis. Furthermore, the expression of histone 3.3 G34W and p62 were down-regulated after denosumab treatment. We speculated histone 3.3 G34W could regulate MS cells phenotype transformation by p62 protein after denosumab treatment..Next we plan to enlarge the GCTB paired sample before and after denosumab treatment to compare morphological change and key protein expression of GCTB tissues. Then we isolate MS cells to obtain H3F3A G34 wild type cells and mutant type cells for mechanism research. We choose GST pull-down, immunoprecipitation, ChIP and RNA interference to assay regulating relationship between histone 3.3 G34W and p62 protein. In the end, we isolate H3F3A G34 mutant type MS cells from GCTB metastasis tissues with denosumab resistance. H3F3A G34 and p62 gene are edited by CRISPR/Cas9. Phenotype transformation is assessed in MS cells treated with denosumab, which is confirmed in nude mice model. In short, based on the research above, we elucidate the mechanism of MS cells phenotype transformation after denosumab treatment. We also add theory basis to generate new strategy for GCTB target therapy.