涎腺多形性腺瘤中PLAG1与不同伙伴基因的融合及相关基因表达调节在肿瘤多向分化中的作用

Pleomorphic adenoma is the most common salivary gland tumor and is characterized by the multiple differentiation in histological and cell phenotypes. Some of the phenotypes are correlated with tumor recurrence or malignant transformation. The mechanism of multiple differentiation is unclear. Pleomorphic adenoma gene 1(PLAG1) is the highly specific gene of the tumor and results from the genetic translocation of the chromosomes. After the translocation, 4 partner genes fused to PLAG1 including CTNNB1、LIFR、CHCD7、TCEA1 have been identified. Whether the different fusion genotypes are correlated with the histological phenotypes and whether the related gene expression alterations can cause the different cell differentiation have not been elucidated yet. Based on our previous studies, in the present project we plan to use the fluorescent in situ hybridization(FISH), RT-PCR, DNA sequencing, real-time PCR and Western Blot to detect the gene fusions involving PLAG1 to CTNNB1、LIFR、CHCD7、TCEA1 and the mRNA and the protein expression of the related genes in different histological patterns of 60 cases of salivary pleomorphic adenoma, and to analyze the relationship of the four fusion genotypes with the gene expression, histological phenotypes and the clinicopathological features, particularly the recurrence and malignant transformation of the tumors. We also plan to choose 10 cases of pleomorphic adenoma with ductal and myoepithelial cell dominated pattern, use laser capture microdissection, real-time PCR and immunohistochemistry to examine the fusion genotypes of PLAG1 and the mRNA/protein expression of PLAG1 and β-catenin in neoplastic ductal and myoepithelial cells respectively within the same tumors. The miR181and miRNA107 expression will be evaluated when there is significant expression difference of PLAG1 between ductal and myoepithelial cells. We will design and synthesize the siRNA targeting the PLAG1 and construct the plasmid vector and transfect it to the cells of pleomorphic adenoma cell line AP-1 and AP-4. The related gene expression alteration and the phenotypic change of the transfected cells will be observed. Finally the correlation between the PLAG1 fusion genotypes and the recurrence and the malignant transformation of the tumor will be testified in clinical tumors. The study will be the first time to investigate the PLAG1 fusion gene function from the multiple histological and cellular differentiation point. It will enrich the mechanism of the tumorigenesis of the pleomorphic adenoma. It will also facilitate the molecular classification, recurrence and malignancy risk predicting of the tumors. In addition, the study may provide more evidence that both the genetic and epigenetic mechanisms are involved in regulating the gene expression.

多形性腺瘤是最常见的涎腺肿瘤，以组织、细胞学多向分化为特征，部分表型与肿瘤复发、恶变相关，肿瘤多向分化机制不明。多形性腺瘤基因1（PLAG1）基因易位是该肿瘤特异性的遗传学改变，PLAG1可与4种不同的伙伴基因融合，4种不同融合型及相关基因表达在肿瘤多向分化中的作用缺乏研究。本课题拟在前期研究基础上，利用荧光原位杂交、RT-qPCR、基因测序、激光捕获显微切割、Western、免疫组化等手段，检测多形性腺瘤不同表型的组织和细胞中PLAG1与CTNNB1、LIFR、CHCD7、TCEA1 伙伴基因融合状况以及与PLAG1、β-catenin、IGF2/ miR181等表达的关系、基因差异表达调节机制，并分析不同基因融合型与组织学表型、复发及恶变关系。研究将首次从组织和细胞多向分化角度探讨PLAG1基因融合的功能，为分子分型、复发和恶变风险预测、遗传学和表观遗传学共同调节基因表达提供实验依据。

多形性腺瘤是最常见的涎腺肿瘤，以组织、细胞学多向分化为特征，部分表型与肿瘤复发、恶变相关，肿瘤多向分化机制不明。本课题检测了77例多形性腺瘤新鲜标本，发生CTNNB1-PLAG1融合的有32例，发生CHCHD7-PLAG1融合的有2例。存在PLAG1融合基因的多形性腺瘤组织病理学类型以黏液软骨样成份多见，细胞成份中腺上皮细胞比例升高（>5%），倾向于多结节状生长，并累及周围组织。我们推断PLAG1融合基因存在与多形性腺瘤的不良生长方式有关，对其肿瘤分子分型有助于预测生物学行为。所有多形性腺瘤中均可见PLAG1和β-catenin蛋白表达，PLAG1几乎只表达于肌上皮细胞，而β-catenin主要表达于腺上皮细胞。PLAG1和β-catenin的蛋白表达与CTNNB1- PLAG1融合基因无显著相关性。在同一例多形性腺瘤中，腺上皮细胞和肌上皮细胞检测到相同的CTNNB1-PLAG1融合基因型。提示在同一PA中在组织学表型上不同、细胞学表型上不同的肿瘤细胞具有相同的基因型，即PA不同组织学、细胞学表型的肿瘤细胞来源于同一多潜能干细胞，这一干细胞具备多向分化的能力。沉默多形性腺瘤细胞系SM-AP1和SM-AP4中PLAG1表达未显著影响细胞增殖活性和侵袭能力。我们通过RT-PCR和FISH方法在SM-AP1和SM-AP4细胞中未检测到PLAG1基因融合，提示PLAG1基因可能更多地参与了肿瘤发生过程，而影响细胞增殖活性、侵袭能力改变的机制还需进一步研究。我们进一步尝试建立一个恶性多形性腺瘤预后预测模型，将334例恶性多形性腺瘤分成两组：训练集（223例）和验证集（111例），在预后预测列线图模型中，包含5个变量：年龄，肿瘤大小，是否有淋巴结转移，侵袭性以及肿瘤部位和组织学亚型的关系，可以综合这5个变量为单独的恶性多形性腺瘤患者的预后进行预测，其精度高于目前仅使用TNM分期预测患者预后的方法。通过本研究，我们确定了PLAG1融合基因在多形性腺瘤中的发生率，并应用于病理诊断中，同时构建的预后预测模型也将为预测患者预后、治疗方案制定和临床随访提供依据。