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。

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.