新的候选抑癌基因LRRC2在甲状腺癌中的功能及其机制研究

By using internet microarray database resources we had screened LRRC2 as a new candidate tumor suppressor gene in thyroid carcinoma. In previous studies we also found LRRC2 expression levels were significantly lower in thyroid carcinoma tissues and cell lines, and low expression of LRRC2 in thyroid and was closely related to the tumor biological behaviors such as cancer proliferation, invasion and metastasis. Thus we speculate LRRC2 may be a key candidate tumor suppressor genes. In this study we firstly using IP-WB to verify LRRC2 direct binding protein, and then to verify LRRC2 play its roles by inhibiting RAS activation of its downstream signaling pathways in vitro an vivo; then reveal thyroid inactivation mechanisms of LRRC2 gene from epigenetic changes and allelic loss of heterozygosity; and finally evaluate LRRC2 / RAS molecular subtype as a survival and prognosis marker with RAS mutations. Our project is expected to determine features and molecular mechanisms of a new candidate tumor suppressor gene LRRC2 and confirm a new subtype classification for thyroid carcinoma.

我们通过基因芯片数据库资源筛选出甲状腺癌候选抑癌基因LRRC2，并在前期研究同样发现LRRC2的表达水平在甲状腺癌组织及甲状腺癌细胞系中显著减低，且LRRC2基因的低表达与甲状腺癌增殖和侵袭转移等生物学行为密切相关。因而我们推测LRRC2可能是一个关键的候选抑癌基因。本研究拟首先采用蛋白质免疫共沉淀技术验证LRRC2的直接结合蛋白，然后通过体内外试验验证LRRC2通过抑制RAS激活及其下游具体信号通路；进而从表观遗传学改变和等位基因的杂合性丢失两个方面，揭示甲状腺癌中LRRC2基因失活的机制；最后通过与RAS基因突变检测结合，全面验证地对LRRC2/RAS分子分型与生存预后的关系。预期本项目将确定候选抑癌基因LRRC2在甲状腺癌中的新功能及具体分子机制，证实LRRC2/RAS分子分型作为甲状腺癌预后标志物的作用。

富含亮氨酸重复单位2（LRRC2）基因可能参与抑制甲状腺癌的发生和发展。结果：LRRC2的表达水平在甲状腺癌组织及甲状腺癌细胞系中显著减低，体内体外功能学试验均发现，LRRC2基因的低表达与甲状腺癌增殖和侵袭转移等生物学行为密切相关。LRRC2可与下游靶蛋白HRAS、NRAS直接结合。阻断LRRC2的下游靶蛋白RAS活性后，过表达LRRC2细胞系的增殖和侵袭转移能力与对照组无差别。而过表达LRRC2细胞系转染过表达RAS质粒后，侵袭运动、增殖能力、RAS信号通路蛋白及其磷酸化蛋白水平较单纯RAS过表达对照组明显减低，低表达LRRC2与甲状腺癌被膜侵犯和淋巴结转移相关，低表达LRRC2组预后较高表达LRRC2组复发中位时间短，低表达LRRC2伴RAS突变组预后较低表达LRRC2不伴RAS突变组复发中位时间短。这些结果提示：LRRC2通过抑制RAS信号通路，抑制甲状腺癌侵袭和增殖能力，发挥潜在的抑癌基因功能。LRRC2低表达预示甲状腺癌高复发风险，LRRC2/RAS分子分型可作为甲状腺癌预后标志物。