Killin通过p21诱导G1期阻滞的作用机制研究

As a p53 target gene Killin, its promoter methylation is closely related to the occurrence of many cancers such as cowden syndrome, breast cancer and thyroid cancer. Killin tumor suppressor mechanism, more in-depth research has its high affinity binding and inhibition of DNA synthesis, and related to cell cycle S and G2 arrest. In recent years, Team found that Killin inhibits the occurrence of tumor closely related to its function in the G1 phase of the cell cycle. Killin is involved in p53-mediated arrest of G1 phase by up-regulating p21. However, the specific function and molecular mechanism of Killin in p53-mediated G1 phase arrest are unclear. Therefore, the project aims to carry out three aspects on the basis of the previous studies: 1) identification of Killin’s function in p53-mediated G1 arrest; 2) revealed the molecules of p53 → Killin → p21 → G1 arrest mechanism; 3）exploring the correlation between the expression of Killin and p21 in clinical colon cancer specimens and its relationship with malignancy. This study will elucidate the specific function and molecular mechanism of Killin in p53-mediated G1 arrest, which will provide new targets and new ideas for the development of new anticancer drugs.

作为p53靶基因Killin，其启动子的甲基化与Cowden综合征，乳腺癌和甲状腺癌等多种癌症的发生紧密相关。关于Killin抑癌机制，较深入的认识主要有其高亲和性结合并抑制DNA合成，并与细胞周期S期和G2期阻滞相关。近年来，项目组研究发现Killin抑制肿瘤发生与其在细胞G1期中的功能紧密相关，Killin能通过上调p21而参与p53介导的G1期阻滞。然而，关于Killin在p53介导的G1期阻滞中具体作用机制尚不明确。因此，本项目拟在前期研究基础上开展三个方面的研究：1）鉴定Killin在p53介导的G1期阻滞中的功能；2）揭示p53→Killin→p21→G1期阻滞的分子机制；3）探究临床结肠癌样本中Killin与p21表达的相关性，及其与肿瘤恶性程度的关系。此项研究有望阐明Killin通过参与p53介导的G1期阻滞而抑制肿瘤发生的新机制，而为新型抗癌药物的研发提供新靶点和新思路。

Killin作为一种抑癌基因，其低表达与多种癌症的发生紧密相关。近年来，众多研究证据表明Killin抑制肿瘤发生与其在细胞周期G2/M和S期中的功能密切相关，而Killin在G1期中的功能有待进一步阐释。为了更深入研究Killin在细胞周期特别是G1期中的功能，本研究通过构建Killin敲除和过表达的细胞模型，确定了Killin参与p53介导的G1期阻滞。通过敲除和过表达Killin，证实其能够上调p21参与G1期阻滞。对过表达Killin后的细胞进行了RNA-seq，蛋白组测序和CO-IP LC-MS后，进行多组学联合分析，阐释了Killin通过蛋白酶体途径上调p21的机制。并且本研究还证实Killin能够通过直接结合p21的启动子区域而上调p21的mRNA水平。进一步研究又发现Killin下调p21Thr145和Ser146的磷酸化水平，进而促进p21的细胞核分布而诱导G1期阻滞。Killin除了调控p21参与G1期阻滞外，本研究还通过RNA-IP和免疫荧光证实了Killin能够直接结合RNA而抑制其合成，进而导致细胞周期停滞的功能。此外，本研究还分析了结肠癌Killin的表达情况，证实结肠癌中Killin表达显著低于正常组织，且Killin表达水平与结肠癌恶性程度呈负相关。综上所述，本研究不仅阐释了Killin参与p53介导的G1期阻滞的机制，还发现了Killin在结肠癌中的表达水平可以作为一种新的肿瘤标志物，具有潜在的临床应用价值。