干预PIPKIγ通过调节γ-catenin影响乳腺癌循环肿瘤细胞簇形成及机制研究

Circulating tumor cell cluster correlates with the high expression of γ-catenin which is present in adherence junctions. PIPKIγ targets the adherence junctions. Both the CTC Cluster and PIPKIγ tell the prognosis of breast cancer. However, their relationship is still elusive until now. Previous experiment revealed that PIPKIγ knockdown may reduce CTC Cluster in breast cancer mouse model with the combined advanced methods: RosetteSep kit and CTCscope. Western blot also showed that γ-catenin expression was changed with PIPKIγ intervention. We further propose that CTC Cluster will be reduced by intervention of PIPKIγ via γ-catenin downregulation and cell adherence junctions decomposition. Next, we will transfect breast cancer cell line 4T1 with GFP or mCherry plasmids. By mixing GFP and mCherry stained cells at 1:1 ratio, we xenograft Balb/c mice by injecting the mixed cells in the mammary gland. This animal model will differentiate well the CTC Cluster and CTC Clone. With PIPKIγ knockdown, we will observe its role in γ-catenin expression, CTC Cluster formation and tumor metastasis in vivo. Together with cultured 4T1 cell line, we will further verify, in vitro, PIPKIγ knockdown exert its regulating effect on cell adherence junction composition and CTC Cluster formation via downregulating γ-catenin expression. At the same time, we collect clinical data of different Luminal types breast cancer patients and analyze the correlation of PIPKIγ and γ-catenin, and their prognostic role in breast cancer. With this study, we want to demonstrate the possible prognostic and therapeutic role of PIPKIγ in breast cancer, providing novel way for breast cancer early diagnosis and therapy.

循环肿瘤细胞簇为乳腺癌转移重要因素，与细胞粘着连接成分γ-catenin高表达相关。PIPKIγ是重要的细胞粘着连接调节因子。预实验首次证实干预PIPKIγ影响细胞γ-catenin表达，PIPKIγ沉默后动物模型中循环肿瘤细胞簇相应减少。为明确PIPKIγ在循环肿瘤细胞簇形成中作用及机制，进一步实验拟予红绿荧光分别标记乳腺癌细胞株，等比混合局部注射制备动物模型，以更好鉴别循环肿瘤细胞簇和CTC克隆，予PIPKIγ沉默或过表达，体内观察干预PIPKIγ对γ-catenin表达、循环肿瘤细胞簇形成及肿瘤转移影响。其次细胞实验研究PIPKIγ沉默是否通过γ-catenin影响细胞粘着连接与细胞簇形成。最后分析临床不同类型乳腺癌PIPKIγ与γ-catenin相关性，二者与循环肿瘤细胞簇形成及肿瘤预后关系；最终阐明干预PIPKIγ通过调节γ-catenin影响循环肿瘤细胞簇形成及乳腺癌转移机制。

循环肿瘤细胞在乳腺癌转移中有重要作用，循环肿瘤细胞簇致转移能力更强，但调控机制不详。本课题主要从细胞实验、动物实验、临床样本三个方向开展，研究进展为从三个角度证明，PIPKIγ与γ-catenin、CTC cluster相关。细胞实验证明，沉默PIPKIγ能够在蛋白及RNA水平下调γ-catenin表达，减少细胞间黏着；动物实验进一步证明，PIPKIγ沉默能下调γ-catenin表达，减少外周循环CTC Cluster形成，抑制肿瘤生长及转移；过表达γ-catenin则可逆转PIPKIγ沉默所致的表型改变。临床数据结果表明，不同luminal分型乳腺癌患者预后不同，外周CTC cluster也不同，而PIPKIγ、γ-catenin与CTC cluster呈明显正相关。γ-catenin在PIPKIγ调控肿瘤转移及CTC cluster形成过程中发挥了重要作用。.课题重要结果为证明三阴性乳腺癌中，PIPKIγ与γ-catenin、CTC cluster呈明显正相关，PIPKIγ是三阴性乳腺癌患者预后的预测因子。基于该结果，在未来转化研究中，可进一步证明PIPKIγ在三阴性乳腺癌中的作用，抑制PIPKIγ可能为三阴性乳腺癌潜在靶点。.课题关键数据为动物实验结果。通过转染shRNA至4T1乳腺癌细胞株沉默PIPKIγ表达，制备乳腺癌动物模型，证明PIPKIγ沉默减少CTC Cluster形成及肿瘤转移；同时在沉默PIPKIγ后过表达γ-catenin，以证明γ-catenin在PIPKIγ调控肿瘤转移中的作用。.该课题研究的主要科学意义为证明了PIPKIγ在乳腺癌中的作用，尤其是在三阴性乳腺癌中，PIPKIγ为判断预后的主要预测因子。PIPKIγ能够通过下调γ-catenin，减少细胞间黏附，抑制肿瘤CTC Cluster形成，进而减少肿瘤转移。PIPKIγ可能为三阴性乳腺癌潜在的治疗靶点之一。