新的潜在卵巢癌耐药信号转导途径上游分子A2M和CRABP2调控铂类耐药机制研究

Platinum-resistant play an important role of poor prognosis in ovarian cancer. A2M and CRABP2 were considered the potential upstream molecules for inhibiting platinum-resistant by using an integrative analysis of system biology and clinical data. we hypothesize a new mechanism that A2M and CRABP2 may inactive signalings of folate metabolism, cell adhesion, FGFRs, MAPKs and Wnt through via cytokines of tumor microenvironment. The aims of this project is to understand molecular mechanism of A2M and CRABP2 of inhibiting platinum-resistant by using three-dimensional co-culture and mouse model. We plan to investigate: 1) Gene silencing and overexpression of A2M and CRABP2 have a noticeable effect on the platinum-resistant phenotype of ovarian cancer cell line SKOV3/DDPⅡ. 2) The screening and verification of cytokines and proteins related folate metabolism, cell adhesion, proliferation and apoptosis in three-dimensional co-culture and mouse model. We hope to find out the downstream cytokines and pathways of A2M and CRABP2, and prove that A2M and CRABP2 are upstream signaling inhibitiors of platinum-resistant in ovarian cancer. It aslo demonstrate that cytokines of microenvironment are related with platinum-resistant.the This results will help to produce a new biological treatment for platinum-resistant reversal.

卵巢癌铂类耐药是患者预后不良的主要原因。A2M和CRABP2是前期整合系统生物学和临床数据得到的两个潜在抑制铂类耐药分子,推测A2M和CRABP2通过调控肿瘤微环境中的细胞因子而失活细胞下游多条铂类耐药相关途径的方式抑制铂类耐药产生。本项目拟用细胞三维共培养和裸鼠移植瘤模型研究A2M和CRABP2调控巢癌铂类耐药的分子机制。观察过表达和沉默A2M/CRABP2基因的卵巢癌耐药细胞株SKOV3/DDPⅡ耐药表型和DMA损伤修复基因的变化，筛选和验证模型中细胞因子及叶酸代谢、细胞粘附、MAPK、FGFRs、 Wnt和FAS途径相关蛋白变化，明确A2M和CRABP2具体调控的细胞因子和下游信号通路，证明A2M和CRABP2是抑制卵巢癌铂类耐药上游关键分子，其调控机制与肿瘤微环境的细胞因子相关，有助于产生新的逆转铂类耐药治疗方案。

卵巢癌铂类耐药是患者预后不良的主要原因。A2M和CRABP2是前期整合系统生物学和临床数据得到的两个潜在抑制铂类耐药分子,推测A2M和CRABP2可能通过调控肿瘤微环境中的细胞因子激活FAS凋亡途径的方式抑制铂类耐药产生。本项目将应用三维共培养模型和裸鼠移植瘤模型研究A2M和CRABP2调抑制巢癌铂类耐药的分子机制，明确A2M和CRABP2具体调控的下游细胞因子和细胞铂类耐药信号通路，研究结果如下：.1.利用课题组收集的卵巢癌组织标本制作组织芯片，将前期筛选出的可能成为卵巢癌铂类耐药信号途径的上游信号分子的A2M和CRABP2进行临床预后相关性分析，初步肯定二者在卵巢癌铂类耐药中发挥潜在的作用。.2. 在前期建立的裸鼠卵巢癌铂类耐药实验动物平台的基础上分别建立的卵巢癌铂类耐药细胞株， A2M蛋白增加SKOV3-GFP/DDPⅡ细胞对顺铂敏感性， 该作用机制是通过诱导肿瘤TGF-β1活化AMPK途径达到。.3．在前期建立的裸鼠卵巢癌铂类耐药实验动物平台的基础上分别建立了裸鼠的卵巢癌铂类敏感和耐药皮下移植肿瘤模型，利用慢病毒表达系统使顺铂耐药的卵巢癌细胞过表达CRABP2基因，证实过表达CRABP2基因后，细胞的顺铂耐药被逆转，细胞的增殖受阻、肿瘤微环境中细胞因子FGF，TNF增加，Fas凋亡信号传导通路重新被显著激活。.4. 体内外基因功能研究证明A2M和CRABP2均活化肿瘤微环境中的细胞因子，激活FAS凋亡信号通路，诱导细胞凋亡，逆转卵巢癌细胞铂类耐药。A2M在人体血浆中表达，研究表明铂类敏感肿瘤机体的A2M维持较高水平，而耐药肿瘤组织A2M急速下降，A2M可作为判别化疗效果的一个潜在临床指标。CRABP2有效的抑制PARP活性，可成为逆转铂类耐药的生物治疗新分子。