CFL1基因Ser3位点磷酸化通路参与调控卵巢癌紫杉醇耐药的机制研究

The acquisition of chemoresistance is a major therapeutic obstacle in the clinical treatment of ovarian cancer.The mechanism of drug resistance have not been discovered. In our previous study, we demonstrated that the protein, phosphorylated cofilin1 (p-CFL1) correlates with taxol resistance in ovarian cancer cells and primary ovarian cancer tissues. However, higher taxol resistance was not observed in the ovarian cancer cells with overexpression of CFL1 by mutant form of CFL1 (Ser3 is replaced for inactive phosphorylation) compared with wild-type form of CFL1. Phosphorylation of Ser3 regulates essential function of cofilin. We assume that phosphorylation of cofilin at the Ser3 can induce paclitaxel resistance in ovarian cancer. Several experiments will be carried out to demonstrate our hypothesis:(1) To indentify the phosphorylation of CFL1 is one of the reason to induce taxol resistance. CFL1 mutants with Ser3 replacement will be transfected into ovarian cells to increase the expression of CFL1 with active/inactive phosphorylated function. Migration, invision, mitotic cycle, growth rate, IC50 and resistant index(RI) of parental cells and mutants will be compared.(2)Select the most related phosphokinases from all the phosphokinase of CFL1 as the candidates,which regulate CFL1 phosphorylation,by molecular biotechnology and phosphorylation assay in vitro. Develope/knock-down the activity of Ser3 in the cells with develop/knock down expression of candidate gene,then the change in cell migration, invision, mitotic cycle, growth rate, and resistant index(RI) will be observed. (3) The expression of phosphokinase of CFL1 will be indentified in the chemosensitve and chemoresistant primary human ovarian cancer tissues by immunohistochemical assay. These data will illuminate the relativity between the phosphorylation of CFL1 and taxol resistance. It will provide new idear on mechanism in drug resistance, and a good experimental basis for candidate for drug resistance reversal.

化疗耐药是卵巢癌治疗中的主要障碍,卵巢癌耐药机制尚不明确。我们前期工作中发现磷酸化cofilin1（CFL1）表达与卵巢癌细胞的紫杉醇耐药呈正相关，且当CFL1磷酸化位点（Ser3）失活后，过表达CFL1的细胞耐药性无改变。Ser3是调控CFL1功能的关键磷酸化位点，由此我们推测CFL1基因Ser3位点磷酸化通路参与调控卵巢癌紫杉醇耐药。本研究拟通过以下工作验证此推论：1）明确CFL1磷酸化对紫杉醇耐药的作用：通过增强/抑制Ser3位点活性改变卵巢癌细胞CFL1磷酸化水平，检测细胞运动、迁移能力和耐药性改变；2）通过分子生物学技术筛选CFL1磷酸化的候选上游调控因子：使细胞过表达/表达沉默候选因子，再抑制/增强Ser3位点活性，检测细胞迁移能力和耐药性变化，明确候选因子对CFL1磷酸化及耐药的影响；3）在临床标本中验证候选因子的表达。本研究为寻找耐药机制提供新思路，为逆转耐药提供候选靶点。

目的 化疗耐药是卵巢癌治疗中的主要障碍。卵巢癌的耐药机制尚不明确。我们在前期工作中发现磷酸化cofilin1（CFL1）在卵巢癌耐药组织和细胞中表达上调。在本研究中我们证实CFL1基因的Ser3位点磷酸化参与了巢癌紫杉醇耐药机制。方法 1)构建Ser3位点磷酸化功能差异的CFL1过表达突变体：野生型（WT）、具有磷酸化功能（S3A）, 磷酸化功能失活（S3D）和CFL1降调质粒shRNA-CFL。检测磷酸化cofilin1表达差异对卵巢癌SKOV3细胞生物学特性和紫杉醇耐药性的影响；2) 筛选出CFL1上游候选磷酸化调控蛋白，并研究它对细胞磷酸化cofilin1的表达和耐药性的影响；3）收集初治卵巢癌化疗患者病理标本及临床病历资料，采用免疫组织化学方法比较化疗敏感和耐药组病理切片中候选调控蛋白的表达差异，及其对卵巢癌预后的价值。结果 1）过表达CFL1的SKOV3细胞，与对照组细胞比较，G0/G1期细胞显著增多（P=0.034），S期细胞显著减少（P=0.031)；细胞凋亡率明显降低（P=0.020）；细胞侵袭性增加；对紫杉醇的耐药性增加。但当ser3位点磷酸化活性被抑制时，以上改变消失；当SKOV3细胞CFL1表达下调时，细胞凋亡率显著升高（P=0.020）；细胞侵袭性和耐药性下降；2）通过实时定量PCR技术筛选出SSH1L、SSH3为与紫杉醇耐药相关的cofilin1磷酸化调控基因。当SKOV3细胞过表达SSH1L或SSH3时，细胞内p-cofilin1表达下调，凋亡率升高，细胞耐药性下降；在敲降的SSH1L细胞内， p-cofilin1表达上调，细胞凋亡率下降、耐药性升高；3）共获得临床病例63例，其中化疗敏感组35例、耐药组28例。SSHIL在化疗敏感组和耐药组卵巢癌组织病理切片中的阳性表达率分别为57.1% (20/35) &17.9% (5/28)， 差异有统计学意义(X2=6.829，P=0.011)；而SSH3在化疗敏感组和耐药组卵巢癌组织病理切片中的表达无显著差异。SSH1L高表达的患者，较低表达组，无进展生存时间（PFS）显著缩短（X2=23.44，P<0.01，95%CI：0.069～0.183)。结论SSHIL调控的Ser3位点的cofilin1的磷酸化与卵巢癌紫杉醇耐药密切相关。