MACC1基因沉默通过ERK通路对卵巢上皮性癌细胞顺铂敏感性的影响

发布时间：2018-06-26 05:04

本文选题：结肠癌转移相关基因1 + 卵巢癌　；参考：《郑州大学》2014年硕士论文

【摘要】：背景作为一种严重威胁女性健康和生命的恶性肿瘤，卵巢癌死亡率居妇科恶性肿瘤第一位。卵巢癌具有起病隐匿、发现晚、转移早、进展快及预后差的特点。目前卵巢癌的发病机理仍不十分清楚。由于卵巢居于盆腔底部，所以卵巢癌缺乏早期特异性症状，患者因腹胀等原因就诊时约70%已是晚期。晚期卵巢上皮性癌行肿瘤细胞减灭术和以铂类为基础的化学治疗。肿瘤细胞减灭术旨在最大限度和范围的切除肿瘤组织，但术后可能残留的病灶仍然无法避免。因此，化疗对晚期卵巢癌患者来说必不可少。但部分患者对化疗药物尤其一线药物顺铂耐药却造成治疗失败。化疗耐药又可分为内在性耐药和获得性耐药两大类：约15％-25％的卵巢癌患者对以铂类为基础的联合化疗方案内在性耐药，即为原发耐药；而至少80％的接受化疗的患者在治疗过程中逐渐对顺铂产生耐药性，称为获得性耐药，即为继发性耐药。化疗耐药严重制约着卵巢癌患者的生活质量和生存期。因此，近年来，科学研究者在不断寻找改善患者化疗敏感性的方法。 2009年，Stein等在结肠癌中发现了一个新的基因，因其与结肠癌的转移密切相关，因而这个基因被称为结肠癌转移相关基因-1(Metastasis-associated in colon cancer-1，MACCl)。MACC1是肝细胞生长因子（hepatocyte growth factor，HGF）／C-MET信号转导途径的一个重要调节因子，通过提高c-met的表达，在结肠癌的侵袭、转移过程中起着重要作用。后来，国内外科学研究者在多种人类恶性肿瘤中发现了MACC1的高表达，比如肝细胞癌、胆囊癌、胃癌、食管癌、乳腺癌等。本课题组前期研究发现，卵巢上皮性癌中检测到异于卵巢良性肿瘤及正常卵巢组织中的MACC1mRNA及蛋白的高表达，并合成了MACC1基因特异性shRNA，成功转染人卵巢癌细胞OVCAR-3，通过一系列功能实验发现转染后OVCAR-3细胞的增殖、迁移、侵袭能力减弱，而凋亡增加；通过RT-PCR和Western Blot发现转染细胞的C-met、p-ERK1/2、cyclinD1、survivin、MMP9、MMP2及VEGFA等表达显著减少，提示MACC1可能通过HGF/C-met信号通路及下游的MAPK通路来调节肿瘤相关的一系列基因表达来参与卵巢癌的恶性进展过程。但MACC1是否参与卵巢癌对顺铂敏感性的调节，目前国内外尚无文献显示。本研究通过RNA干扰（RNA interferring，RNAi）技术分别抑制人上皮性卵巢癌细胞亲本株SKOV3和相应的顺铂耐药株SKOV3/DDP细胞中MACC1表达，应用RT-PCR检测细胞中MACC1基因的mRNA的表达情况，同时采用western blot方法检测MACC1、ERK1/2、p-ERK1/2、caspase-3及cleaved caspase-3蛋白的表达，MTT法检测细胞增殖及顺铂敏感性变化，Transwell法研究细胞体外侵袭能力的变化，探讨MACC1基因抑制对上皮性卵巢癌细胞顺铂敏感性的影响，以及MACC1基因抑制后ERK通路蛋白的变化，试图进一步了解MACC1是否通过ERK通路对卵巢癌顺铂敏感性产生影响，并为寻求临床上有效逆转卵巢癌耐药提供新的思路。目的通过RNA干扰技术分别抑制SKOV3和SKOV3/DDP细胞中MACC1表达，，分析MACC1基因抑制后卵巢癌细胞增殖、凋亡及顺铂敏感性的变化，分析MACC1影响卵巢癌上述生物学行为的分子机制，为临床上逆转卵巢癌耐药提供一个新的思路。方法 1.前期设计的针对MACC1mRNA的小发卡状RNA转染卵巢癌细胞SKOV3，RT-PCR及Western blot分别检测MACC1mRNA和蛋白表达，并检测ERK1/2及p-ERK1/2蛋白含量的变化以及ERK通路抑制剂PD98059对上述蛋白水平的影响。MTT法评价细胞对顺铂化疗敏感性的变化。 2.前期设计的针对MACC1mRNA的小发卡状RNA转染卵巢癌耐药细胞SKOV3/DDP，RT-PCR及Western blot检测MACC1mRNA和蛋白表达，并检测ERK1/2及p-ERK1/2、caspase-3及cleaved caspase-3蛋白含量的变化以及ERK通路抑制剂PD98059对上述蛋白水平的影响。MTT法、FCM法和Transwell实验分别评价细胞增殖能力和顺铂化疗敏感性、凋亡的变化及细胞体外迁移能力的变化。 3.统计学处理：利用SPSS17.0软件进行统计分析。采用单因素方差分析比较各组细胞MACC1mRNA及蛋白、下游蛋白、细胞IC50、细胞凋亡率差异，采用LSD-t法行两两比较。检验水准=0.05。结果 1.在SKOV3中，转染MACC1shRNA的细胞可检测到MACC1mRNA和蛋白的低表达；p-ERK1/2表达明显降低，细胞的顺铂半数抑制浓度（IC50）明显低于空白对照组和转染空质粒组。在此基础上加入PD98059后p-ERK1/2表达及IC50改变更为明显。 2. MACC1沉默的SKOV3/DDP细胞中，MACC1mRNA和蛋白表达降低，p-ERK1/2及caspase-3表达降低，cleaved caspase-3升高。同时，MACC1沉默的SKOV3/DDP细胞增殖和迁移能力减弱、凋亡增加、对顺铂敏感性提高。PD98059能够进一步促进以上变化。结论 1、MACC1可能参与卵巢癌对顺铂的敏感性调节 2、MACC1可能通过下游的ERK通路调节卵巢癌的增殖、凋亡、迁移及顺铂敏感性
[Abstract]:background
As a malignant tumor that seriously threatens the health and life of women, the mortality of ovarian cancer is the first in gynecologic malignancies. Ovarian cancer has the characteristics of insidious onset, late discovery, early metastasis, rapid progress and poor prognosis. The pathogenesis of ovarian cancer is still not very clear. "Heterosexual symptoms, about 70% of patients with abdominal distention are advanced. Advanced ovarian epithelial cancer is treated with tumor cell subtraction and platinum based chemotherapy. Tumor cell subtraction is designed to maximize and range the tumor tissue, but the possible residual focus remains unavoidable. Therefore, chemotherapy for advanced ovary." Cancer patients are essential. But some patients are resistant to chemotherapy, especially cisplatin, which can be divided into two major categories: intrinsic and acquired resistance: approximately 15%-25% of ovarian cancer patients are resistant to platinum based combined chemotherapy, that is, primary drug resistance; at least 80% of patients receiving chemotherapy are gradually resistant to cisplatin during the treatment, known as acquired resistance, namely, secondary resistance. Chemotherapeutic resistance severely restricts the quality of life and life of ovarian cancer patients. Therefore, in recent years, scientific researchers are constantly looking for ways to improve chemotherapy sensitivity.
In 2009, Stein found a new gene in colon cancer, which was closely related to the metastasis of colon cancer, so the gene called the colon cancer metastasis related gene -1 (Metastasis-associated in colon cancer-1, MACCl).MACC1 is a hepatocyte growth factor (hepatocyte growth factor, HGF) / signal transduction pathway. Important regulatory factors play an important role in the invasion and metastasis of colon cancer by improving the expression of c-met. Later, researchers at home and abroad found high expression of MACC1 in many human malignant tumors, such as hepatocellular carcinoma, gallbladder cancer, gastric cancer, esophagus cancer, breast cancer, etc. The high expression of MACC1mRNA and protein in the ovarian benign tumor and normal ovarian tissue was detected, and the MACC1 gene specific shRNA was synthesized, and the human ovarian cancer cell OVCAR-3 was transfected successfully. Through a series of functional experiments, the proliferation, migration, emplacement ability and apoptosis of OVCAR-3 cells after transfection were found to be increased by RT-PCR and Weste. RN Blot found that the expression of C-met, p-ERK1/2, cyclinD1, survivin, MMP9, MMP2 and VEGFA in transfected cells decreased significantly, suggesting that MACC1 may regulate a series of tumor related gene expressions through the HGF/C-met signaling pathway and downstream MAPK pathway to participate in the malignant progression of ovarian cancer. There is no literature at home and abroad at present.
In this study, the expression of MACC1 in human epithelial ovarian cancer cell line SKOV3 and corresponding cisplatin resistant strain SKOV3/DDP cells was suppressed by RNA interference (RNA interferring, RNAi). RT-PCR was used to detect the expression of mRNA in the MACC1 gene in the cells, and the Western blot method was used to detect the expression of mRNA. The expression of ED caspase-3 protein, the change of cell proliferation and cisplatin sensitivity by MTT method, the change of cell invasiveness in vitro by Transwell method, the effect of MACC1 gene inhibition on cisplatin sensitivity of epithelial ovarian cancer cells, and the changes of ERK pathway protein after the inhibition of MACC1 gene, and try to further understand whether MACC1 is through ERK pass. It has an effect on cisplatin sensitivity of ovarian cancer and provides a new way of thinking to effectively reverse the drug resistance of ovarian cancer.
objective
The expression of MACC1 in SKOV3 and SKOV3/DDP cells was inhibited by RNA interference technique, and the changes of proliferation, apoptosis and cisplatin sensitivity of ovarian cancer cells after the inhibition of MACC1 gene were analyzed. The molecular mechanism of MACC1 affecting the biological behavior of ovarian cancer was analyzed, which provided a new idea for clinical reversal of ovarian cancer tolerance.
Method
1. a small hairpin RNA transfected into ovarian cancer cell SKOV3, RT-PCR and Western blot to detect the expression of MACC1mRNA and protein respectively, and to detect the changes of ERK1/2 and p-ERK1/2 protein content and the effect of ERK pathway inhibitor PD98059 on the above protein level, and the changes in sensitivity of cell to cisplatin chemotherapy were evaluated in 1..
2. the small hairpin RNA transfected to MACC1mRNA, SKOV3/DDP, RT-PCR and Western blot were designed to detect the expression of MACC1mRNA and protein, and the changes of ERK1/2 and p-ERK1/2, caspase-3 and cleaved caspase-3 protein content and the effect of the inhibitor on the protein level were detected. Well assay was used to evaluate cell proliferation and cisplatin chemosensitivity, apoptosis and cell migration in vitro.
3. statistical analysis: statistical analysis was carried out by SPSS17.0 software. Single factor analysis of variance was used to compare MACC1mRNA and protein, downstream protein, cell IC50, cell apoptosis rate, and LSD-t method was used to compare 22. Test level =0.05.
Result
1. in SKOV3, the cells transfected with MACC1shRNA could detect the low expression of MACC1mRNA and protein, the expression of p-ERK1/2 was obviously decreased, and the half inhibitory concentration of cisplatin (IC50) was significantly lower than that of the blank control group and the transfected empty plasmid group. On this basis, the expression of p-ERK1/2 and the change of IC50 were more obvious after adding PD98059.
In 2. MACC1 silent SKOV3/DDP cells, the expression of MACC1mRNA and protein decreased, the expression of p-ERK1/2 and caspase-3 decreased, and cleaved caspase-3 increased. At the same time, the proliferation and migration ability of MACC1 silenced SKOV3/DDP cells decreased, the apoptosis increased, and the sensitivity of cisplatin was enhanced by.PD98059.
conclusion
1, MACC1 may be involved in the sensitivity of ovarian cancer to cisplatin.
2, MACC1 may regulate the proliferation, apoptosis, migration and cisplatin sensitivity of ovarian cancer through the downstream ERK pathway.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

【参考文献】