【作者】 张楠；

【导师】 王明臣；

【作者基本信息】 郑州大学 ， 生物化学与分子生物学， 2021， 硕士

【摘要】 背景和目的乳腺癌是全世界女性最常见的恶性肿瘤。在女性中,乳腺癌占所有癌症发病率的25%,占所有癌症死亡人数的15%。尽管外科手术和化学疗法已经取得了显著进展,但晚期乳腺癌患者的长期生存率仍然很低,这主要归因于乳腺癌的复发或远处转移。尽管在乳腺癌的病因学和发病学研究中,已发现大量致癌基因和抑癌基因参与了乳腺癌的发生发展,但是仍需深入探索揭示乳腺癌发生和发展的分子机制,希冀获取人乳腺癌干预防治的潜在分子靶点,这具有重要的理论及实用意义。目前研究表明,乳腺癌的发病机制很复杂,由多种细胞信号分子介导并存在交叉对话(cross-talk),例如丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)、磷酸肌醇3-激酶(Phosphoinositide3-kinase,PI3K)以及蛋白激酶B(Protein kinase B,Akt)等信号分子皆关联乳腺癌的发生发展。在人类诸多肿瘤研究中,PI3K/Akt信号途径通常表达异常。该通路中某些信号分子的突变亦可导致通路功能和细胞生物学行为的改变。它不仅可以调节肿瘤细胞的存活和增殖,而且与肿瘤的侵袭、转移、耐药性等密切相关。在肿瘤发生的分子机制研究中,Ras家族在肿瘤细胞生物学行为中的作用成为研究热点,且Ras家族对靶基因的调控机制和作用途径更是阐明肿瘤发展机制的研究重点。Ras家族是Ras超家族的一个重要分支。Ras超家族蛋白介导多种细胞生物学效应,例如细胞增殖、凋亡等。Ras超家族蛋白与乳腺癌之间的相关性研究表明,Ras超家族蛋白与患者的病理数据和预后密切相关。RhebL1系Ras超家族成员之一,其与乳腺癌细胞生物学行为改变的关系是本课题研究的重要切入点。本研究旨在分析人乳腺癌MCF-7和MDA-MB-231细胞系中RhebL1的表达水平,探讨RhebL1对人乳腺癌细胞增殖、凋亡、侵袭与转移等生物学行为的影响,深入揭示RhebL1影响乳腺癌细胞生物学行为及乳腺癌发生发展的分子机制,期望为乳腺癌的有效干预防治提供新的靶点。方法1 RhebL1在人乳腺癌细胞中的表达1.1采用实时荧光定量PCR法在m RNA水平检测乳腺癌细胞系MCF-7、MDA-MB-231和正常乳腺上皮细胞系MCF-10A中RhebL1的表达情况。1.2采用Western Blot法在蛋白水平检测乳腺癌细胞系MCF-7、MDA-MB-231和正常乳腺上皮细胞系MCF-10A中RhebL1的表达情况。2 RhebL1对乳腺癌细胞恶性生物学行为的影响及机制研究2.1建立RhebL1稳转敲减乳腺癌细胞系,利用CCK8法检测乳腺癌细胞增殖情况;利用裸鼠皮下成瘤法检测RhebL1在体内对乳腺癌细胞增殖的影响;利用流式细胞术检测细胞凋亡水平变化;利用划痕实验检测细胞迁移能力变化;利用Transwell法检测细胞侵袭能力变化。2.2 Western Blot实验技术检测在肿瘤生物学功能中发挥重要作用的相关蛋白p21及p27。2.3有变化趋势的蛋白与PI3K/Akt信号通路密切相关,我们用Western Blot检测PI3K/Akt通路相关蛋白Bax、Bcl2、PI3K、p-Akt及Akt。2.4应用PI3K/Akt信号通路的蛋白激酶抑制剂LY294002进行干预处理,应用CCK8、Transwell实验方法来检测LY294002对乳腺癌细胞增殖和迁移的作用,应用Western Blot检测相关蛋白变化。2.5使用Akt抗体和RhebL1抗体进行Co-IP实验,揭示RhebL1与Akt的相互作用。结果1 RhebL1在人乳腺癌细胞中的表达RhebL1在正常乳腺细胞和乳腺癌细胞中均有表达,且在乳腺癌细胞中的表达明显高于正常乳腺细胞(P<0.05)。2 RhebL1对乳腺癌细胞恶性生物学行为的影响及机制研究2.1下调RhebL1的表达抑制乳腺癌细胞的增殖、迁移及侵袭能力在乳腺癌细胞中下调RhebL1的表达能明显抑制细胞活力;敲低RhebL1的乳腺癌细胞凋亡率明显升高;敲低RhebL1后的乳腺癌细胞迁移和侵袭能力显著降低(P<0.05)。2.2下调RhebL1的表达抑制PI3K/Akt通路的活性下调乳腺癌细胞中RhebL1时能明显调控PI3K/Akt通路相关蛋白活性,Bcl2、PI3K和p-Akt蛋白表达水平明显降低,Bax蛋白表达水平显著升高(P<0.05);Akt蛋白表达情况无明显差异(P>0.05)。2.3 RhebL1通过Akt调控PI3K/Akt信号通路PI3K/Akt信号通路的蛋白酶抑制剂LY294002抑制了乳腺癌细胞的增殖和侵袭能力。应用PI3K/Akt信号通路的蛋白酶抑制剂LY294002后,乳腺癌细胞中Bcl2、PI3K和p-Akt蛋白表达水平明显降低,Bax、p21和p27蛋白表达水平显著升高(P<0.05);Akt蛋白表达情况无明显差异(P>0.05)。免疫共沉淀实验表明,RhebL1与Akt存在互作。结论1.RhebL1在乳腺癌细胞中高表达。2.下调RhebL1能影响乳腺癌细胞增殖、凋亡、迁移及侵袭等生物学行为。3.RhebL1干预影响人乳腺癌细胞生物学行为的改变可能是通过介导PI3K/Akt信号通路来实现的。更多还原

【Abstract】 Background and Objective:Breast cancer is the most common malignant tumor in women all over the world,accounting for 25% of all cancer incidences in women and 15% of all cancer deaths.Despite advances in surgery and chemotherapy,the long-term survival rate of patients with advanced breast cancer is still very low,mainly due to tumor recurrence or distant metastasis.Although a large number of oncogenes and tumor suppressor genes have been found to be involved in the development of breast cancer,in order to clarify the potential targets of breast cancer treatment,it is still necessary to explore the potential molecular mechanisms in the development and development of breast cancer.Current studies have shown that the pathogenesis of breast cancer is complex,mediated by a variety of cell signaling molecules and cross-talk,such as mitogen-activated protein kinase(MAPK),phosphoinositide3-kinase(PI3K)and protein kinase B(Akt)are all related to the occurrence and development of breast cancer.In many human tumor studies,the PI3K/Akt signaling pathway is usually expressed abnormally.Mutations in certain signaling molecules in this pathway can also lead to changes in pathway function and cell biological behavior.It can not only regulate the survival and proliferation of tumor cells,but is also closely related to tumor invasion,metastasis,and drug resistance.With the continuous research on breast cancer and other tumors expanding,people have eventually be aware of the role of the Ras superfamily in tumorigenesis.The Ras superfamily proteins mediate various cellular effects,such as cell proliferation and apoptosis.Researches on the correlation between ras superfamily proteins and breast cancer have illustrated that ras superfamily proteins are closely related to the pathological data and prognosis of patients,among which.RhebL1 is a member of the Ras superfamily.The association of RhebL1 with breast cancer is the research priority.The objective of this study is to clarify the correlation between RhebL1 expression level and breast cancer,to distinguish the effect of RhebL1 on breast cancer cell proliferation,invasion and apoptosis,and to explore the influence of RhebL1 on breast cancer cell biological behaviors and the mechanism of occurrence and development in breast cancer,in the interest of providing new targets for the diagnosis and treatment of breast cancer.Methods:1 Expression of RhebL1 in human breast cancer cells1.1 RT-q PCR technology was used to detect the expression of RhebL1 in breast cancer cell lines MCF-7,MDA-MB-231 and normal breast epithelial cell line MCF-10 A at the m RNA level.1.2 Western Blot technology was used to detect the expression of RhebL1 in breast cancer cell lines MCF-7,MDA-MB-231 and normal breast epithelial cell line MCF-10 A at the protein level.2 The Effect of RhebL1 on the Malignant Biological Behavior of Breast Cancer Cells and Its Mechanism2.1 Establish a stable RhebL1 knockdown breast cancer cell line.The CCK8 method is used to detect breast cancer cell proliferation.By subcutaneously injecting breast cancer cells into nude mice,the effect of RhebL1 on the proliferation of breast cancer cells in vivo was detected.Flow cytometry is used to detect changes in the level of apoptosis.Transwell methodand scarification was used to evaluate influence of RhebL1 expression on breast cancer cell invasion and metastasis.2.2 Western Blot experimental technology is used to detect important proteins p21 and p27 that play an important role in tumor biological functions.2.3 Proteins with changing trends are closely related to the PI3K/Akt signaling pathway.We use Western Blot to detect PI3K/Akt pathway-related proteins2.4 We use the protein kinase inhibitor LY294002 of the PI3K/Akt signaling pathway to intervene,after which,CCK8 and Transwell are carried out to detect its effects on breast cancer cell proliferation and migration,with Western Blot performed to detect the changes of related proteins in the purpose of clarifying the mechanism of RhebL1 in breast cancer.2.5 Co-IP was performed using Akt antibody and RhebL1 antibody to reveal the interaction between RhebL1 and Akt.Results:1 Expression of RhebL1 in human breast cancer cells RhebL1 is expressed in both normal breast cells and breast cancer cells,with the expression in breast cancer cells significantly surpassing that in normal breast cells(P<0.05).2 The Effect of RhebL1 on the Malignant Biological Behavior of Breast Cancer Cells and Its Mechanism2.1 Down-regulating the expression of RhebL1 inhibits the proliferation,migration and invasion of breast cancer cells Knockdown of RhebL1 can inhibit markedly the viability of MCF-7 and MDA-MB-231 cells;knockdown of RhebL1 considerably increases the apoptosis rate of breast cancer cells;knockdown of RhebL1 substantially reduces breast cancer’s capabilities in migration and invasion.(P<0.05)2.2 Down-regulating the expression of RhebL1 inhibits the activity of the PI3K/Akt pathway Down-regulating RhebL1 in breast cancer cells can significantly regulate the activity of PI3K/Akt pathway related proteins.The expression levels of Bcl2,PI3 K and p-Akt protein are significantly reduced(P<0.05).And the expression level of Bax protein is significantly increased(P<0.05).There is no significant difference in Akt protein expression(P >0.05).2.3 RhebL1 regulates PI3K/Akt signaling pathway through Akt PI3K/Akt signaling pathway protease inhibitor LY294002 inhibits the proliferation and invasion of breast cancer cells.After application of PI3K/Akt signaling pathway protease inhibitor LY294002,the expression levels of Bcl2,PI3 K and p-Akt proteins in breast cancer cells were significantly reduced.And the expression levels of Bax,p21 and p27 proteins were significantly increased(P <0.05).There is no significant difference in Akt protein expression(P >0.05).Co-IP shows that RhebL1 interacts with Akt.Conclusion:1.RhebL1 is highly expressed in breast cancer cells.2.Down-regulation of RhebL1 can affect the biological behaviors of breast cancer cell proliferation,apoptosis and invasion.3.RhebL1 promotes the proliferation and migration of breast cancer through the PI3K/Akt signaling pathway.更多还原