CD74调控乳腺癌细胞迁移的分子机制
发布时间：2018-09-07 10:05
【摘要】：研究背景CD74又称为Ii,是一个多功能的细胞因子。作为MHC class Ⅱ的恒定链,CD74参与MHC class Ⅱ所介导的抗原呈递过程,不仅在内质网中辅助MHC class Ⅱ的正确折叠,调控MHC class Ⅱ出内质网,也能调控MHC class Ⅱ从高尔基体转运到抗原加工小室。作为MIF的受体,CD74不仅参与MIF对炎症因子的调控,也参与MIF所介导的抑制细胞凋亡,促进存活过程。此外,CD74也与多种人类疾病相关,其中包括自身免疫疾病,如系统性红斑狼疮,也包括动脉粥样硬化、阿尔茨海默病以及癌症。研究表明CD74在包括乳腺癌在内的多种肿瘤中过表达,并且与透明细胞肾细胞癌、胃癌和胰腺癌的侵袭有关。近期研究表明CD74在乳腺癌中过表达,并且与乳腺癌的淋巴结转移和三阴性乳腺癌呈线性相关,但尚未见相关分子机制报道。CD44是透明质酸受体,当透明质酸与CD44结合后可以促进CD44直接或通过接头蛋白与受体酪氨酸激酶相互作用,并激活下游信号通路,如PI3K-AKT信号通路和MAPK级联反应,主要参与调控细胞凋亡、存活和增殖等,并与肿瘤细胞的抗药性相关。CD44还能够通过接头蛋白与Ras超家族的小GTPase蛋白相互作用,促进RHOA、RAC等RHO GTPases的活性,参与调控细胞骨架的组装或重排。肿瘤细胞的干性也与CD44相关,高表达CD44的人类肿瘤细胞具有恶性程度高以及耐药性的特点,乳腺癌细胞中所分离出来的CD44+ CD24-抗原表型的细胞亚群所表现出来的干细胞特性通过上调EMT相关的细胞因子(如TWIST、SNAIL等)来实现。RHO GTPases属于小GTPase Ras超家族的成员,根据氨基酸序列的相似性划分为8个亚家族,其中RHOA、RAC1和CDC42获得广泛研究。RHOA通过其下游效应蛋白ROCK调控肌球蛋白轻链的磷酸化,磷酸化的肌球蛋白轻链可以增强肌球蛋白ATPase的活性来诱导肌球蛋白收缩。另外,RHOA也可以通过ROCK调控下游LIMK的磷酸化,激活的LIMK对其主要下游底物CFL进行磷酸化,调控细胞骨架组装或重排。PAK是RAC1和CDC42的共同下游效应蛋白,激活的PAK通过LIMK调控CFL的磷酸化,并影响细胞骨架的稳定性。此外,RAC1的效应蛋白WAVE可以对ARP2/3进行调控,促进actin的聚合和actin骨架形成。CDC42则通过WASP来调控ARP2/3介导的actin骨架的组装。目前已经发现RHO GTPases在多种人类肿瘤中过表达,并且与肿瘤的侵袭和转移相关。CFL是一个actin结合蛋白,具有两种形式,无切割活性的Ser3位点磷酸化形式和具有活性的非磷酸化形式。活化的CFL倾向于与F-actin中负极端的GDP·actin结合,并对F-actin切割,促进F-actin解聚,产生大量的G-actin单体以及游离的F-actin末端,而G-actin会在结合GTP后被添加到正在延伸的F-actin中,促进F-actin聚合,所产生的游离F-actin末端则会被ARP2/3复合物再次利用,促进F-actin的成核、延伸或actin网状分支的形成。因此CFL对细胞骨架具有聚合和解聚的双重调控作用。本研究在于明确CD74是否通过调控细胞骨架相关蛋白,影响乳腺癌细胞的侵袭和转移。研究方法1.通过免疫组化和临床数据分析,检测CD74在乳腺癌细胞中的分布以及与乳腺癌的关系。2.细胞侵袭实验和细胞迁移实验检测CD74敲低后对乳腺癌细胞侵袭和迁移能力的影响。3.TRITC-phalloidin对F-actin进行染色,激光共聚焦显微镜(CLSM)下检测CD74敲低或过表达时对乳腺癌细胞细胞突出形成的影响。4.抑制或过表达CD74后,western blot检测细胞骨架调控相关蛋白的变化。5.免疫荧光和免疫共沉淀实验检测CD74与CD44的相互作用。6.CD74或/和CD44敲低,western blot检测CD74和CD44对CFL1磷酸化水平的影响。7.CD74 或/和 CD44 敲低,TRITC-phalloidin 对 F-actin 进行染色,CLSM 下检测乳腺癌细胞细胞突出形成的变化。8.过表达CD74的情况下敲低CD44,western blot检测CD74与CD44的调控关系。9.敲低CD74的情况下加入MG132,western blot检测CD74对CD44稳定性的影响。10.利用特异siRNA或抑制剂抑制MIF,western blot检测MIF对CFL1磷酸化水平的影响。11.免疫共沉淀实验检测MIF过表达减弱CD74与CD44的结合。12.乳腺癌细胞中转染RHO GTPase家族成员相关质粒,western blot检测RHO GTPase家族成员对CD74介导的CFL1磷酸化影响。13.细胞迁移实验检测RHOA对CD74介导的乳腺癌细胞迁移能力的影响。14.利用慢病毒包装体系制备病毒,并感染MDA-MB-231细胞,筛选获得CD74稳定敲低细胞系。15.裸鼠移植瘤实验检测CD74对肿瘤形成和转移的影响。实验结果1.BIDC中CD74在质膜和细胞质中的表达水平普遍偏高,CD74与乳腺癌以及乳腺癌的临床阶段和淋巴结转移关系密切。2.在MDA-MB-231细胞中利用含CD74 shRNA的质粒敲低CD74,细胞侵袭实验表明敲低CD74可以抑制MDA-MB-231细胞的侵袭能力。3.在MDA-MB-231细胞中利用含CD74 shRNA的质粒敲低CD74,细胞划痕实验表明敲低CD74可以抑制MDA-MB-231细胞的迁移能力。4.MDA-MB-231 细胞(或 T47D 细胞)中敲低 CD74,TRITC-phalloidin 对 F-actin进行染色,CLSM下观察细胞突出的形成,结果表明CD74敲低后细胞突出的形成受到抑制。5.在MCF-7细胞中过表达CD74,TRITC-phalloidin对F-actin进行染色,CLSM下观察细胞突出的形成,结果表明过表达CD74可以促进MCF-7细胞细胞突出的形成。6.利用含CD74 shRNA的质粒在CD74高表达的MDA-MB-231细胞中敲低CD74,检测到与细胞骨架调控相关的ROCK1和p-CFL1蛋白水平的下调。T47D细胞中重复上述实验,获得了一致结果。7.利用pcDNA3.1-CD74质粒在低表达CD74的MCF-7细胞中过表达CD74,检测到与细胞骨架调控相关的ROCK1和p-CFL1蛋白水平的上调。HCC1806细胞中重复上述实验,获得了一致结果。8.免疫荧光实验表明,MDA-MB-231和T47D细胞中AlexaFluor 568染料标记的CD74与AlexaFluor488染料标记的CD44分别存在共定位。9.在 MDA-MB-231 细胞(或 T47D 细胞)中过表达 pcDNA3.1-Flag-CD74,免疫共沉淀实验表明CD74和CD44存在相互作用。10.MDA-MB-231细胞中利用细胞内源的CD44进行免疫共沉淀实验同样证实CD74和CD44存在相互作用。11.利用 CD44 特异的 siRNA 在 MDA-MB-231 细胞中敲低 CD44,western blot检测到CD74、ROCK1和p-CFL1蛋白水平的下调。T47D细胞中重复上述实验获得了一致结果。12.MDA-MB-231 细胞中利用含 CD74 shRNA 的质粒(pLT-shCD74#1 和pLT-shCD74#2)敲低 CD74 或/和利用 CD44 特异的 siRNA 敲低 CD44,western blot检测到CD74或/和CD44的敲低会引起CFL1磷酸化水平的下调。T47D细胞中重复上述实验获得了一致结果。13.MDA-MB-231 细胞中利用含 CD74 shRNA 的质粒(pLT-shCD74#1和pLT-shCD74#2)敲低CD74或/和利用CD44特异的siRNA敲低CD44,TRITC-phalloidin对F-actin进行染色,CLSM下观察细胞突出的形成,结果表明敲低CD74或/和CD44可以抑制MDA-MB-231细胞细胞突出的形成。T47D细胞中重复上述实验获得了一致结果。14.在MDA-MB-231细胞(或T47D细胞)中利用pcDNA3.1-CD74质粒过表达CD74并利用CD44特异的siRNA敲低CD44,western blot结果表明CD44的下调能够抑制由于CD74过表达所引起的CFL1磷酸化水平的上调。15.MDA-MB-231细胞中敲低CD74能够引起CD44蛋白水平的下调,而当加入不同浓度的MG132后CD44的下调受到抑制。16.利用MIF特异的siRNA或MIF特异性的抑制剂ISO-1抑制MIF,以及通过pcDNA3.1-MIF质粒过表达MIF,结果表明MIF蛋白水平与CFL1的磷酸化水平呈负相关。17.HEK 293FT 细胞中同时过表达 pcDNA3.1-Flag-CD74 和 pcDNA3.1-MIF,免疫共沉淀实验结果表明MIF过表达的情况下,CD74与CD44的相互作用减弱。18.MCF-7细胞中CD74过表达引起的CFL1磷酸化水平上调可以被RHO GD1或RHOAN19所抑制:MDA-MB-231细胞中CD74敲低所引起的CFL1磷酸化水平的下调可以被RHOAL63所抑制。19.MDA-MB-231细胞中敲低CD74并过表达RHOAL63,细胞迁移实验表明RHOAL63能够抑制CD74敲低引起的细胞迁移能力的下调。MCF-7细胞中过表达CD74和RHOAN19,细胞迁移实验表明RHOAN19能够抑制CD74过表达引起的细胞迁移能力的上调,免疫荧光实验表明RHOAN19能够抑制CD74过表达条件下细胞突出的形成。20.裸鼠分为3组,分别进行腹部皮下注射MDA-MB-231pLT-shCTRL,MDA-MB-231 pLT-shCD74#1 或 MDA-MB-231 pLT-shCD74#2 细胞,成瘤实验表明CD74的敲低能够抑制肿瘤的生长,且MDA-MB-231 pLT-shCTRL对照细胞注射组裸鼠发现一例肝转移。21.裸鼠分为3组,分别进行尾静脉注射MDA-MB-231 pLT-shCTRL,MDA-MB-231 pLT-shCD74#1 或 MDA-MB-231 pLT-shCD74#2 细胞,MDA-MB-231 pLT-shCTRL对照细胞注射组裸鼠发现2例肺出现转移结节。结论1.CD74与乳腺癌以及乳腺癌的临床阶段和淋巴结转移之间关系密切。2.CD74敲低能够抑制乳腺癌细胞迁移和侵袭能力。3.CD74的表达水平与细胞突出的形成正相关。4.CFL1的磷酸化水平与CD74的表达呈正相关。5.乳腺癌细胞中CD74与CD44存在共定位。6.CD74和CD44共同调控CFL1的磷酸化水平和细胞突出的形成,并且CD74位于CD44的上游。7.CD74可能通过抑制CD44蛋白酶体途径的降解,促进CD44的稳定性。8.乳腺癌细胞中,MIF过表达减弱CD74与CD44的结合,从而抑制CD74对CFL1磷酸化水平的调控。9.RHOA介导CD74对CFL1磷酸化水平的调控和细胞突出的形成。10.抑制CD74能够抑制乳腺癌肿瘤的形成。11.抑制CD74能够抑制乳腺癌细胞的肺转移。综合以上结果,我们的研究证实:乳腺癌细胞中,CD74与肿瘤的迁移、侵袭以及细胞突出的形成相关,CD74与CD44相互作用通过RHOA介导的CFL1磷酸化促进肿瘤的形成和转移。另外,我们的研究结果也表明CD74可能通过抑制CD44蛋白酶体途径的降解来提高CD44的稳定性:MIF过表达减弱CD74与CD44的结合,从而抑制CD74所介导的CFL1磷酸化信号通路。
[Abstract]:Background CD74, also known as Ii, is a multifunctional cytokine. As a constant chain of MHC class II, CD74 participates in the antigen presentation process mediated by MHC class II. It not only assists the correct folding of MHC class II in endoplasmic reticulum, regulates MHC class II out of endoplasmic reticulum, but also regulates the transport of MHC class II from Golgi to antigen processing. Chamber. As a receptor for MIF, CD74 is involved not only in the regulation of inflammatory factors by MIF, but also in the MIF-mediated inhibition of apoptosis and promotion of survival. In addition, CD74 is also associated with a variety of human diseases, including autoimmune diseases, such as systemic lupus erythematosus, as well as atherosclerosis, Alzheimer's disease and cancer. CD74 is overexpressed in a variety of tumors including breast cancer and is associated with the invasion of clear cell renal cell carcinoma, gastric cancer and pancreatic cancer. Recent studies have shown that CD74 is overexpressed in breast cancer and is linearly associated with lymph node metastasis and triple-negative breast cancer, but no relevant molecular mechanism has been reported. Hyaluronic acid receptors, when combined with CD44, promote the interaction of CD44 with receptor tyrosine kinases directly or through adaptor proteins, and activate downstream signaling pathways, such as PI3K-AKT signaling pathway and MAPK cascade, which are mainly involved in regulating apoptosis, survival and proliferation, and are related to drug resistance of tumor cells. Over-adaptor proteins interact with small GTPase proteins of the Ras superfamily, promote the activity of RHO GTPases such as RHOA, RAC, and participate in the regulation of cytoskeleton assembly or rearrangement. The dryness of tumor cells is also associated with CD44. Human tumor cells with high expression of CD44 have high malignancy and drug resistance. CD4 isolated from breast cancer cells is highly resistant to the disease. The stem cell characteristics of the 4+CD24-antigen phenotype are achieved by up-regulating EMT-related cytokines (such as TWIST, SNAIL, etc.). RHO GTPases belong to the small GTPase Ras superfamily and are divided into eight subfamilies according to the similarity of amino acid sequences. RHOA, RAC1 and CDC22 have been extensively studied through their downstream. ROCK regulates the phosphorylation of myosin light chains, and phosphorylated myosin light chains can enhance the activity of myosin ATPase to induce myosin contraction. In addition, RHOA can also regulate the phosphorylation of downstream LIMK through ROCK, and the activated LIMK phosphorylates its main downstream substrate CFL to regulate cytoskeleton assembly or rearrangement. PAK is a common downstream effector protein of RAC1 and CDC22. Activated PAK regulates CFL phosphorylation and cytoskeletal stability through LIMK. In addition, WAVE, an effector protein of RAC1, can regulate ARP2/3 and promote actin aggregation and actin skeleton formation. CDC42 regulates ARP2/3-mediated actin skeleton assembly through WASP. It was found that RHO GTPases were overexpressed in a variety of human tumors and were associated with tumor invasion and metastasis. CFL is an actin-binding protein with two forms, non-cleavage active Ser3 phosphorylation and active non-phosphorylation. Activated CFL tends to bind to negative extreme GDP. Cut, promote F-actin depolymerization, produce a large number of G-actin monomers and free F-actin terminal, and G-actin will be added to the extended F-actin after binding to GTP, promote F-actin polymerization, resulting in free F-actin terminal will be reused by ARP2/3 complex, promote F-actin nucleation, extension or actin network branch formation. The purpose of this study is to determine whether CD74 affects the invasion and metastasis of breast cancer cells by regulating cytoskeleton-related proteins. Methods 1. The distribution of CD74 in breast cancer cells and its relationship with breast cancer were detected by immunohistochemistry and clinical data analysis. Invasion and migration assays were used to detect the effect of CD74 knockdown on invasion and migration of breast cancer cells. 3. TRITC-phalloidin stained F-actin and laser confocal microscopy (CLSM) was used to detect the effect of CD74 knockdown or overexpression on the formation of breast cancer cell protrusion. 4. Western blot was used to detect the effect of CD74 knockdown or overexpression on the formation of breast cancer cells. Immunofluorescence and immunoprecipitation assay were used to detect the interaction between CD74 and CD44.6.CD74 or/and CD44 knock-down, Western blot to detect the effect of CD74 and CD44 on the phosphorylation level of CFL-1.7.CD74 or/and CD44 knock-down, TRITC-phalloidin to stain F-actin, and CLSM to detect breast cancer cells. Changes in cell protrusion formation. 8. Overexpression of CD74 knocked down CD44, Western blot detected the regulatory relationship between CD74 and CD44. 9. Add MG132 when CD74 was knocked down, Western blot detected the effect of CD74 on the stability of CD44. 10. Inhibiting MIF by specific siRNA or inhibitors, Western blot detected the effect of MIF on the phosphorylation level of CFL1. Co-precipitation assay was used to detect the binding of CD74 to CD44. 12. The RHO GTPase family members related plasmids were transfected into breast cancer cells. The effect of RHO GTPase family members on CD74-mediated phosphorylation of CFL1 was detected by Western blot. 13. Cell migration assay was used to detect the effect of RHOA on CD74-mediated migration of breast cancer cells. CD74 stably knock down cell line was screened by virus packaging system and infected MDA-MB-231 cells. 15. The effect of CD74 on tumor formation and metastasis was detected in nude mice xenograft tumor experiment. Results 1. The expression of CD74 in plasma membrane and cytoplasm was generally high in BIDC, CD74 was associated with breast cancer and clinical stage and lymph node metastasis of breast cancer. CD74 was knocked down by CD74 shRNA plasmid in MDA-MB-231 cells. Invasion experiments showed that CD74 could inhibit the invasion of MDA-MB-231 cells. 3. CD74 was knocked down by CD74 shRNA plasmid in MDA-MB-231 cells. Scratch experiments showed that CD74 could inhibit the migration of MDA-MB-231 cells. MDA-MB-231 cells (or T47D cells) knocked down CD74, TRITC-phalloidin stained F-actin, CLSM observed the formation of cell prominence, the results showed that CD74 knocked down the formation of cell prominence was inhibited. 5. Overexpression of CD74 in MCF-7 cells, TRITC-phalloidin stained F-actin, CLSM observed the formation of cell prominence, results Overexpression of CD74 could promote the formation of MCF-7 cells. 6. CD74 was knocked down by plasmid containing CD74 shRNA in MDA-MB-231 cells with high expression of CD74. The levels of ROCK 1 and p-CFL1 related to cytoskeleton regulation were detected. The same results were obtained in T47D cells by repeating the above experiments. Overexpression of CD74 in MCF-7 cells with low expression of CD74 resulted in an up-regulation of ROCK1 and p-CFL1 protein levels associated with cytoskeleton regulation. The same results were obtained in HCC1806 cells by repeating the above experiments. 8. Immunofluorescence assay showed that AlexaFluor 568 dye-labeled CD74 and AlexaFluor 488 dye-labeled CD4 were detected in MDA-MB-231 and T47D cells. Overexpression of pcDNA3.1-Flag-CD74 in MDA-MB-231 cells (or T47D cells) and interaction between CD74 and CD44 were detected by immunoprecipitation assay. 10. Immunocoprecipitation assay using CD44 in MDA-MB-231 cells also confirmed the interaction between CD74 and CD44. CD44 was knocked down in MDA-MB-231 cells, and CD74, ROCK1 and p-CFL1 protein levels were detected by Western blot. Repeated experiments in T47D cells yielded consistent results. 12. In MDA-MB-231 cells, CD74 shRNA plasmids (pLT-shCD74 # 1 and pLT-shCD74 # 2) were used to knock down CD74 or/or CD44 with CD44-specific siRNA. Repeated experiments in T47D cells yielded consistent results. 13. In MDA-MB-231 cells, plasmids containing CD74 shRNA (pLT-shCD74 # 1 and pLT-shCD74 # 2) were used to knock down CD74 or / or CD44 with CD44-specific siRNA. TRITC-phalloidin stained F-actin. The results showed that knocking down CD74 or / and CD44 could inhibit the formation of MDA-MB-231 cells. The same results were obtained in T47D cells by repeating the above experiments. 14. CD74 was overexpressed in MDA-MB-231 cells (or T47D cells) by pcDNA3.1-CD74 plasmid and CD44 was knocked down by CD44-specific siRNA. The results of blot showed that the down-regulation of CD44 could inhibit the up-regulation of CFL1 phosphorylation caused by overexpression of CD74. 15. The down-regulation of CD44 protein could be induced by knocking down CD74 in MDA-MB-231 cells. The down-regulation of CD44 was inhibited by adding different concentrations of MG132. 16. MIF-specific siRNA or MIF-specific inhibitor ISO-1 inhibited MIF. The results showed that the level of MIF protein was negatively correlated with the phosphorylation level of CFL 1. 17. Overexpression of pcDNA3.1-Flag-CD74 and pcDNA3.1-MIF was observed in HEK 293FT cells, and the interaction between CD74 and CD44 was weakened by co-immunoprecipitation. Upregulation of CFL1 phosphorylation induced by expression can be inhibited by RHO GD1 or RHOAN19: Downregulation of CFL1 phosphorylation induced by CD74 knockdown in MDA-MB-231 cells can be inhibited by RHOAL63. 19. MDA-MB-231 cells knockdown CD74 and overexpression of RHOAL63. Migration experiments showed that RHOAL63 could inhibit cell migration induced by CD74 knockdown. Overexpression of CD74 and RHOAN19 in MCF-7 cells showed that RHOAN19 could inhibit the up-regulation of cell migration induced by overexpression of CD74. Immunofluorescence assay showed that RHOAN19 could inhibit the formation of cell protrusion induced by overexpression of CD74. 20. Nude mice were divided into three groups and subcutaneously injected with MDA-MB-231pLT-s. HCTRL, MDA-MB-231 pLT-shCD74 # 1, or MDA-MB-231 pLT-shCD74 # 2 cells, tumorigenesis experiments showed that CD74 knockdown inhibited tumor growth, and MDA-MB-231 pLT-shCTRL control cell injection group found a liver metastasis. 21. Nude mice were divided into three groups, which were injected with MDA-MB-231 pLT-shCTRL, MDA-MB-231 pLT-shCD74 # or MDA-MB-231 pLT-shCTRL respectively. Two metastatic nodules were found in the lungs of nude mice injected with B-231 pLT-shCD74# 2 cells and MDA-MB-231 pLT-shCTRL control cells. Conclusion 1. CD74 is closely related to the clinical stage and lymph node metastasis of breast cancer and breast cancer. 2. CD74 knockdown can inhibit the migration and invasion of breast cancer cells. 3. CD74 expression level and cell prominence. The phosphorylation level of CFL1 was positively correlated with the expression of CD74. 5. CD74 and CD44 co-located in breast cancer cells. 6. CD74 and CD44 co-regulated the phosphorylation level of CFL1 and the formation of cell prominence, and CD74 was located upstream of CD44. 7. CD74 may promote the stability of CD44 by inhibiting the degradation of CD44 proteasome pathway. In breast cancer cells, MIF overexpression weakens the binding of CD74 to CD44, thereby inhibiting the regulation of CD74 on CFL-1 phosphorylation. 9. RHOA mediates the regulation of CD74 on CFL-1 phosphorylation and the formation of cell protrusion. 10. Inhibition of CD74 can inhibit the formation of breast cancer tumors. 11. Inhibition of CD74 can inhibit lung metastasis of breast cancer cells. Our study confirms that CD74 is associated with tumor migration, invasion and protrusion formation in breast cancer cells. The interaction between CD74 and CD44 promotes tumor formation and metastasis through RHOA-mediated phosphorylation of CFL1. In addition, our results also suggest that CD74 may increase CD44 expression by inhibiting the degradation of CD44 proteasome pathway. Stability: Overexpression of MIF weakens the binding of CD74 to CD44, thereby inhibiting CD74-mediated phosphorylation of CFL1.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9
本文编号：2227943
[Abstract]:Background CD74, also known as Ii, is a multifunctional cytokine. As a constant chain of MHC class II, CD74 participates in the antigen presentation process mediated by MHC class II. It not only assists the correct folding of MHC class II in endoplasmic reticulum, regulates MHC class II out of endoplasmic reticulum, but also regulates the transport of MHC class II from Golgi to antigen processing. Chamber. As a receptor for MIF, CD74 is involved not only in the regulation of inflammatory factors by MIF, but also in the MIF-mediated inhibition of apoptosis and promotion of survival. In addition, CD74 is also associated with a variety of human diseases, including autoimmune diseases, such as systemic lupus erythematosus, as well as atherosclerosis, Alzheimer's disease and cancer. CD74 is overexpressed in a variety of tumors including breast cancer and is associated with the invasion of clear cell renal cell carcinoma, gastric cancer and pancreatic cancer. Recent studies have shown that CD74 is overexpressed in breast cancer and is linearly associated with lymph node metastasis and triple-negative breast cancer, but no relevant molecular mechanism has been reported. Hyaluronic acid receptors, when combined with CD44, promote the interaction of CD44 with receptor tyrosine kinases directly or through adaptor proteins, and activate downstream signaling pathways, such as PI3K-AKT signaling pathway and MAPK cascade, which are mainly involved in regulating apoptosis, survival and proliferation, and are related to drug resistance of tumor cells. Over-adaptor proteins interact with small GTPase proteins of the Ras superfamily, promote the activity of RHO GTPases such as RHOA, RAC, and participate in the regulation of cytoskeleton assembly or rearrangement. The dryness of tumor cells is also associated with CD44. Human tumor cells with high expression of CD44 have high malignancy and drug resistance. CD4 isolated from breast cancer cells is highly resistant to the disease. The stem cell characteristics of the 4+CD24-antigen phenotype are achieved by up-regulating EMT-related cytokines (such as TWIST, SNAIL, etc.). RHO GTPases belong to the small GTPase Ras superfamily and are divided into eight subfamilies according to the similarity of amino acid sequences. RHOA, RAC1 and CDC22 have been extensively studied through their downstream. ROCK regulates the phosphorylation of myosin light chains, and phosphorylated myosin light chains can enhance the activity of myosin ATPase to induce myosin contraction. In addition, RHOA can also regulate the phosphorylation of downstream LIMK through ROCK, and the activated LIMK phosphorylates its main downstream substrate CFL to regulate cytoskeleton assembly or rearrangement. PAK is a common downstream effector protein of RAC1 and CDC22. Activated PAK regulates CFL phosphorylation and cytoskeletal stability through LIMK. In addition, WAVE, an effector protein of RAC1, can regulate ARP2/3 and promote actin aggregation and actin skeleton formation. CDC42 regulates ARP2/3-mediated actin skeleton assembly through WASP. It was found that RHO GTPases were overexpressed in a variety of human tumors and were associated with tumor invasion and metastasis. CFL is an actin-binding protein with two forms, non-cleavage active Ser3 phosphorylation and active non-phosphorylation. Activated CFL tends to bind to negative extreme GDP. Cut, promote F-actin depolymerization, produce a large number of G-actin monomers and free F-actin terminal, and G-actin will be added to the extended F-actin after binding to GTP, promote F-actin polymerization, resulting in free F-actin terminal will be reused by ARP2/3 complex, promote F-actin nucleation, extension or actin network branch formation. The purpose of this study is to determine whether CD74 affects the invasion and metastasis of breast cancer cells by regulating cytoskeleton-related proteins. Methods 1. The distribution of CD74 in breast cancer cells and its relationship with breast cancer were detected by immunohistochemistry and clinical data analysis. Invasion and migration assays were used to detect the effect of CD74 knockdown on invasion and migration of breast cancer cells. 3. TRITC-phalloidin stained F-actin and laser confocal microscopy (CLSM) was used to detect the effect of CD74 knockdown or overexpression on the formation of breast cancer cell protrusion. 4. Western blot was used to detect the effect of CD74 knockdown or overexpression on the formation of breast cancer cells. Immunofluorescence and immunoprecipitation assay were used to detect the interaction between CD74 and CD44.6.CD74 or/and CD44 knock-down, Western blot to detect the effect of CD74 and CD44 on the phosphorylation level of CFL-1.7.CD74 or/and CD44 knock-down, TRITC-phalloidin to stain F-actin, and CLSM to detect breast cancer cells. Changes in cell protrusion formation. 8. Overexpression of CD74 knocked down CD44, Western blot detected the regulatory relationship between CD74 and CD44. 9. Add MG132 when CD74 was knocked down, Western blot detected the effect of CD74 on the stability of CD44. 10. Inhibiting MIF by specific siRNA or inhibitors, Western blot detected the effect of MIF on the phosphorylation level of CFL1. Co-precipitation assay was used to detect the binding of CD74 to CD44. 12. The RHO GTPase family members related plasmids were transfected into breast cancer cells. The effect of RHO GTPase family members on CD74-mediated phosphorylation of CFL1 was detected by Western blot. 13. Cell migration assay was used to detect the effect of RHOA on CD74-mediated migration of breast cancer cells. CD74 stably knock down cell line was screened by virus packaging system and infected MDA-MB-231 cells. 15. The effect of CD74 on tumor formation and metastasis was detected in nude mice xenograft tumor experiment. Results 1. The expression of CD74 in plasma membrane and cytoplasm was generally high in BIDC, CD74 was associated with breast cancer and clinical stage and lymph node metastasis of breast cancer. CD74 was knocked down by CD74 shRNA plasmid in MDA-MB-231 cells. Invasion experiments showed that CD74 could inhibit the invasion of MDA-MB-231 cells. 3. CD74 was knocked down by CD74 shRNA plasmid in MDA-MB-231 cells. Scratch experiments showed that CD74 could inhibit the migration of MDA-MB-231 cells. MDA-MB-231 cells (or T47D cells) knocked down CD74, TRITC-phalloidin stained F-actin, CLSM observed the formation of cell prominence, the results showed that CD74 knocked down the formation of cell prominence was inhibited. 5. Overexpression of CD74 in MCF-7 cells, TRITC-phalloidin stained F-actin, CLSM observed the formation of cell prominence, results Overexpression of CD74 could promote the formation of MCF-7 cells. 6. CD74 was knocked down by plasmid containing CD74 shRNA in MDA-MB-231 cells with high expression of CD74. The levels of ROCK 1 and p-CFL1 related to cytoskeleton regulation were detected. The same results were obtained in T47D cells by repeating the above experiments. Overexpression of CD74 in MCF-7 cells with low expression of CD74 resulted in an up-regulation of ROCK1 and p-CFL1 protein levels associated with cytoskeleton regulation. The same results were obtained in HCC1806 cells by repeating the above experiments. 8. Immunofluorescence assay showed that AlexaFluor 568 dye-labeled CD74 and AlexaFluor 488 dye-labeled CD4 were detected in MDA-MB-231 and T47D cells. Overexpression of pcDNA3.1-Flag-CD74 in MDA-MB-231 cells (or T47D cells) and interaction between CD74 and CD44 were detected by immunoprecipitation assay. 10. Immunocoprecipitation assay using CD44 in MDA-MB-231 cells also confirmed the interaction between CD74 and CD44. CD44 was knocked down in MDA-MB-231 cells, and CD74, ROCK1 and p-CFL1 protein levels were detected by Western blot. Repeated experiments in T47D cells yielded consistent results. 12. In MDA-MB-231 cells, CD74 shRNA plasmids (pLT-shCD74 # 1 and pLT-shCD74 # 2) were used to knock down CD74 or/or CD44 with CD44-specific siRNA. Repeated experiments in T47D cells yielded consistent results. 13. In MDA-MB-231 cells, plasmids containing CD74 shRNA (pLT-shCD74 # 1 and pLT-shCD74 # 2) were used to knock down CD74 or / or CD44 with CD44-specific siRNA. TRITC-phalloidin stained F-actin. The results showed that knocking down CD74 or / and CD44 could inhibit the formation of MDA-MB-231 cells. The same results were obtained in T47D cells by repeating the above experiments. 14. CD74 was overexpressed in MDA-MB-231 cells (or T47D cells) by pcDNA3.1-CD74 plasmid and CD44 was knocked down by CD44-specific siRNA. The results of blot showed that the down-regulation of CD44 could inhibit the up-regulation of CFL1 phosphorylation caused by overexpression of CD74. 15. The down-regulation of CD44 protein could be induced by knocking down CD74 in MDA-MB-231 cells. The down-regulation of CD44 was inhibited by adding different concentrations of MG132. 16. MIF-specific siRNA or MIF-specific inhibitor ISO-1 inhibited MIF. The results showed that the level of MIF protein was negatively correlated with the phosphorylation level of CFL 1. 17. Overexpression of pcDNA3.1-Flag-CD74 and pcDNA3.1-MIF was observed in HEK 293FT cells, and the interaction between CD74 and CD44 was weakened by co-immunoprecipitation. Upregulation of CFL1 phosphorylation induced by expression can be inhibited by RHO GD1 or RHOAN19: Downregulation of CFL1 phosphorylation induced by CD74 knockdown in MDA-MB-231 cells can be inhibited by RHOAL63. 19. MDA-MB-231 cells knockdown CD74 and overexpression of RHOAL63. Migration experiments showed that RHOAL63 could inhibit cell migration induced by CD74 knockdown. Overexpression of CD74 and RHOAN19 in MCF-7 cells showed that RHOAN19 could inhibit the up-regulation of cell migration induced by overexpression of CD74. Immunofluorescence assay showed that RHOAN19 could inhibit the formation of cell protrusion induced by overexpression of CD74. 20. Nude mice were divided into three groups and subcutaneously injected with MDA-MB-231pLT-s. HCTRL, MDA-MB-231 pLT-shCD74 # 1, or MDA-MB-231 pLT-shCD74 # 2 cells, tumorigenesis experiments showed that CD74 knockdown inhibited tumor growth, and MDA-MB-231 pLT-shCTRL control cell injection group found a liver metastasis. 21. Nude mice were divided into three groups, which were injected with MDA-MB-231 pLT-shCTRL, MDA-MB-231 pLT-shCD74 # or MDA-MB-231 pLT-shCTRL respectively. Two metastatic nodules were found in the lungs of nude mice injected with B-231 pLT-shCD74# 2 cells and MDA-MB-231 pLT-shCTRL control cells. Conclusion 1. CD74 is closely related to the clinical stage and lymph node metastasis of breast cancer and breast cancer. 2. CD74 knockdown can inhibit the migration and invasion of breast cancer cells. 3. CD74 expression level and cell prominence. The phosphorylation level of CFL1 was positively correlated with the expression of CD74. 5. CD74 and CD44 co-located in breast cancer cells. 6. CD74 and CD44 co-regulated the phosphorylation level of CFL1 and the formation of cell prominence, and CD74 was located upstream of CD44. 7. CD74 may promote the stability of CD44 by inhibiting the degradation of CD44 proteasome pathway. In breast cancer cells, MIF overexpression weakens the binding of CD74 to CD44, thereby inhibiting the regulation of CD74 on CFL-1 phosphorylation. 9. RHOA mediates the regulation of CD74 on CFL-1 phosphorylation and the formation of cell protrusion. 10. Inhibition of CD74 can inhibit the formation of breast cancer tumors. 11. Inhibition of CD74 can inhibit lung metastasis of breast cancer cells. Our study confirms that CD74 is associated with tumor migration, invasion and protrusion formation in breast cancer cells. The interaction between CD74 and CD44 promotes tumor formation and metastasis through RHOA-mediated phosphorylation of CFL1. In addition, our results also suggest that CD74 may increase CD44 expression by inhibiting the degradation of CD44 proteasome pathway. Stability: Overexpression of MIF weakens the binding of CD74 to CD44, thereby inhibiting CD74-mediated phosphorylation of CFL1.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9
【参考文献】
相关期刊论文 前4条
1 Tasuku Matsuoka;Masakazu Yashiro;;Rho/ROCK signaling in motility and metastasis of gastric cancer[J];World Journal of Gastroenterology;2014年38期
2 Toshiyuki Ishiwata;Yoko Matsuda;Zenya Naito;;Nestin in gastrointestinal and other cancers: Effects on cells and tumor angiogenesis[J];World Journal of Gastroenterology;2011年04期
3 Nitsan Maharshak;Sivan Cohen;Frida Lantner;Gili Hart;Richard Bucala;Idit Shachar;;CD74 is a survival receptor on colon epithelial cells[J];World Journal of Gastroenterology;2010年26期
4 Ellen J Beswick;Victor E Reyes;;CD74 in antigen presentation,inflammation,and cancers of the gastrointestinal tract[J];World Journal of Gastroenterology;2009年23期
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