發(fā)布時(shí)間：2018-09-07 10:05

【摘要】：研究背景CD74又稱為Ii,是一個(gè)多功能的細(xì)胞因子。作為MHC class Ⅱ的恒定鏈,CD74參與MHC class Ⅱ所介導(dǎo)的抗原呈遞過(guò)程,不僅在內(nèi)質(zhì)網(wǎng)中輔助MHC class Ⅱ的正確折疊,調(diào)控MHC class Ⅱ出內(nèi)質(zhì)網(wǎng),也能調(diào)控MHC class Ⅱ從高爾基體轉(zhuǎn)運(yùn)到抗原加工小室。作為MIF的受體,CD74不僅參與MIF對(duì)炎癥因子的調(diào)控,也參與MIF所介導(dǎo)的抑制細(xì)胞凋亡,促進(jìn)存活過(guò)程。此外,CD74也與多種人類(lèi)疾病相關(guān),其中包括自身免疫疾病,如系統(tǒng)性紅斑狼瘡,也包括動(dòng)脈粥樣硬化、阿爾茨海默病以及癌癥。研究表明CD74在包括乳腺癌在內(nèi)的多種腫瘤中過(guò)表達(dá),并且與透明細(xì)胞腎細(xì)胞癌、胃癌和胰腺癌的侵襲有關(guān)。近期研究表明CD74在乳腺癌中過(guò)表達(dá),并且與乳腺癌的淋巴結(jié)轉(zhuǎn)移和三陰性乳腺癌呈線性相關(guān),但尚未見(jiàn)相關(guān)分子機(jī)制報(bào)道。CD44是透明質(zhì)酸受體,當(dāng)透明質(zhì)酸與CD44結(jié)合后可以促進(jìn)CD44直接或通過(guò)接頭蛋白與受體酪氨酸激酶相互作用,并激活下游信號(hào)通路,如PI3K-AKT信號(hào)通路和MAPK級(jí)聯(lián)反應(yīng),主要參與調(diào)控細(xì)胞凋亡、存活和增殖等,并與腫瘤細(xì)胞的抗藥性相關(guān)。CD44還能夠通過(guò)接頭蛋白與Ras超家族的小GTPase蛋白相互作用,促進(jìn)RHOA、RAC等RHO GTPases的活性,參與調(diào)控細(xì)胞骨架的組裝或重排。腫瘤細(xì)胞的干性也與CD44相關(guān),高表達(dá)CD44的人類(lèi)腫瘤細(xì)胞具有惡性程度高以及耐藥性的特點(diǎn),乳腺癌細(xì)胞中所分離出來(lái)的CD44+ CD24-抗原表型的細(xì)胞亞群所表現(xiàn)出來(lái)的干細(xì)胞特性通過(guò)上調(diào)EMT相關(guān)的細(xì)胞因子(如TWIST、SNAIL等)來(lái)實(shí)現(xiàn)。RHO GTPases屬于小GTPase Ras超家族的成員,根據(jù)氨基酸序列的相似性劃分為8個(gè)亞家族,其中RHOA、RAC1和CDC42獲得廣泛研究。RHOA通過(guò)其下游效應(yīng)蛋白R(shí)OCK調(diào)控肌球蛋白輕鏈的磷酸化,磷酸化的肌球蛋白輕鏈可以增強(qiáng)肌球蛋白ATPase的活性來(lái)誘導(dǎo)肌球蛋白收縮。另外,RHOA也可以通過(guò)ROCK調(diào)控下游LIMK的磷酸化,激活的LIMK對(duì)其主要下游底物CFL進(jìn)行磷酸化,調(diào)控細(xì)胞骨架組裝或重排。PAK是RAC1和CDC42的共同下游效應(yīng)蛋白,激活的PAK通過(guò)LIMK調(diào)控CFL的磷酸化,并影響細(xì)胞骨架的穩(wěn)定性。此外,RAC1的效應(yīng)蛋白WAVE可以對(duì)ARP2/3進(jìn)行調(diào)控,促進(jìn)actin的聚合和actin骨架形成。CDC42則通過(guò)WASP來(lái)調(diào)控ARP2/3介導(dǎo)的actin骨架的組裝。目前已經(jīng)發(fā)現(xiàn)RHO GTPases在多種人類(lèi)腫瘤中過(guò)表達(dá),并且與腫瘤的侵襲和轉(zhuǎn)移相關(guān)。CFL是一個(gè)actin結(jié)合蛋白,具有兩種形式,無(wú)切割活性的Ser3位點(diǎn)磷酸化形式和具有活性的非磷酸化形式�；罨腃FL傾向于與F-actin中負(fù)極端的GDP·actin結(jié)合,并對(duì)F-actin切割,促進(jìn)F-actin解聚,產(chǎn)生大量的G-actin單體以及游離的F-actin末端,而G-actin會(huì)在結(jié)合GTP后被添加到正在延伸的F-actin中,促進(jìn)F-actin聚合,所產(chǎn)生的游離F-actin末端則會(huì)被ARP2/3復(fù)合物再次利用,促進(jìn)F-actin的成核、延伸或actin網(wǎng)狀分支的形成。因此CFL對(duì)細(xì)胞骨架具有聚合和解聚的雙重調(diào)控作用。本研究在于明確CD74是否通過(guò)調(diào)控細(xì)胞骨架相關(guān)蛋白,影響乳腺癌細(xì)胞的侵襲和轉(zhuǎn)移。研究方法1.通過(guò)免疫組化和臨床數(shù)據(jù)分析,檢測(cè)CD74在乳腺癌細(xì)胞中的分布以及與乳腺癌的關(guān)系。2.細(xì)胞侵襲實(shí)驗(yàn)和細(xì)胞遷移實(shí)驗(yàn)檢測(cè)CD74敲低后對(duì)乳腺癌細(xì)胞侵襲和遷移能力的影響。3.TRITC-phalloidin對(duì)F-actin進(jìn)行染色,激光共聚焦顯微鏡(CLSM)下檢測(cè)CD74敲低或過(guò)表達(dá)時(shí)對(duì)乳腺癌細(xì)胞細(xì)胞突出形成的影響。4.抑制或過(guò)表達(dá)CD74后,western blot檢測(cè)細(xì)胞骨架調(diào)控相關(guān)蛋白的變化。5.免疫熒光和免疫共沉淀實(shí)驗(yàn)檢測(cè)CD74與CD44的相互作用。6.CD74或/和CD44敲低,western blot檢測(cè)CD74和CD44對(duì)CFL1磷酸化水平的影響。7.CD74 或/和 CD44 敲低,TRITC-phalloidin 對(duì) F-actin 進(jìn)行染色,CLSM 下檢測(cè)乳腺癌細(xì)胞細(xì)胞突出形成的變化。8.過(guò)表達(dá)CD74的情況下敲低CD44,western blot檢測(cè)CD74與CD44的調(diào)控關(guān)系。9.敲低CD74的情況下加入MG132,western blot檢測(cè)CD74對(duì)CD44穩(wěn)定性的影響。10.利用特異siRNA或抑制劑抑制MIF,western blot檢測(cè)MIF對(duì)CFL1磷酸化水平的影響。11.免疫共沉淀實(shí)驗(yàn)檢測(cè)MIF過(guò)表達(dá)減弱CD74與CD44的結(jié)合。12.乳腺癌細(xì)胞中轉(zhuǎn)染RHO GTPase家族成員相關(guān)質(zhì)粒,western blot檢測(cè)RHO GTPase家族成員對(duì)CD74介導(dǎo)的CFL1磷酸化影響。13.細(xì)胞遷移實(shí)驗(yàn)檢測(cè)RHOA對(duì)CD74介導(dǎo)的乳腺癌細(xì)胞遷移能力的影響。14.利用慢病毒包裝體系制備病毒,并感染MDA-MB-231細(xì)胞,篩選獲得CD74穩(wěn)定敲低細(xì)胞系。15.裸鼠移植瘤實(shí)驗(yàn)檢測(cè)CD74對(duì)腫瘤形成和轉(zhuǎn)移的影響。實(shí)驗(yàn)結(jié)果1.BIDC中CD74在質(zhì)膜和細(xì)胞質(zhì)中的表達(dá)水平普遍偏高,CD74與乳腺癌以及乳腺癌的臨床階段和淋巴結(jié)轉(zhuǎn)移關(guān)系密切。2.在MDA-MB-231細(xì)胞中利用含CD74 shRNA的質(zhì)粒敲低CD74,細(xì)胞侵襲實(shí)驗(yàn)表明敲低CD74可以抑制MDA-MB-231細(xì)胞的侵襲能力。3.在MDA-MB-231細(xì)胞中利用含CD74 shRNA的質(zhì)粒敲低CD74,細(xì)胞劃痕實(shí)驗(yàn)表明敲低CD74可以抑制MDA-MB-231細(xì)胞的遷移能力。4.MDA-MB-231 細(xì)胞(或 T47D 細(xì)胞)中敲低 CD74,TRITC-phalloidin 對(duì) F-actin進(jìn)行染色,CLSM下觀察細(xì)胞突出的形成,結(jié)果表明CD74敲低后細(xì)胞突出的形成受到抑制。5.在MCF-7細(xì)胞中過(guò)表達(dá)CD74,TRITC-phalloidin對(duì)F-actin進(jìn)行染色,CLSM下觀察細(xì)胞突出的形成,結(jié)果表明過(guò)表達(dá)CD74可以促進(jìn)MCF-7細(xì)胞細(xì)胞突出的形成。6.利用含CD74 shRNA的質(zhì)粒在CD74高表達(dá)的MDA-MB-231細(xì)胞中敲低CD74,檢測(cè)到與細(xì)胞骨架調(diào)控相關(guān)的ROCK1和p-CFL1蛋白水平的下調(diào)。T47D細(xì)胞中重復(fù)上述實(shí)驗(yàn),獲得了一致結(jié)果。7.利用pcDNA3.1-CD74質(zhì)粒在低表達(dá)CD74的MCF-7細(xì)胞中過(guò)表達(dá)CD74,檢測(cè)到與細(xì)胞骨架調(diào)控相關(guān)的ROCK1和p-CFL1蛋白水平的上調(diào)。HCC1806細(xì)胞中重復(fù)上述實(shí)驗(yàn),獲得了一致結(jié)果。8.免疫熒光實(shí)驗(yàn)表明,MDA-MB-231和T47D細(xì)胞中AlexaFluor 568染料標(biāo)記的CD74與AlexaFluor488染料標(biāo)記的CD44分別存在共定位。9.在 MDA-MB-231 細(xì)胞(或 T47D 細(xì)胞)中過(guò)表達(dá) pcDNA3.1-Flag-CD74,免疫共沉淀實(shí)驗(yàn)表明CD74和CD44存在相互作用。10.MDA-MB-231細(xì)胞中利用細(xì)胞內(nèi)源的CD44進(jìn)行免疫共沉淀實(shí)驗(yàn)同樣證實(shí)CD74和CD44存在相互作用。11.利用 CD44 特異的 siRNA 在 MDA-MB-231 細(xì)胞中敲低 CD44,western blot檢測(cè)到CD74、ROCK1和p-CFL1蛋白水平的下調(diào)。T47D細(xì)胞中重復(fù)上述實(shí)驗(yàn)獲得了一致結(jié)果。12.MDA-MB-231 細(xì)胞中利用含 CD74 shRNA 的質(zhì)粒(pLT-shCD74#1 和pLT-shCD74#2)敲低 CD74 或/和利用 CD44 特異的 siRNA 敲低 CD44,western blot檢測(cè)到CD74或/和CD44的敲低會(huì)引起CFL1磷酸化水平的下調(diào)。T47D細(xì)胞中重復(fù)上述實(shí)驗(yàn)獲得了一致結(jié)果。13.MDA-MB-231 細(xì)胞中利用含 CD74 shRNA 的質(zhì)粒(pLT-shCD74#1和pLT-shCD74#2)敲低CD74或/和利用CD44特異的siRNA敲低CD44,TRITC-phalloidin對(duì)F-actin進(jìn)行染色,CLSM下觀察細(xì)胞突出的形成,結(jié)果表明敲低CD74或/和CD44可以抑制MDA-MB-231細(xì)胞細(xì)胞突出的形成。T47D細(xì)胞中重復(fù)上述實(shí)驗(yàn)獲得了一致結(jié)果。14.在MDA-MB-231細(xì)胞(或T47D細(xì)胞)中利用pcDNA3.1-CD74質(zhì)粒過(guò)表達(dá)CD74并利用CD44特異的siRNA敲低CD44,western blot結(jié)果表明CD44的下調(diào)能夠抑制由于CD74過(guò)表達(dá)所引起的CFL1磷酸化水平的上調(diào)。15.MDA-MB-231細(xì)胞中敲低CD74能夠引起CD44蛋白水平的下調(diào),而當(dāng)加入不同濃度的MG132后CD44的下調(diào)受到抑制。16.利用MIF特異的siRNA或MIF特異性的抑制劑ISO-1抑制MIF,以及通過(guò)pcDNA3.1-MIF質(zhì)粒過(guò)表達(dá)MIF,結(jié)果表明MIF蛋白水平與CFL1的磷酸化水平呈負(fù)相關(guān)。17.HEK 293FT 細(xì)胞中同時(shí)過(guò)表達(dá) pcDNA3.1-Flag-CD74 和 pcDNA3.1-MIF,免疫共沉淀實(shí)驗(yàn)結(jié)果表明MIF過(guò)表達(dá)的情況下,CD74與CD44的相互作用減弱。18.MCF-7細(xì)胞中CD74過(guò)表達(dá)引起的CFL1磷酸化水平上調(diào)可以被RHO GD1或RHOAN19所抑制:MDA-MB-231細(xì)胞中CD74敲低所引起的CFL1磷酸化水平的下調(diào)可以被RHOAL63所抑制。19.MDA-MB-231細(xì)胞中敲低CD74并過(guò)表達(dá)RHOAL63,細(xì)胞遷移實(shí)驗(yàn)表明RHOAL63能夠抑制CD74敲低引起的細(xì)胞遷移能力的下調(diào)。MCF-7細(xì)胞中過(guò)表達(dá)CD74和RHOAN19,細(xì)胞遷移實(shí)驗(yàn)表明RHOAN19能夠抑制CD74過(guò)表達(dá)引起的細(xì)胞遷移能力的上調(diào),免疫熒光實(shí)驗(yàn)表明RHOAN19能夠抑制CD74過(guò)表達(dá)條件下細(xì)胞突出的形成。20.裸鼠分為3組,分別進(jìn)行腹部皮下注射MDA-MB-231pLT-shCTRL,MDA-MB-231 pLT-shCD74#1 或 MDA-MB-231 pLT-shCD74#2 細(xì)胞,成瘤實(shí)驗(yàn)表明CD74的敲低能夠抑制腫瘤的生長(zhǎng),且MDA-MB-231 pLT-shCTRL對(duì)照細(xì)胞注射組裸鼠發(fā)現(xiàn)一例肝轉(zhuǎn)移。21.裸鼠分為3組,分別進(jìn)行尾靜脈注射MDA-MB-231 pLT-shCTRL,MDA-MB-231 pLT-shCD74#1 或 MDA-MB-231 pLT-shCD74#2 細(xì)胞,MDA-MB-231 pLT-shCTRL對(duì)照細(xì)胞注射組裸鼠發(fā)現(xiàn)2例肺出現(xiàn)轉(zhuǎn)移結(jié)節(jié)。結(jié)論1.CD74與乳腺癌以及乳腺癌的臨床階段和淋巴結(jié)轉(zhuǎn)移之間關(guān)系密切。2.CD74敲低能夠抑制乳腺癌細(xì)胞遷移和侵襲能力。3.CD74的表達(dá)水平與細(xì)胞突出的形成正相關(guān)。4.CFL1的磷酸化水平與CD74的表達(dá)呈正相關(guān)。5.乳腺癌細(xì)胞中CD74與CD44存在共定位。6.CD74和CD44共同調(diào)控CFL1的磷酸化水平和細(xì)胞突出的形成,并且CD74位于CD44的上游。7.CD74可能通過(guò)抑制CD44蛋白酶體途徑的降解,促進(jìn)CD44的穩(wěn)定性。8.乳腺癌細(xì)胞中,MIF過(guò)表達(dá)減弱CD74與CD44的結(jié)合,從而抑制CD74對(duì)CFL1磷酸化水平的調(diào)控。9.RHOA介導(dǎo)CD74對(duì)CFL1磷酸化水平的調(diào)控和細(xì)胞突出的形成。10.抑制CD74能夠抑制乳腺癌腫瘤的形成。11.抑制CD74能夠抑制乳腺癌細(xì)胞的肺轉(zhuǎn)移。綜合以上結(jié)果,我們的研究證實(shí):乳腺癌細(xì)胞中,CD74與腫瘤的遷移、侵襲以及細(xì)胞突出的形成相關(guān),CD74與CD44相互作用通過(guò)RHOA介導(dǎo)的CFL1磷酸化促進(jìn)腫瘤的形成和轉(zhuǎn)移。另外,我們的研究結(jié)果也表明CD74可能通過(guò)抑制CD44蛋白酶體途徑的降解來(lái)提高CD44的穩(wěn)定性:MIF過(guò)表達(dá)減弱CD74與CD44的結(jié)合,從而抑制CD74所介導(dǎo)的CFL1磷酸化信號(hào)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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R737.9

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