【作者】 张征峥；

【导师】 魏林； 宋淑霞；

【作者基本信息】 河北医科大学 ， 免疫学， 2014， 博士

【摘要】 目的：乳腺癌是导致女性癌症死亡的第二大主要原因，其发病率及死亡率在我国及全世界呈上升趋势。在中国，每年因乳腺癌死亡的人数约40,000人。近几十年来，尽管放疗、化疗及多种生物治疗手段有了长足发展，然而，乳腺癌转移、复发及化疗耐药等仍是乳腺癌治疗的难题。越来越多证据表明，微小RNA（microRNA, miRNA）参与了肿瘤的发生发展，积极探索乳腺癌侵袭转移机制、寻找新的治疗靶点，克服化疗耐药，将为临床治疗乳腺癌提供重要的实验依据。MicroRNA-155是一类多功能microRNA。人类miR-155基因，由BIC基因编码，位于21q21.3号染色体。最早发现，miR-155在多种病理生理过程中发挥重要作用，如造血系统分化、免疫、炎症、病毒感染、肿瘤、心血管疾病及唐氏综合症等。作为一种重要的oncomiR，在人类多种肿瘤中，miR-155过度表达，且与肿瘤的增殖、凋亡、转移及预后不良密切相关。在小鼠乳腺上皮细胞模型中，miR-155可靶向Rho A促进TGF-β诱导的EMT过程。此外，miR-155在促进肿瘤细胞增殖及抗凋亡中也发挥重要作用。通过对一系列人类乳腺癌细胞株的研究发现，在高表达miR-155的乳腺癌细胞株HS578T中，通过miR-155ASO抑制miR-155表达，可导致细胞周期阻滞，诱导细胞凋亡；而在低表达miR-155的BT474乳腺癌细胞株中，异位高表达miR-155可促进细胞增殖，增强化疗抵抗。在MCF-7细胞中，miR-155高表达同样促进了细胞的增殖，而抑制miR-155表达可减慢细胞增殖速度，诱导肿瘤细胞放疗抵抗。许多研究表明，miR-155不仅参与肿瘤的发生发展，同样，在肿瘤预后及放化疗抵抗中同样发挥重要作用。在肺癌中，miR-155的高表达与患者预后不良有关。缺氧可诱导非小细胞肺癌细胞株A549及H460中miR-155的表达增加，抑制miR-155的表达，可提高该细胞的放疗敏感性。虽然以往大量研究表明，miR-155做为oncomiR促进了肿瘤的发生发展，然而，在另外一些肿瘤中，miR-155却发挥抑瘤作用。在人宫颈癌细胞株Caski中，miR-155过表达可抑制EGF诱导的EMT转化,降低了侵袭/转移能力，抑制了细胞增殖，提高了Caski对顺铂的化疗敏感性。Xiang等在建立的以4T1为肿瘤模型的动物实验中发现，稳转miR-155的4T1细胞在小鼠体内转移能力明显降低，然而，若直接将肿瘤细胞注射至尾静脉，miR-155却明显促进了肿瘤的肺转移。LI等在通过分析胃癌细胞株中miRNA的表达变化发现，与永生化的胃上皮细胞GES-1相比，在其研究的胃癌细胞株（MKN-45、MKN-28、SGC-7901、NCI-N87、AGS）中miR-155表达均降低。外源转染miR-155可明显抑制胃癌的转移侵袭能力。划痕实验及transwell迁移侵袭实验结果显示，转染miR-155mimics组细胞迁移侵袭能力降低。miR-155在不同肿瘤中的作用众说纷纭，究竟是什么原因导致miR-155的作用有如此大的不同？还有待于我们进一步研究。为此，本研究以小鼠乳腺癌细胞株4T1为模型，通过建立高表达及低敲miR-155的4T1细胞株，研究miR-155对4T1细胞增殖、转移、侵袭、凋亡等生物学行为的影响，进而探寻miR-155影响4T1转移侵袭的靶点。化疗是乳腺癌治疗的常用治疗手段，本课题通过进一步研究乳腺癌一线化疗药物阿霉素作用于4T1细胞后miR-155的表达变化，探讨了高表达miR-155对阿霉素作用的影响，旨在进一步了解miR-155在小鼠乳腺癌细胞株4T1进展、治疗中的作用，以期为临床治疗肿瘤提供新的治疗视角。方法：1应用慢病毒载体，建立miR-155高表达稳转细胞株（4T1-miR155），应用miR-155inhibitor，建立瞬时转染低敲miR-155的4T1细胞模型（4T-miR155-inh），Taqman探针法实时定量PCR检测miR-155表达。2倒置显微镜下观察转染细胞形态，以转染相应无关miR（4T1-NC）为对照，应用MTS法检测高表达miR-155对肿瘤细胞增殖的影响。Westernblot检测miR-155与EMT相关的蛋白（E-cadherin、vimentin）分子的表达的关系，并通过免疫荧光予以证实。应用低敲miR-155的4T1细胞模型，Western blot检测与EMT相关的蛋白（E-cadherin、vimentin）分子的表达，MTS法检测细胞增殖情况。利用Transwell小室检测miR-155在4T1细胞迁移中的作用，平板克隆形成实验检测高表达miR-155的4T1细胞在体外的克隆形成能力，实时定量PCR检测CXCR4表达，通过caspase3/9活性检测及TUNNEL实验检测miR-155对凋亡的影响，最后通过体内实验观察荷瘤小鼠肿瘤大小及生存期，研究高表达miR-155对4T1细胞在体内外各种生物学行为的影响。3为进一步探讨miR-155在4T1中的作用机制，Western blot检测与增殖、凋亡、及细胞周期相关蛋白（p-AKT、p-ERK1/2、FOXO3a及p-FOXO3a、p27、p21、p15）的表达，PI染色检测细胞周期变化。4应用乳腺癌一线化疗药物阿霉素进行体内外实验，探讨miR-155与化疗药物的关系。阿霉素以终浓度0μg/mL、0.2μg/mL、0.4μg/mL、0.8μg/mL、1.6μg/mL，作用于4T1细胞，48h后MTS检测各孔OD值，确定阿霉素体外肿瘤细胞的作用浓度。阿霉素以选定浓度作用于4T1细胞，检测阿霉素作用后miR-155的表达变化。5MTS增殖实验检测阿霉素作用前后4T1-NC及4T1-miR155的增殖情况，caspase3/9活性检测及TUNNEL实验检测miR-155对凋亡的影响，PI染色检测阿霉素处理后各组细胞周期变化。结果：1成功建立高表达及低敲4T1细胞模型，Taqman探针法实时定量PCR检测miR-155表达，结果显示，在高表达模型中，与4T1-NC相比，4T1-miR-155细胞中miR-155上调759±39倍；在低敲细胞模型中，与相应对照相比，低敲模型中miR-155下降81%。2倒置显微镜下观察细胞形态发现，过表达miR-155的4T1-miR-155细胞由梭型的间质样表型变为鹅卵石样的上皮样表型，进一步应用Western blot检测与EMT相关分子的表达发现，与对照组相比，过表达miR-155细胞上皮标志物E-caherin表达上调，而间质标志物vimentin表达下降，免疫荧光结果与该结果一致。抑制miR-155表达时，对上皮标志物E-caherin的影响无统计学差异，而间质标志物vimentin表达增加。3MTS检测各组细胞增殖能力，结果发现，与对照组相比，过表达miR-155细胞增殖能力明显降低，而抑制miR-155表达对增殖无影响。4划痕实验及Transwell小室检测肿瘤细胞迁移能力，结果发现，与对照组相比，过表达miR-155细胞迁移能力显著下降。实时定量PCR检测CXCR4表达，结果显示，4T1-miR155组细胞CXCR4表达显著降低。5平板克隆形成实验结果显示，将细胞以300个/孔接种于6孔板，两周后观察克隆形成情况，结果发现，4T1-miR155组细胞克隆形成能力降低。6caspase3/9活性检测及TUNNEL实验结果显示，过表达miR-155并未诱导细胞发生凋亡。7动物实验观察荷瘤小鼠肿瘤大小及生存期，结果显示，接种4T1-miR155组细胞的小鼠肿瘤生长缓慢，生存期明显延长。8Western blot研究发现，过表达miR-155活化AKT， p-AKT水平增加，p-AKT进一步使下游FOXO3a发生磷酸化，p-FOXO3a表达水平增加。9细胞周期结果显示，与4T1-NC相比，4T1-miR155细胞G0/G1期比例增加（4T1-miR15566.96±1.40%vs4T1-NC36.98±1.56%），过表达miR-155诱导细胞周期G0/G1期阻滞。10MTS检测OD值，通过SPSS软件分析4T1IC50值为（0.8±0.12）μg/mL。体外实验以0.7μg/mL作为阿霉素的实验用浓度。4T1细胞经阿霉素作用48h，实时定量PCR结果显示，miR-155表达增加（4.5±1.5）倍。11MTS增殖实验结果显示，4T1-NC经阿霉素处理后，增殖速度明显减慢（P<0.01），而4T1-miR155组经阿霉素处理后，在第0h、24h、48h，其增殖速度与未处理组差别无显著性意义，在第72h，增殖速度显著低于未经阿霉素处理的4T1-miR155组（P<0.01）。12Caspase3/9活性检测结果显示，与未经阿霉素处理的4T1-NC相比，4T1-NC经阿霉素处理后， caspase3活性明显增强(P<0.05)，caspase9活性无改变。4T-miR155组经阿霉素处理前后，Caspase3/9活性无改变(P>0.05)。13TUNEL检测结果显示，经阿霉素处理后，4T1-NC大量细胞出现凋亡，而4T1-miR155组细胞凋亡细胞明显少于4T1-NC。14细胞周期结果显示，经阿霉素处理后4T1-NC细胞G0/G1期比例显著减少，G2/M期比例显著增加，与未经DOX处理的对照细胞相比，P<0.01，差别具有显著性；而4T1-miR155组细胞经阿霉素处理后，与未经阿霉素处理的4T1-miR155相比，G0/G1期比例降低，S期比例增加，G0/G1期与S期的变化均有统计学差异（P<0.01）。4T1-miR155组与4T1-NC对照组相比，经阿霉素处理后，处于G0/G1期的细胞明显增加（P<0.01），而处于G2/M期的细胞数显著下降（P<0.01）。结论：1成功建立miR-155高表达及低敲的4T1细胞模型。2miR-155过表达可促进4T1向上皮方向转化，细胞增殖、迁移、侵袭能力下降，体外肿瘤克隆形成能力下降，体内小鼠生存期延长；敲低miR-155则相反。3miR-155可通过诱导细胞周期G0/G1期阻滞及通过AKT/FOXO3a途径影响肿瘤增殖及迁移能力。4miR-155诱导4T1细胞休眠。5在4T1细胞中，miR-155对阿霉素的化疗具有拮抗作用。更多还原

【Abstract】 Objective: Breast cancer is the second leading cause of cancer death inwomen and its incidence is increasing in many countries. In China, it isestimated40，000people die from breast cancer every year. Even though earlydetection methods and treatment options greatly improved, the resistance ofcancer cells to chemotherapeutic agents or radiation therapy frequently resultsin the subsequent recurrence and metastasis of cancer. About30%of all breastcancer patients who are successfully treated at early stages are suffering arelapse accompanied by metastasis and chemoresistance. Hence, anadvancement of the treatment by avoiding drug resistance and a betterprediction of chemotherapy efficacy would improve the clinical outcome forbreast cancer patients.MicroRNAs (miRNAs) are endogenous, short (about19~24nucleotideslong) strands of non-coding RNA that regulate the expression of multiplegenes. It is reported that miRNAs dysregulated in many cancers, such asbreast, prostate, colon and lung. Thereby, microRNAs can function asoncomiRs or tumor-suppressor-miRs depending on their respective targetgenes. MiR-155is a kind of miRNA with multifunction and produced fromthe processing of the B-Cell Integration Cluster (BIC), which is located atchromosome21q21.3. Early studies found that miR-155plays a critical role invarious physiological and pathological processes such as hematopoieticlineage differentiation, immunity, inflammation, viral infections, cancer,cardiovascular disease, and Down syndrome. As an important miRNA,miR-155overexpressed in many human tumors and is associated withproliferation, apoptosis, metastasis and poor prognosis. In normal murinemammary gland (NMuMG) epithelial cells model, miR-155plays animportant role in TGF-beta-induced EMT and cell migration and invasion by targeting RhoA. Furthermore, miR-155also plays a critical role in promotingproliferation or anti-apoptosis in cancer. Ectopic overexpression of miR-155in BT-474induces cell survival and chemoresistance to multiple agents,whereas knockdown of miR-155in HS578T renders cells to apoptosis andenhances chemosensitivity. The same results were also conducted inMDA-MB-231and MCF-7. Furthermore, other studies have shown thatmiR-155also play important role in prognosis and chemoresistance orradioresistance. For instance, miR-155is overexpressed in lung cancer, whichcorrelates with poor patient prognosis. Researchers found that increased levelsof miR-155radioprotects lung cancer cell lines such as A549or H460, whileinhibition of miR-155radiosensitizes these cells.Although a number of studies show that miR-155acts as oncomiRpromoting the development of tumor, however, in other tumors, miR-155hasthe opposite effect. It is reported that miR-155overexpression suppressedEGF-induced EMT, decreased migration/invasion capacities, inhibited cellproliferation and increased the chemosensitivity to DDP in human Caskicervical cancer cells. Xiang X et.al reported that stable overexpression ofmiR-155in4T1breast tumor cells reduces significantly the aggressiveness oftumor cell dissemination as a result of preventing epithelial-to-mesenchymaltransition (EMT) of tumor cells in vivo. While when tumor cells are injecteddirectly into the bloodstream, miR-155remarkably promotes macroscopictumor formation in the lung. Another studies also found that miR-155wassignificantly downregulated in gastric cancer cell lines compared with animmortalized gastric epithelial cell line (GES-1). Overexpression of miR-155in SGC-7901and MKN-45gastric cancer cells dramatically suppressed cellmigration, invasion and adhesion in vitro. Taken together, these data suggestthat miR-155may function as a tumor suppressor. What is the reason that therole of miR-155varied according to different tumors? It is to be our furtherresearch.To investigate the effects of microRNA-155on the proliferation,metastasis, invasion and apoptosis of4T1cells, lentiviral vector containing miR-155precusor was used to get the ectopic overexpression miR-155in4T1cells. Chemotherapy is a common treatment in breast cancer. In this study,doxorubicin was used to explore the relationship between miR-155and thechemotherapeutics.Methods：1The Lentiviral carrying miR-155precusor was packaged with themethod of transfecting293T cell lines in vitro. Then,4T1cells were infectedwith the fresh virus according to kit instructions. Then4T1-miR155cell linethat stably overexpression miR155were selected.4T-miR155-inh cell linewith transiently downregulated miR-155was obtained by using Lipofectamine2000. miR-155expression levels were detected by Taqman probe quantitativereal-time PCR.2Cell morphology was observed by light microscope. Cell proliferationwas detected by MTS. E-cadherin, vimentin expression level were detected byWestern Blot and immunofluorescence. The growth and migration of cellswere detected by transwell, wound healing assay and colony formation.CXCR4mRNA was detected by real time PCR. The apoptosis was determinedby TUNEL and Caspase3/9activity assay. Furthermore, Tumor sizes andsurvival were observed after inoculation with4T1-miR155or4T1-NC in vivo.3To explore the role of miR-155in4T1, we detected the protein level ofp-AKT, p-ERK1/2, FOXO3a as well as p-FOXO3a by Western blot. The cellcycle distribution was analyzed by flow cytometry.44T1cells were treated with different concentration of doxorubicin(DOX) at a final concentration of0μg/mL,0.2μg/mL,0.4μg/mL,0.8μg/mLand1.6μg/mL. MTS assay was performed to determine the IC50of DOX in4T1cells. miR-155expression was detected in4T1cells after DOX treated byquantitative real time PCR.5Cell proliferation assay was detected by MTS in4T1-NC or4T1-miR155after treated with DOX or not. The apoptosis was determined byTUNEL and Caspase3/9assay. Cell cycle assay was carried out by flowcytometry analysis. Results:1A stable overexpression of miR-155(4T1-miR155) and a transientknockdown miR-155cell line4T1-miR155-inh were obtained. miR-155levelwas759±39fold increased in4T1-miR155compaerd to control (4T1-NC) anddecreased81%in4T1-miR155-inh cells, respectively.2The appearance of4T1-miR155cells was changed from spindle type tocobblestone-like epithelial morphology. The expression level of E-cadherinwas significantly higher and vimentin was lower in4T1-miR155comparedwith4T1-NC. While in4T1-miR155-inh cells, there is no statistic differencein E-cadherin expression but vimentin was higher compared with control.3Compared with4T1-NC, cell proliferation was significantly decreasedin4T1-miR-155while there is no significant change in4T1-miR155-inh cells.4To investigate whether miR-155has a direct role in cell migration,transwell assay and wound healing assay were performed. Overexpression ofmiR-155impeded the migration of4T1cells significantly. Real-time PCRresults indicate that the expression level of CXCR4is higher in4T1-controlcells than in4T1-miR-155cells.5The colony formation ability of4T1-miR155was significantlydecreased than4T1-NC.6Caspase3/9activity and TUNEL assay revealed that miR-155did notinduce4T1cell apoptosis.7Overexpression of miR-155restrains tumor growth and prolongssurvival time of tumor-bearing mice in vivo.8The results of western blot analysis indicate that the level of p-AKTand p-FOXO3a in4T1-miR155cell line increased.9Flow cytometry revealed that G0/G1phrase cell proportion (%) in4T1-miR-155cells was significant higher than that in4T1-NC (4T1-miR15566.96±1.40%vs4T1-NC36.98±1.56%).10MTS assays showed that IC50of DOX in4T1cells were（0.8±0.12）μg/mL.0.7μg/mL was used as the final concentration in experiment. Whentreated with DOX at a final concentration of0.7μg/mL, the expression of miR-155was (4.5±1.5) folds increased in4T1cells.11In proliferation experiments, after treated with DOX, the inhibition ofcell growth was more pronounced in4T1-NC cells than4T1-miR155cells.12Caspase3activity of4T1-NC cells was higher after DOX treatedwhile no difference in4T1-miR155. There is no change of caspase9in bothof the two cells after DOX treated.13After treated with DOX, most of4T1-NC cells appeared apoptosiswhile4T1-miR155did not.14Cell cycle analysis showed that the G0/G1phase proportion decreasedand the G2/M phase proportion increased in4T1-NC cells after DOX treated(P<0.01). While in4T1-miR155，the G0/G1phase proportion decreased butthe G2/M phase proportion did not change. In addition, compared with4T1-NC group, the proportion of G0/G1phase increased and G2/M phasedecreased in4T1-miR155group (P<0.01).Conclusions:1A model that overexpressing or knocking down miR-155in4T1cellswas successfully obtained.2Overexpression of miR-155dramatically suppressed cell proliferation,migration, colony formation and EMT in vitro, prolonged animal survival.While knocking down expression of miR-155play the opposite role.3miR-155suppresses proliferation and migration by AKT/FOXO3apathway and induces G0/G1arrest.4miR-155induced dormancy in4T1cells.5miR-155showed antagonism to DOX in4T1cells.更多还原