【作者】 韩宇；

【导师】 刘建国；

【作者基本信息】 吉林大学 ， 外科学， 2019， 博士

【摘要】 骨肉瘤(Osteosarcoma,OS)是一种常见的原发性恶性肿瘤,具有较高的发病率,严重的威胁着我们的健康。骨肉瘤的增殖速率较快,使得骨肉瘤患者的预后差,同时5年内的生存率较低。因此,研究导致骨肉瘤快速増殖的原因,进而揭示骨肉瘤快速增殖的分子机制对提高骨肉瘤患者预后具有十分重要的作用。目前缺乏针对FOSL1引发骨肉瘤的具体致病机制研究,特别是FOSL1是以何种方式帮助骨肉瘤细胞增殖还不清楚,因此研究其在骨肉瘤发病过程中的调控机制十分重要。肿瘤基因伴随人的一生,虽然有很多很多的基因锁,却不断的被恶劣的生活习惯开启。所以肿瘤的发生是多因素,多阶段,多基因相互作用的结果。相关研究证明lncRNA-UFC1在肿瘤及炎症等方向发挥着重要作用,可以调控相关病情的发展。但是lncRNA-UFC1在骨肉瘤中是否也同样发挥着作用,是通过何种方式发挥作用的仍没有文献报道。因此,多方面深入的研究骨肉瘤的发病机制,阐明骨肉瘤细胞内相关基因的调控机制对于骨肉瘤的临床治疗具有十分必要。在本课题的第一部分研究中,主要目的是探讨FOSL1通过ERK/AP-1信号通路对骨肉瘤细胞增殖、侵袭和迁移的影响。我们首先利用qRT-PCR和Western-blot技术对新鲜骨肉瘤组织中FOSL1相关分子及ERK/AP-1的相关分子在表达和转录水平上进行定量分析,同时利用免疫组化技术分析了骨肉瘤组织中FOSL1的表达情况。进一步,我们检测了si-FOSL1和过表达FOSL1对C-JUN,C-FOS,ERK1/2,p-ERK1/2,p38MAPK和p-p38MAPK在转录和蛋白表达水平的影响。接下来,通过流式细胞技术和CCK-8(Cell counting kit-8)实验,观察FOSL1对骨肉瘤细胞细胞周期及增殖能力的影响,利用CB17联合免疫缺陷小鼠进行动物实验,观察接种si-FOSL1和过表达FOSL1的骨肉瘤细胞株的成瘤能力。最后,通过Transwell细胞侵袭实验,检测了FOSL1对骨肉瘤细胞侵袭能力的影响,通过细胞划痕实验,检测了FOSL1对骨肉瘤细胞迁移能力的影响。结果显示:(1)在骨肉瘤组织中,FOSL1相关分子及ERK/AP-1的相关分子的表达显著上调。(2)PCR及Western-blot显示si-FOSL1组C-JUN,C-FOS,p-ERK1/2和p-p38MAPK的表达水平下降,而过表达FOSL1组表达升高。(3)加入ERK/AP-1的抑制剂后,p-ERK1/2,p-p38MAPK在过表达FOSL1组的表达水平降低。(4)流式细胞检测及CCK-8实验证实了FOSL1可以通过影响细胞周期进而影响骨肉瘤细胞的增殖能力。(5)动物实验发现si-FOSL1组和过表达FOSL1组的肿瘤形成能力分别显著降低和升高。(6)Transwell细胞侵袭实验发现si-FOSL1组可以明显抑制骨肉瘤细胞的侵袭能力,过表达FOSL1组侵袭能力增加。(7)细胞划痕实验发现si-FOSL1组可以明显抑制骨肉瘤细胞的迁移能力,而过表达FOSL1组迁徙能力增强。综上实验得出结论:FOSL1通过ERK/AP-1信号通路影响骨肉瘤细胞增殖、侵袭和迁移的能力,并可能为骨肉瘤治疗提供了新的靶点。在本课题的第二部分研究中,我们主要对lncRNA-UFC1对骨肉瘤的调控作用进行了初步的研究。首先,我们利用qRT-PCR技术对骨肉瘤组织及癌旁组织中lncRNA-UFC1的表达水平进行了检测,通过qRT-PCR技术分析了lncRNA-UFC1在MG-63,U20S和SaOs-2三种细胞系中的表达情况来筛选细胞系以进行后续实验。接下来,我们通过MTT实验、克隆形成实验及原位杂交实验来观察过表达和敲低lncRNA-UFC1后,骨肉瘤细胞的增殖情况。最后,我们进行了体内动物实验,向小鼠体内注射过表达和敲低lncRNA-UFC1的SaOs2细胞,观察其在小鼠体内骨肉瘤增殖的能力。实验结果显示:(1)qRT-PCR结果显示lncRNA-UFC1在骨肉瘤组织中表达显著升高。(2)MTT实验、克隆形成实验及原位杂交实验均证实lncRNA-UFC1在体外可以促进骨肉瘤细胞的増殖。(3)动物实验进一步证实了lncRNA-UFC1在体内也可以促进骨肉瘤细胞的増殖。由此我们得出结论:lncRNA-UFC1可以促进骨肉瘤细胞的増殖,对骨肉瘤的调控起到重要的作用。更多还原

【Abstract】 Osteosarcoma(OS)is a common primary malignant tumor.It has a high incidence and seriously threatens our health.The proliferation rate of osteosarcoma is very fast,which makes the prognosis of osteosarcoma poor and the survival rate within 5 years is low.Therefore,the molecular mechanism of rapid proliferation of osteosarcoma play a very important role.The mechanism of osteosarcoma is very important.However,there is a lack of research on the specific pathogenesis of osteosarcoma caused by FOSL1,especially the way that FOSL1 can help osteosarcoma cell proliferation is unclear.It is very important to study the mechanism of osteosarcoma.At the same time,the pathogenesis of osteosarcoma must be the result of the interaction of multiple genes.Relevant studies have proved that lncRNA-UFC1 plays an important role in tumor and inflammation and can regulate the development of related diseases.However,whether lncRNA-UFC1 also plays a role in osteosarcoma,and how it plays a role has not been reported in the literature.Therefore,in-depth studies on the pathogenesis of osteosarcoma and the regulation mechanism of relevant genes in osteosarcoma cells are necessary for the clinical treatment of osteosarcoma.In the first part of this study,the main purpose was to explore the mechanism of the effect of FOSL1 on osteosarcoma cells,and to explore the effect of FOSL1-mediated ERK/AP-1 signaling pathway on the proliferation,invasion and migration of osteosarcoma cells.We first quantitatively analyze FOSL1 related molecules and ERK/ AP-1 related molecules in fresh osteosarcoma tissues and corresponding para cancerous tissues at the expression and transcription levels by qRT-PCR and Western-blot,meanwhile analyze the expression of FOSL1 in osteosarcoma tissues by immunohistochemistry.Furthermore,we examined the effects of si-FOSL1 on the transcription and protein expression levels of C-JUN,C-FOS,ERK1/2,p-ERK1/2,p38 MAPK and p-p38 MAPK.Next,the inhibitory effect of si-FOSL1 on the proliferation capacity of osteosarcoma cells was observed by CCK-8(Cell counting kit-8)experiments,and tumor formation ability of si-FOSL1 cell line inoculated in immunodeficient mice was observed by CB17 severe combined immunodeficient mice.After that,Flow cytometry detected the changes in the cell cycle of si-FOSL1.Finally,the influence of FOSL1 on the invasion ability of osteosarcoma cells was detected by Transwell cell invasion test,and the influence of FOSL1 on the migration ability of osteosarcoma cells was detected by cell scratch test.The results showed that: 1.In osteosarcoma tissues,the expressions of FOSL1 and ERK/ ap-1 related molecules were significantly up-regulated.2.PCR and Western-blot results showed that si-FOSL1 could reduce the expression levels of C-JUN,C-FOS,p-ERK1/2 and p-p38 MAPK.3.The expression levels of p-ERK1/2 and p-p38 MAPK in the overexpressed FOSL1 group were decreased after the addition of ERK/AP-1 inhibitors.4.Flow cytometry and CCK-8 experiments confirmed that FOSL1 could affect the proliferation of osteosarcoma cells by affecting cell cycle.5.The tumorigenic ability of the si-FOSL1 group was significantly decreased in vivo.6.Transwell cell invasion assay showed that si-FOSL1 group could significantly inhibit the invasion ability of osteosarcoma cells,which increased in the overexpression of FOSL1 group.7.Cell scratch assay showed that si-FOSL1 group could significantly inhibit the migration ability of osteosarcoma cells,which increased in the overexpression group.It is concluded that FOSL1 affects the ability of osteosarcoma cells to proliferate,invade and migrate through ERK/ ap-1signaling pathway,and may provide a new target for the treatment of osteosarcoma.In the second part of this study,we conducted a preliminary study on the regulation of lncRNA-UFC1 on osteosarcoma.First,the expression levels of lncRNA-UFC1 in osteosarcoma and adjacent tissues were detected by qRT-PCR.The expression levels of lncRNA-UFC1 in MG-63,U20 S and SaOs-2 cell lines were analyzed by qRT-PCR to screen cell lines for subsequent experiments.Next,we used MTT test,clonal formation test and in situ hybridization test to observe the proliferation of osteosarcoma cells after overexpression and knockdown of lncRNA-UFC1.Finally,we inject SaOs2 cells with overexpression and knockdown of lncRNA-UFC1 into mice to observe their proliferation capacity of osteosarcoma in vivo.The results show: 1.qRT-PCR results showed that lncRNA-UFC1 expression was significantly increased in osteosarcoma tissues.2.MTT assay,clonal formation assay and in situ hybridization assay all confirmed that lncRNA-UFC1 could promote the proliferation of osteosarcoma cells in vitro.3.Animal experiments furtherconfirmed that lncRNA-UFC1 can also promote the proliferation of osteosarcoma cells in vivo.It was concluded that lncRNA-UFC1 could promote the proliferation of osteosarcoma cells and play an important role in the regulation of osteosarcoma.更多还原