SATB1及miR-34a对皮肤基底癌细胞的影响及机制研究

发布时间：2018-04-28 18:17

本文选题：SATB1 + miR-34a　；参考：《北京协和医学院》2012年博士论文

【摘要】：过量的紫外线作用于皮肤基底细胞会诱发DNA损伤,引起原癌基因异常表达或抑癌基因突变,从而导致细胞周期失控,凋亡受阻,继而发生细胞形态改变和永生化,进一步导致恶性肿瘤发生。伴随着全球工业化和臭氧层的破坏,紫外线(UV)照射量逐渐增加,基底细胞癌的发病率逐年上升,但目前临床治疗方法仍存在许多弊端,所以,从紫外线诱发基底细胞癌的分子机制方面着手探索开发一种新的诊断防治方法是我们的研究目标。在紫外线诱导的DNA损伤和细胞凋亡调控分子网络中,处于核心位置的要数抑癌基因p53和原癌基因Bcl-2家族,二者也是导致基底细胞癌发生的关键分子。作为p53细胞调控网络的一个重要成员,miR-34a不但受p53的直接调控,还能够作用于某些p53活性调节基因,进而参与p53通路的反馈调节,并且多项研究均表明miR-34a在肿瘤发生过程中发挥重要作用,这些作用主要通过影响其靶分子来实现的。核基质结合蛋白SATB1则与Bcl-2的表达和功能的发挥存在紧密联系,并且综合以往的研究以及我们实验室的前期工作可以看出,SATB1广泛影响并参与了小至癌基因的表达以及细胞凋亡大至肿瘤转移等一系列癌症迁移的过程。故我们推测miR-34a与SATB1均与紫外线诱发的基底细胞癌相关,并且二者可能存在相互作用关系。为了考察miR-34a与SATB1在紫外线所诱发的基底细胞癌中的功能和作用机制,本研究首先收集了多例基底细胞癌临床样本,采用Real-time PCR和免疫组化的方法分别从mRNA水平和蛋白水平检测发现SATB1相对于正常对照组在基底细胞癌样本中表达明显上升,miR-34a表达水平则有所下降。随后在细胞水平上又选取了目前普遍认可的基底细胞癌细胞A431作为模型,借助流式细胞术,MTT法以及trans-well等手段证明miR-34a与SATB1对于基底细胞癌细胞的浸润有影响应明确。接着我们选取紫外线为刺激方式照射正常基底细胞模型Hacat,照射前对细胞分别进行过表达和干扰miR-34a与SATB1,发现SATB1可以加剧紫外线引发的DNA损伤程度并能够降低紫外线作用下细胞的存活率,与之相反miR-34a可以减缓紫外线对正常基底细胞Hacat造成的不良影响。为了深入解析二者对于皮肤细胞在紫外线作用下所表现出的相反作用的机制,我们先用生物信息学的方法预测发现SATB1是(?)niR-34a的靶基因,随后借助双荧光素酶报告基因系统,Real-time PCR和Western blotting的方法验证了miR-34a通过靶向结合于SATB13'UTR区的种子序列而抑制该基因的表达,从而实现对SATB1功能的反向调节。同时,我们发现SATB1也可以上调miR-34a的表达,随后我们利用报告基因系统以及序列删除和突变技术结合染色质免疫共沉淀技术(Chromatin Immunoprecipitation, ChIP)和EMSA实验确定pri-miR-34a的启动子区域256-579nt能够被SATB1激活,而其中AT富含片段对于二者的识别和结合是必须的。而过量的紫外线刺激能够增强SATB1特异性识别并结合miR-34a启动子256-579nt序列的能力,进而实现对1miR-34a的上调。由此证实miR-34a和SATB1可以形成反馈作用环路,该环路在紫外线诱发的基底细胞癌的发生中发挥着一定的功能,例如影响皮肤基底癌细胞的增殖及浸润。本研究首次阐明了SATB1与miR-34a之间存在反馈调控环路,该环路在紫外线诱发的基底细胞癌的发生中发挥着一定的功能,为基底细胞癌的临床诊断与防治提供了新的线索。
[Abstract]:Excessive ultraviolet radiation induces DNA damage in the basal cells of the skin, causing abnormal expression of the proto oncogene or mutation of the tumor suppressor gene, which causes the cell cycle to lose control and the apoptosis is blocked, and then the cell morphology changes and immortalization, which further causes the malignant tumor to occur. With the global industrialization and the destruction of the ozone layer, ultraviolet (UV) illumination However, the incidence of basal cell carcinoma is increasing year by year, but there are still many drawbacks in the clinical treatment methods. Therefore, it is our goal to explore and develop a new method of diagnosis and control from the molecular mechanism of ultraviolet induced basal cell carcinoma.
In the molecular network of DNA damage and apoptosis induced by ultraviolet rays, the key location is the tumor suppressor gene p53 and the proto oncogene Bcl-2 family. The two are also the key molecules that lead to the occurrence of basal cell carcinoma. As an important member of the p53 cell regulation network, miR-34a is not only directly regulated by p53, but also can act on some of them. P53 activity regulates genes and then participates in the feedback regulation of the p53 pathway, and a number of studies have shown that miR-34a plays an important role in the development of tumors. These effects are mainly achieved by affecting their target molecules. The nuclear matrix binding protein SATB1 is closely related to the expression and function of Bcl-2, and is integrated with the previous research. The early work of our laboratory shows that SATB1 has widely influenced and participated in a series of cancer migration processes, such as the expression of small to cancer gene and the process of cancer migration, such as cell apoptosis and tumor metastasis. Therefore, we speculate that both miR-34a and SATB1 are associated with ultraviolet induced basal cell carcinoma, and the two may have interactions.
In order to investigate the function and mechanism of miR-34a and SATB1 in the basal cell carcinoma induced by ultraviolet rays, this study first collected a number of clinical samples of basal cell carcinoma. Using Real-time PCR and immunohistochemical methods, the SATB1 phase was detected from the mRNA level and protein level respectively in the normal control group in the basal cell carcinoma samples. The expression level was obviously increased and the expression level of miR-34a decreased. Then the commonly recognized basal cell carcinoma cell A431 was selected at the cell level as a model. The influence of miR-34a and SATB1 on the infiltration of basal cell carcinoma cells should be confirmed by flow cytometry, MTT and trans-well. The exterior line is stimulated by the normal basal cell model Hacat. The cells are overexpressed and interfered with miR-34a and SATB1 before irradiation. It is found that SATB1 can aggravate the DNA damage caused by ultraviolet radiation and reduce the survival rate of cells under ultraviolet radiation. On the contrary, miR-34a can slow down the ultraviolet radiation of the normal basal cell Hacat. In order to analyze the adverse effects of the two people on the ultraviolet radiation of the skin cells, we first predicted that SATB1 was the target gene of (?) niR-34a by bioinformatics, and then the dual luciferase reporter gene system, Real-time PCR and Western blotting were used to verify miR-3 4A inhibits the expression of the gene by targeting the sequence of seeds in the SATB13'UTR region to achieve the reverse regulation of the function of SATB1. At the same time, we found that SATB1 can also increase the expression of miR-34a. Then we use the reporting gene system and sequence deletion and mutation technology to combine chromatin immunoprecipitation (Chromatin Immuno). Precipitation, ChIP) and EMSA experiments determine that 256-579nt in the promoter region of the pri-miR-34a can be activated by SATB1, in which the AT is rich in the recognition and combination of the two, and the excess ultraviolet stimulus can enhance the SATB1 specificity recognition and combine the miR-34a promoter 256-579nt sequence, and then realize the 1miR-34a. Up - regulation. This confirms that miR-34a and SATB1 can form a feedback loop that plays a role in the occurrence of ultraviolet induced basal cell carcinoma, such as the proliferation and infiltration of basal cancer cells in the skin.
In this study, the feedback loop between SATB1 and miR-34a has been clarified for the first time. This loop plays a certain function in the occurrence of UV induced basal cell carcinoma, which provides a new clue for the clinical diagnosis and prevention of basal cell carcinoma.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.5

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