miR-125b在皮肤鳞状细胞癌中的作用及其机制研究

发布时间：2019-03-22 13:49

【摘要】：皮肤鳞状细胞癌(简称皮肤鳞癌)是我国常见的皮肤恶性肿瘤之一,容易发生转移和复发,是导致非黑色素瘤皮肤癌患者死亡的主要病因。因此,寻找其发病过程中发挥关键作用的分子对鳞癌的诊断和治疗是必不可少的。微小RNA (microRNA或miRNA)是一类内源性的单链非编码小RNA分子,在转录后水平调控基因的表达。miRNA的异常表达与肿瘤的发生以及发展密不可分。本课题主要研究miR-125b对皮肤鳞癌细胞的作用及其机制,并对影响miR-125b表达的因素进行初步探讨。本研究分为四部分进行： 1.首先运用低密度芯片检测皮肤鳞癌组织中差异性表达的miRNAs,运用实时定量PCR检测miR-125b在皮肤鳞癌、光化性角化病和健康人皮肤组织以及不同分化程度的皮肤鳞癌组织中的表达。最后运用原位杂交检测皮肤鳞癌组织中miR-125b的表达水平。 2.运用脂质体介导方法将人工合成的miRNA前体转染皮肤鳞癌UT-SCC-7和A431细胞系,升高细胞内miR-125b的表达水平,观察细胞生长增殖、侵袭和迁移以及细胞凋亡的变化情况。 3.运用芯片和生物信息学方法预测miR-125b的潜在靶基因,并运用荧光素酶报告基因系统、实时定量PCR和western印迹实验对靶基因进行验证。同时运用实时定量PCR检测皮肤鳞癌组织中靶基因的表达,并分析靶基因与miR-125b表达水平的相关性。运用RNA干扰技术敲降细胞内靶基因的表达,观察细胞生长增殖、侵袭和迁移以及细胞凋亡的变化情况。最后研究miR-125b下游调控可能涉及的分子机制,运用实时定量PCR检测miR-125b对下游基因的影响,阐述miR-125b在皮肤鳞癌细胞中可能参与的调控网络。 4.通过生物信息学方法对miR-125b的启动子及其上游区域进行分析,利用药物5-aza对鳞癌细胞进行处理,运用实时定量PCR检测药物处理后细胞内miR-125b的表达,探讨miR-125b的表观遗传学改变。此外,利用KGF(角质细胞生长因子)刺激细胞,检测miR-125b的表达是否受影响。通过对本课题的研究,可以得到以下几个结论： 1. miR-125b在皮肤鳞癌组织中低表达,但是miR-125b可能与肿瘤的分化程度无关。 2.细胞内miR-125b的表达升高后,造成细胞克隆形成能力降低、细胞周期G1/S进程阻滞、细胞增殖减慢以及侵袭和迁移能力均降低,而细胞凋亡增加。 3.MMP-13和MAP2K7是miR-125b的直接靶基因,miR-125b抑制MMP-13和MAP2K7的表达；同时MMP-13和MAP2K7与miR-125b的表达呈负相关。除此之外,TGFBR2、TP53INP1、MMP-7、cyclinD1和c-Jun等基因也受miR-125b的调控。敲降细胞内MMP-13的表达后,细胞克隆形成、侵袭和迁移能力降低,但细胞的增殖和凋亡不受影响。敲降MAP2K7的表达后,细胞的增殖和克隆形成能力降低,细胞凋亡增加。通过对下游基因的分析,推测miR-125b可能参与TGF-β、NF-kappaB. JNK和Wnt信号通路的调控。 4. miR-125b的启动子区域呈现密集的CpG岛,并且利用5-aza对鳞癌细胞进行去甲基化处理后miR-125b的表达水平升高,说明受到甲基化的调控。同时KGF刺激细胞后miR-125b的表达受抑制。
[Abstract]:Cutaneous squamous cell carcinoma (SCC) is one of the most common skin malignant tumors in China. It is easy to transfer and relapse. It is the main cause of the death of non-melanoma skin cancer patients. Therefore, it is necessary to find the molecules that play a key role in the diagnosis and treatment of squamous cell carcinoma. MicroRNA (microRNA or miRNA) is a kind of endogenous single-chain non-coding small RNA molecule, and the table of the gene is regulated after transcription. The abnormal expression of the miRNA and the occurrence of the tumor and the development of the miRNA are not The purpose of this study is to study the role of miR-125b on skin squamous cell carcinoma and its mechanism, and to investigate the factors that affect the expression of miR-125b. This study is divided into four parts. 1. First, using low-density chip to detect the differentially expressed miRNAs in skin squamous cell carcinoma (SCC), real-time quantitative PCR was used to detect the expression of miR-125b in the squamous cell carcinoma of the skin, the actinic keratosis and the skin tissue of healthy people and the different degree of differentiation of the squamous cell carcinoma of the skin. The expression of miR-125b in the tissue of skin squamous cell carcinoma was detected by in situ hybridization. 2. The expression level of miR-125b in the cell was increased by using the liposome-mediated method to transfect the human skin squamous cell carcinoma (UT-SCC-7) and the A431 cell line (A431), and the cell growth, proliferation, invasion and migration and the cell were observed. 3. The potential target genes of miR-125b were predicted by using the method of chip and bioinformatics, and the luciferase reporter gene system, real-time quantitative PCR and western blot were used. The target gene was verified by real-time quantitative PCR, and the target gene and miR-125 were analyzed. B. the expression of the target gene in the cell is knocked down by using the RNA interference technique, and the growth, the invasion and the migration of the cell are observed, and the expression of the target gene in the cell is observed by using the RNA interference technique. The effect of miR-125b on the downstream gene was detected by real-time quantitative PCR, and the expression of miR-125b in the skin squamous cell carcinoma was described. and 4. the promoter of the miR-125b and the upstream region of the miR-125b are analyzed by a bioinformatics method, the scale cancer cells are treated by using the drug 5-aza, and the expression of the miR-125b in the cells after the drug treatment is detected by the real-time quantitative PCR, and the miR-1 25b. In addition, the cells were stimulated with KGF (Keratinocyte Growth Factor) to detect miR-1. The expression of 25b is affected. By this subject The following conclusions can be obtained by the study:1. miR-125b is low-expressed in the skin squamous cell carcinoma tissue, but miR-1 25b may not be related to the degree of differentiation of the tumor.2. After the expression of miR-125b in the cell is increased, the cell clone formation ability is reduced, the cell cycle G1/ S process block, the cell proliferation is slowed down, and the invasion 3. MMP-13 and MAP2K7 are the direct target genes of miR-125b, and miR-125b inhibits the expression of MMP-13 and MAP2K7; and MMP-13 and MAP 2K7 was negatively correlated with the expression of miR-125b. In addition, TGFBR2, TP53INP1, MMP-7, cyclinD1 and c- The gene of Jun et al is also regulated by miR-125b. After the expression of MMP-13 in the cell, the cell clones form, invade and move. The cell proliferation and apoptosis are not affected. After the expression of MAP2K7, the cells The proliferation and clone formation ability of the cells is reduced, and the cell apoptosis is increased. By analyzing the downstream gene, it is presumed that the miR-125b May be involved in TGF-1, NF-ka Regulation of the ppaB. JNK and Wnt signaling pathways.4. The promoter region of miR-125b presents a dense CpG island and miR-1 is miR-1 after demethylation of the squamous cell with 5-aza. The expression level of 25b is increased and the regulation of methylation is indicated. At the same time K
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R739.5

【共引文献】