miR-100在食管鳞癌中的调控作用及机制研究

发布时间：2017-12-26 16:25

本文关键词：miR-100在食管鳞癌中的调控作用及机制研究　出处：《北京工业大学》2015年博士论文　论文类型：学位论文

更多相关文章： 食道癌 EC109 mi R-100 CXCR7 FGFR3

【摘要】：食道癌是发生在食管上皮组织的恶性肿瘤。发病率占肿瘤发病率的第八位,而死亡率则占肿瘤死亡率的第六位。80%死亡病例来自发展中国家。食道癌依据病理特性分为两个亚型:鳞癌(Esophageal squamous cell carcinoma,ESCC)和腺癌(Esophageal adenocarcinoma,EAC),鳞癌是主要的亚型,全球95%的食道癌的病理分型为鳞癌。食道鳞癌主要是产生于食道的鳞状上皮,而腺癌主要来源于食道与胃交界处的腺体。流行病学调查认为过热饮食、吸烟和嗜酒是造成发病的主要原因。MicroRNA是近年来发现的一类小的非编码的RNA,对基因的表达起负向调节作用。它通过诱导mRNA的降解和抑制翻译等方式抑制转录后的基因表达。miRNA通过碱基互补配对识别靶基因并诱导其降解。miRNA的识别位点通常位于mRNA的3’UTR区。研究发现miRNA在肿瘤组织中异常表达。最近针对食管癌的miRNA表达谱的分析发现正常食管细胞系、ESC和EAC之间的microRNA表达谱各不相同。而miR-100的表达低调同时出现在食道癌的两个亚型之中。在食管鳞癌细胞中,miR-100的表达水平显著低于正常食管上皮细胞,但鲜有见其作用靶基因和调节机理的报道。本研究通过构建miR-100表达质粒和脂质体转染的方法将miR-100转入食管鳞癌细胞系EC109中,筛选出稳定高表达miR-100的克隆,在此基础上进一步进行了microRNA抑癌机理的研究。我们发现miR-100能够调节食管癌细胞的某些生物学特性,如其抑制了食管癌细胞的增殖,研究表明miR-100可调节食道癌细胞的细胞周期并抑制食管癌细胞从G1期进入S期,减少了食管癌细胞在S期和G2/M期的细胞比例,从而降低癌细胞的增殖;此外,miR-100还具有抑制食管癌细胞的迁移和侵袭的能力,促进了癌细胞的凋亡;体外软琼脂成瘤性试验和裸鼠荷瘤试验,均发现miR-100具有明显的抑制肿瘤生长的作用。本研究还进一步鉴定了miR-100的作用靶点,揭示了miR-100抑制食道癌细胞生长,促进癌细胞凋亡和抑制其致瘤性的的作用机理。Luciferase报告基因检测试验和qRT-PCR以及Western blot的分析结果显示,miR-100可显著抑制趋化因子受体CXCR7(CXC chemokine receptor7)和成纤维细胞生长因子受体FGFR3(fibroblast growth factor receptor 3)的表达。CXCR7是趋化因子受体家族的一员。CXCR7在多种肿瘤组织中高表达如肺癌、乳腺癌、前列腺癌和肝癌等。CXCR7的高表达可以促进细胞的生长、迁移并在实验动物体内促进肿瘤生长。CXCR7有两个配体分别为CXCL11和CXCL12。研究发现CXCL12与CXCR7的相互作用可以促进细胞的迁移并与胰腺癌患者的预后相关。FGFR3是成纤维细胞生长因子受体家族成员。他是一个跨膜蛋白,分膜外、跨膜和膜内区组成,膜外区含有3个免疫球蛋白相似区,胞内区具有酪氨酸激酶活性。与配体结合后FGFR3的可以激活下游通路并参与调节细胞的生长、分化、血管形成、损伤修复和胚胎发育等生理过程。在肿瘤中激活FGFR3通路可以促进细胞的生长、迁移和。实验结果表明miR-100可通过抑制CXCR7和FGFR3的表达实现调控食道癌细胞生长、凋亡、迁移侵润和致瘤性的作用。本研究为食道癌的基于miRNA的基因治疗提供了新的思路。
[Abstract]:Cancer of the esophagus is a malignant tumor occurring in the epithelial tissue of the esophagus. The incidence of the disease accounted for eighth of the tumor incidence, while the mortality rate accounted for sixth of the tumor mortality. 80% of the deaths are from developing countries. Esophageal cancer is divided into two subtypes based on pathological characteristics: Esophageal squamous cell carcinoma (ESCC) and adenocarcinoma (Esophageal adenocarcinoma, EAC), squamous cell carcinoma is the main subtype, and 95% of the world's esophageal cancer is classified as squamous cell carcinoma. Squamous carcinoma of the esophagus is mainly a squamous epithelium that occurs in the esophagus, while adenocarcinoma is mainly derived from the glands at the junction of the esophagus and stomach. Epidemiological studies suggest that overheated diet, smoking and drinking are the main causes of the disease. MicroRNA is a kind of small non coding RNA found in recent years, which plays a negative regulatory role in gene expression. It inhibits the post transcriptional gene expression by inducing the degradation of mRNA and inhibition of translation. MiRNA can identify the target gene and induce its degradation by complementary base pairs. The loci of miRNA are usually located in the 3 'UTR area of mRNA. The abnormal expression of miRNA in tumor tissues was found. The recent analysis of miRNA expression profiles for esophageal cancer found that the microRNA expression profiles between normal esophageal cell lines, ESC and EAC were different. The low profile of the expression of miR-100 appeared in the two subtypes of esophageal cancer. In squamous cell carcinoma of the esophagus, the expression of miR-100 was significantly lower than that of normal esophageal epithelial cells, but few of the target genes and regulatory mechanisms were reported. In this study, we constructed miR-100 expression plasmid and liposome transfection method to transfer miR-100 into esophageal squamous cell carcinoma cell line EC109, and screened stable and highly expressed miR-100 clones. Based on that, we further studied the mechanism of microRNA inhibiting cancer. We found that miR-100 can regulate some biological characteristics of esophageal carcinoma cells, it inhibited the proliferation of esophageal carcinoma cells, cell cycle studies show that miR-100 can regulate the esophageal cancer cells and inhibition of esophageal cancer cells from G1 phase into S phase, decreased in S phase and G2/ M phase cells proportion of esophageal cancer cells, thereby reducing cancer cell proliferation; in addition, miR-100 also inhibited esophageal cancer cell migration and invasion ability, promote the apoptosis of cancer cells in soft agar; tumorigenicity test and tumor growth test showed that miR-100 has obvious effect of inhibiting tumor. This study further identified the target of miR-100, and revealed the mechanism of miR-100 inhibiting the growth of esophageal cancer cells, promoting the apoptosis and inhibiting tumorigenicity of esophageal cancer cells. Luciferase reporter gene detection test and qRT-PCR and Western blot analysis showed that miR-100 could significantly inhibit the expression of chemokine receptor CXCR7 (CXC chemokine receptor7) and fibroblast growth factor receptor FGFR3 (fibroblast FGFR3 3). CXCR7 is a member of the chemokine receptor family. CXCR7 is highly expressed in a variety of tumor tissues such as lung cancer, breast cancer, prostate cancer and liver cancer. The high expression of CXCR7 can promote cell growth, migration and promote tumor growth in experimental animals. The two ligands of CXCR7 are CXCL11 and CXCL12, respectively. The study found that the interaction of CXCL12 and CXCR7 can promote cell migration and be associated with the prognosis of pancreatic cancer patients. FGFR3 is a member of the fibroblast growth factor receptor family. He is a transmembrane protein consisting of the outer membrane, the transmembrane and the inner membrane. The outer region contains 3 similar regions of immunoglobulin, and the intracellular region has tyrosine kinase activity. After binding to ligands, FGFR3 can activate downstream pathways and participate in regulating physiological processes such as cell growth, differentiation, angiogenesis, injury repair, and embryonic development. Activation of the FGFR3 pathway in the tumor can promote cell growth, migration and migration. The results show that miR-100 can regulate the growth, apoptosis, migration and tumorigenicity of esophageal cancer cells by inhibiting the expression of CXCR7 and FGFR3. This study provides a new way of thinking for miRNA based gene therapy for esophageal cancer.
【学位授予单位】：北京工业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.1

【相似文献】