miR-a和miR-503在食管癌发生发展中的作用及机制研究
发布时间：2018-10-17 18:44
【摘要】：食管癌是恶性程度最高的癌种之一,其死亡率占世界癌症相关死亡率的第六位。其中,中国的食管癌患者数量居世界首位。根据病理学分类,可将食管癌分为两种亚型,即食管鳞状细胞癌(ESCC)和食管腺癌(EAC),而中国的食管癌患者绝大部分是食管鳞状细胞癌(90%)。目前,食管鳞状细胞癌的临床治疗手段很有限,中国食管癌患者的五年生存率低于20%。因此,进一步探究食管鳞癌的发病机理、筛选食管鳞癌诊断的分子标志物、寻找有效的药物靶点具有重要的临床意义。microRNA是一类长度约18～25nt的小分子单链非编码RNA,是由具有约70～90nt发夹结构的单链RNA前体经过Dicer酶加工生成。microRNA主要通过其seed region与靶基因mRNA的3'UTR碱基互补配对,导致靶基因mRNA的翻译抑制或降解,从而对靶基因在转录后水平进行调控。越来越多的研究结果表明,microRNA在细胞的增殖、分化、凋亡、恶性转化及肿瘤转移等过程中发挥重要作用。本文的第一部分,对91例食管鳞癌及癌旁样本进行小RNA测序、分析并验证测序结果,发现miR-a在食管癌组织中的表达量明显高于癌旁。利用细胞模型和裸鼠模型研究miR-a在食管癌中的功能,发现miR-a可促进食管癌细胞的侵袭和迁移,且高表达miR-a食管癌细胞可促进裸鼠的肺转移。mRNA芯片分析、数据库预测结合荧光素酶报告基因实验证实SMAD7是miR-a的靶基因。在食管癌细胞中敲降SMAD7,可模拟miR-a对食管癌细胞侵袭、迁移的促进作用,在过表达miR-a的食管癌细胞中同时过表达SMAD7可部分回复miR-a对食管癌细胞侵袭、迁移的促进作用;在食管癌细胞中,敲降SMAD7的表达可促进SMAD2/3入核,而过表达miR-a也可促进SMAD2/3入核;以上结果说明miR-a通过靶向SMAD7促进SMAD2/3入核从而促进细胞的侵袭迁移。在血浆中检测miR-a的表达情况,发现miR-a在食管癌患者血浆中的表达明显高于在健康人血浆中的表达,这提示miR-a可能作为食管鳞癌的一个新的诊断标志物。本文第二部分,通过qPCR检测71例食管鳞癌样本中miR-503的表达,发现miR-503在食管癌组织中的表达明显低于配对的癌旁组织。细胞功能研究发现,miR-503可抑制食管癌细胞的增殖、侵袭、迁移及并导致细胞周期G1/S期的阻滞。数据库预测、Western blot、结合荧光素酶报告基因实验表明CyclinD1是miR-503的靶基因。过表达miR-503可以抑制CyclinD1 mRNA和蛋白水平的表达。回复实验结果表明CyclinD1可以部分回复miR-503对细胞恶性表型的抑制作用。qPCR结果显示,CyclinD1 mRNA在食管癌组织中的表达低于其在配对的癌旁正常组织中的表达,且与miR-503的表达呈负相关。以上结果说明miR-503通过靶向CyclinD1抑制食管癌细胞的恶性表型,提示miR-503可作为食管鳞癌的一个新的治疗靶点。
[Abstract]:Esophageal cancer is one of the most malignant cancer species, and its mortality rate is the sixth highest in the world. Among them, the number of esophageal cancer patients in China ranks first in the world. According to the pathological classification, esophageal carcinoma can be divided into two subtypes: esophageal squamous cell carcinoma (ESCC) and adenocarcinoma of esophagus (EAC),). The majority of esophageal cancer patients in China are esophageal squamous cell carcinoma (90%). At present, the clinical treatment of esophageal squamous cell carcinoma is very limited. The five-year survival rate of esophageal cancer patients in China is lower than 20%. Therefore, we should further explore the pathogenesis of esophageal squamous cell carcinoma and screen molecular markers for the diagnosis of esophageal squamous cell carcinoma. It is of great clinical significance to find effective drug targets. MicroRNA is a class of small molecular single-stranded non-coding RNA, with about 18~25nt in length. The precursor of single-stranded RNA with about 70~90nt hairpin structure is produced by Dicer enzyme. MicroRNA is mainly produced by Dicer enzyme. Seed region is complementary to the 3'UTR base of target gene mRNA. It leads to the inhibition or degradation of target gene mRNA, which regulates the target gene at posttranscriptional level. More and more studies have shown that microRNA plays an important role in cell proliferation, differentiation, apoptosis, malignant transformation and tumor metastasis. In the first part of this paper, 91 cases of esophageal squamous cell carcinoma and its adjacent samples were sequenced by small RNA sequencing. The results showed that the expression of miR-a in esophageal carcinoma tissues was significantly higher than that in adjacent tissues. Cell model and nude mouse model were used to study the function of miR-a in esophageal carcinoma. It was found that miR-a could promote invasion and migration of esophageal carcinoma cells, and high expression of miR-a could promote lung metastasis in nude mice. Database prediction combined with luciferase reporter gene experiment confirmed that SMAD7 is the target gene of miR-a. Knockdown of SMAD7, in esophageal cancer cells could mimic the role of miR-a in promoting the invasion and migration of esophageal cancer cells. Overexpression of SMAD7 at the same time in esophageal cancer cells with overexpression of miR-a could partly restore the role of miR-a in promoting the invasion and migration of esophageal cancer cells. In esophageal cancer cells, knockdown SMAD7 expression can promote SMAD2/3 entry, and overexpression of miR-a can also promote SMAD2/3 entry, which suggests that miR-a promotes the invasion and migration of SMAD2/3 by targeting SMAD7. The expression of miR-a in patients with esophageal cancer was significantly higher than that in healthy controls, which suggested that miR-a might be a new diagnostic marker for esophageal squamous cell carcinoma. In the second part, the expression of miR-503 in 71 cases of esophageal squamous cell carcinoma was detected by qPCR. It was found that the expression of miR-503 in esophageal carcinoma tissues was significantly lower than that in matched paracancerous tissues. Cell function studies showed that miR-503 could inhibit proliferation, invasion, migration and arrest of G 1 / S phase in esophageal carcinoma cells. Database predictive, Western blot, binding luciferase reporter gene experiment showed that CyclinD1 is the target gene of miR-503. Overexpression of miR-503 can inhibit the expression of CyclinD1 mRNA and protein. The results showed that CyclinD1 could partially restore the inhibitory effect of miR-503 on cell malignant phenotype. QPCR results showed that the expression of CyclinD1 mRNA in esophageal carcinoma was lower than that in matched adjacent normal tissues and was negatively correlated with the expression of miR-503. These results suggest that miR-503 inhibits the malignant phenotype of esophageal carcinoma cells by targeting CyclinD1, suggesting that miR-503 may be a new therapeutic target for esophageal squamous cell carcinoma.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1
本文编号：2277609
[Abstract]:Esophageal cancer is one of the most malignant cancer species, and its mortality rate is the sixth highest in the world. Among them, the number of esophageal cancer patients in China ranks first in the world. According to the pathological classification, esophageal carcinoma can be divided into two subtypes: esophageal squamous cell carcinoma (ESCC) and adenocarcinoma of esophagus (EAC),). The majority of esophageal cancer patients in China are esophageal squamous cell carcinoma (90%). At present, the clinical treatment of esophageal squamous cell carcinoma is very limited. The five-year survival rate of esophageal cancer patients in China is lower than 20%. Therefore, we should further explore the pathogenesis of esophageal squamous cell carcinoma and screen molecular markers for the diagnosis of esophageal squamous cell carcinoma. It is of great clinical significance to find effective drug targets. MicroRNA is a class of small molecular single-stranded non-coding RNA, with about 18~25nt in length. The precursor of single-stranded RNA with about 70~90nt hairpin structure is produced by Dicer enzyme. MicroRNA is mainly produced by Dicer enzyme. Seed region is complementary to the 3'UTR base of target gene mRNA. It leads to the inhibition or degradation of target gene mRNA, which regulates the target gene at posttranscriptional level. More and more studies have shown that microRNA plays an important role in cell proliferation, differentiation, apoptosis, malignant transformation and tumor metastasis. In the first part of this paper, 91 cases of esophageal squamous cell carcinoma and its adjacent samples were sequenced by small RNA sequencing. The results showed that the expression of miR-a in esophageal carcinoma tissues was significantly higher than that in adjacent tissues. Cell model and nude mouse model were used to study the function of miR-a in esophageal carcinoma. It was found that miR-a could promote invasion and migration of esophageal carcinoma cells, and high expression of miR-a could promote lung metastasis in nude mice. Database prediction combined with luciferase reporter gene experiment confirmed that SMAD7 is the target gene of miR-a. Knockdown of SMAD7, in esophageal cancer cells could mimic the role of miR-a in promoting the invasion and migration of esophageal cancer cells. Overexpression of SMAD7 at the same time in esophageal cancer cells with overexpression of miR-a could partly restore the role of miR-a in promoting the invasion and migration of esophageal cancer cells. In esophageal cancer cells, knockdown SMAD7 expression can promote SMAD2/3 entry, and overexpression of miR-a can also promote SMAD2/3 entry, which suggests that miR-a promotes the invasion and migration of SMAD2/3 by targeting SMAD7. The expression of miR-a in patients with esophageal cancer was significantly higher than that in healthy controls, which suggested that miR-a might be a new diagnostic marker for esophageal squamous cell carcinoma. In the second part, the expression of miR-503 in 71 cases of esophageal squamous cell carcinoma was detected by qPCR. It was found that the expression of miR-503 in esophageal carcinoma tissues was significantly lower than that in matched paracancerous tissues. Cell function studies showed that miR-503 could inhibit proliferation, invasion, migration and arrest of G 1 / S phase in esophageal carcinoma cells. Database predictive, Western blot, binding luciferase reporter gene experiment showed that CyclinD1 is the target gene of miR-503. Overexpression of miR-503 can inhibit the expression of CyclinD1 mRNA and protein. The results showed that CyclinD1 could partially restore the inhibitory effect of miR-503 on cell malignant phenotype. QPCR results showed that the expression of CyclinD1 mRNA in esophageal carcinoma was lower than that in matched adjacent normal tissues and was negatively correlated with the expression of miR-503. These results suggest that miR-503 inhibits the malignant phenotype of esophageal carcinoma cells by targeting CyclinD1, suggesting that miR-503 may be a new therapeutic target for esophageal squamous cell carcinoma.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1
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