【摘要】：食管癌是惡性程度最高的癌種之一,其死亡率占世界癌癥相關(guān)死亡率的第六位。其中,中國的食管癌患者數(shù)量居世界首位。根據(jù)病理學(xué)分類,可將食管癌分為兩種亞型,即食管鱗狀細胞癌(ESCC)和食管腺癌(EAC),而中國的食管癌患者絕大部分是食管鱗狀細胞癌(90%)。目前,食管鱗狀細胞癌的臨床治療手段很有限,中國食管癌患者的五年生存率低于20%。因此,進一步探究食管鱗癌的發(fā)病機理、篩選食管鱗癌診斷的分子標(biāo)志物、尋找有效的藥物靶點具有重要的臨床意義。microRNA是一類長度約18～25nt的小分子單鏈非編碼RNA,是由具有約70～90nt發(fā)夾結(jié)構(gòu)的單鏈RNA前體經(jīng)過Dicer酶加工生成。microRNA主要通過其seed region與靶基因mRNA的3'UTR堿基互補配對,導(dǎo)致靶基因mRNA的翻譯抑制或降解,從而對靶基因在轉(zhuǎn)錄后水平進行調(diào)控。越來越多的研究結(jié)果表明,microRNA在細胞的增殖、分化、凋亡、惡性轉(zhuǎn)化及腫瘤轉(zhuǎn)移等過程中發(fā)揮重要作用。本文的第一部分,對91例食管鱗癌及癌旁樣本進行小RNA測序、分析并驗證測序結(jié)果,發(fā)現(xiàn)miR-a在食管癌組織中的表達量明顯高于癌旁。利用細胞模型和裸鼠模型研究miR-a在食管癌中的功能,發(fā)現(xiàn)miR-a可促進食管癌細胞的侵襲和遷移,且高表達miR-a食管癌細胞可促進裸鼠的肺轉(zhuǎn)移。mRNA芯片分析、數(shù)據(jù)庫預(yù)測結(jié)合熒光素酶報告基因?qū)嶒炞C實SMAD7是miR-a的靶基因。在食管癌細胞中敲降SMAD7,可模擬miR-a對食管癌細胞侵襲、遷移的促進作用,在過表達miR-a的食管癌細胞中同時過表達SMAD7可部分回復(fù)miR-a對食管癌細胞侵襲、遷移的促進作用;在食管癌細胞中,敲降SMAD7的表達可促進SMAD2/3入核,而過表達miR-a也可促進SMAD2/3入核;以上結(jié)果說明miR-a通過靶向SMAD7促進SMAD2/3入核從而促進細胞的侵襲遷移。在血漿中檢測miR-a的表達情況,發(fā)現(xiàn)miR-a在食管癌患者血漿中的表達明顯高于在健康人血漿中的表達,這提示miR-a可能作為食管鱗癌的一個新的診斷標(biāo)志物。本文第二部分,通過qPCR檢測71例食管鱗癌樣本中miR-503的表達,發(fā)現(xiàn)miR-503在食管癌組織中的表達明顯低于配對的癌旁組織。細胞功能研究發(fā)現(xiàn),miR-503可抑制食管癌細胞的增殖、侵襲、遷移及并導(dǎo)致細胞周期G1/S期的阻滯。數(shù)據(jù)庫預(yù)測、Western blot、結(jié)合熒光素酶報告基因?qū)嶒灡砻鰿yclinD1是miR-503的靶基因。過表達miR-503可以抑制CyclinD1 mRNA和蛋白水平的表達。回復(fù)實驗結(jié)果表明CyclinD1可以部分回復(fù)miR-503對細胞惡性表型的抑制作用。qPCR結(jié)果顯示,CyclinD1 mRNA在食管癌組織中的表達低于其在配對的癌旁正常組織中的表達,且與miR-503的表達呈負相關(guān)。以上結(jié)果說明miR-503通過靶向CyclinD1抑制食管癌細胞的惡性表型,提示miR-503可作為食管鱗癌的一個新的治療靶點。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.1

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