miR-193a-5p下调ERBB2表达抑制食管鳞状细胞癌侵袭的机制研究

发布时间：2018-05-22 16:57

本文选题：食管鳞状细胞癌 + miR-193a-5p　；参考：《郑州大学》2017年博士论文

【摘要】：1背景及目的食管癌(esophageal cancer)是目前发病率、死亡率都较高的消化道恶性肿瘤之一,目前全球癌症发病率中排名第9位,总死亡率占到第6位。在发达国家,食管癌的发病率排名第20位,但其死亡排第11位。在发展中国家,食管癌发病率排名第8位,死亡率排在第5位。食管癌已从1990年的第七大癌症上升至2013年的第六大癌症,衰老和不断增长的人群是食管癌病例增加的驱动因素。在食管癌中,鳞状细胞癌和腺癌为主要病理类型,其中鳞状细胞癌在我国占90%以上。世界每年约有40万人死于食管癌,而我国是世界上食管癌的高发地区之一,平均每年因该疾病的死亡人数大于15万。由于食管癌的早期症状不明显并且缺乏早期诊断,多数患者在首次诊断时已处于疾病晚期,食管癌切除术后5年生存率9.5-45%,肿瘤侵袭和转移是导致食管癌患者死亡的主要因素,侵袭和转移主要依赖于原癌基因的激活和肿瘤抑制基因的失活,以及细胞增殖和凋亡等各种因素协作的共同结果。如果我们可以从遗传学和表观遗传学的角度研究食管癌的发生和发展,它将为食管癌的诊断和治疗提供新的靶点以及新的策略。人类表皮生长因子受体2(human epidermal growth factor receptor 2,ERBB2)是人表皮生长因子受体家族的成员之一,位于染色体17q12-21.32,编码相对分子质量为185,000跨膜受体样蛋白,属于跨膜酪氨酸激酶受体蛋白,具有酪氨酸激酶活性,该家族成员的过表达与乳腺癌、前列腺癌等肿瘤疾病的不良后果有关。已明确证明ERBB2的扩增已经显示在某些侵袭型乳腺癌的进展中起重要作用,ERBB2蛋白已成为30%乳腺癌患者的重要生物标志物和治疗靶标。ERBB2在细胞生长、分化和迁移中起重要作用,其过表达促进肿瘤生长、转移和肿瘤血管生成。在食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)中,已报道有ERBB2过表达,ERBB2被认为是食管癌的潜在治疗靶点。然而,ERBB2如何调节ESCC发生的分子机制仍然不明确。Micro RNA(miRNA)是近年来发现的小的非编码RNA分子,含有约22个核苷酸,广泛分布于真核生物中。虽然大多数miRNA位于细胞内,但也已经在细胞外环境中发现了一些miRNA,称之为循环miRNA或细胞外miRNA。目前最常见对靶基因的调控是miRNA通过与一些蛋白结合形成miRNA诱导沉默复合物,进而引导与信使RNA(Messenger RNA,m RNA)的3'非编码区(3'-UTR)完全或不完全配对,识别特异的m RNA后使其降解或阻遏其转录后翻译,即基因表达的转录后抑制靶基因的表达。miRNA通过调节靶基因的表达,在细胞增殖、凋亡、分化等过程中起着重要作用,并且在维持细胞的正常功能中起重要作用。研究人员已经发现,miRNA失调与许多疾病有关,如与肿瘤密切相关。本课题首先采用免疫组化方法探讨ERBB2在食管鳞状细胞癌中的表达及其与临床病理特征之间的关系;阐明miR-193a-5p抑制ERBB2翻译的分子机制揭示ERBB2的全新调控方式;进一步检测食管癌组织及患者血清中miR-193a-5p的表达水平,评价miR-193a-5p作为食管癌预后指标的价值。2方法及结果2.1免疫组化检测ERBB2在食管鳞癌中的表达及意义我们采用免疫组化探讨ERBB2在食管鳞状细胞癌中的表达及其与临床病理特征之间的关系,食管鳞癌组织中的ERBB2表达率(35/68,51.5%)高于正常食管组织8.8%(6/68)(χ2=33.03 P0.01)。ERBB2的表达与肿瘤淋巴结转移和肿瘤分期相关,差异有统计学意义(P0.01),与年龄、性别无统计学意义(P0.05)。2.2 miR-193a-5p在翻译水平抑制ERBB2表达我们通过QRT-PCR及Western blot两种方法检测了ERBB2在正常食管粘膜细胞Het-1A及食管癌细胞株KYSE150、KYSE410、ECA-109、TE-2、TE-13中的mRNA和蛋白表达情况。发现ERBB2的mRNA水平和蛋白表达水平在食管癌细胞系中的表达与正常细胞不同。我们推断在食管癌中ERBB2存在转录后水平的调控机制来抑制其表达,通过三个主要micro RNA预测软件,Targetscan,miRanda和Micro Inspector,预测与ERBB2 3'URT区域结合的潜能,发现miR-193a-5p与ERBB2的3'URT区有一个结合位点,位于转录终止密码子下游的41nt。通过转染miR-193a-5p mimics来上调miR-193a-5p的表达水平,然后检测ERBB2的蛋白表达水平和m RNA表达水平,发现miR-193a-5p能够显著下调ERBB2蛋白水平而对RNA水平无明显影响。结果提示miR-193a-5p能够通过与ERBB2 3'URT区结合进而抑制ERBB2翻译下调其表达。2.3 miR-193a-5p在组织及血清中的表达及临床指标关系通过QRT-PCR检测35例中miR-193a-5p的表达水平,通过配对T检验比较癌组织和癌旁组织中miR-193a-5p的表达水平,结果表明食管鳞状细胞癌组织中的miR-193a-5p表达水平显著低于癌旁正常组织。根据临床资料,我们将标本分为淋巴结转移组和非淋巴结转移组,通过Wilcoxon秩和检验,发现淋巴结转移组中miR-193a-5p的表达水平显著低于无转移组(P0.05),结果表明miR-193a-5p可能参与抑制食管癌的转移过程。将ERBB2的表达水平分类为ERBB2高表达组和ERBB2低表达组,通过Wilcoxon秩和检验,发现ERBB2高表达组中miR-193a-5p的表达水平显著低于ERBB2低表达组(P0.05),提示在食管鳞状细胞癌组织中ERBB2的表达水平与miR-193a-5p的表达水平呈负相关。通过对数秩检验比较miR-193a-5p高表达组和低表达组的存活情况,结果表明miR-193a-5p高表达组的预后显著高于miR-193a-5p低表达组(P0.05)。对35例冰冻食管鳞状细胞癌组织和血清提取了miRNA,通过QRT-PCR检测miR-193a-5p的表达水平,并通过Pearson相关性分析血清中miR-193a-5p和组织中miR-193a-5p之间的相关性,结果表明血清中miR-193a-5p的表达水平与组织中miR-193a-5p的表达水平呈正相关。3结论3.1 ERBB2免疫组化研究结果,ERBB2的表达水平增高则预示食管癌的预后较差,参考学者在乳腺癌中的相关报道,ERBB2可能参与了肿瘤的侵袭及转移等相关机制,ERBB2上调是食管癌预后不良的危险因素之一。3.2在食管鳞状细胞癌中ERBB2在转录后水平的表达被抑制,通过分析可能的调控miRNA,miR-193a-5b结合ERBB2 3'URT区域并抑制ERBB2的翻译和沉默其表达,揭示ERBB2的新调节机制。3.3食管鳞状细胞癌组织中ERBB2的表达水平与miR-193a-5p的表达水平呈负相关,miR-193a-5p表达水平与患者转移和预后不良呈负相关。血清中miR-193a-5p表达水平可以反映组织中miR-193a-5p的表达水平,提示我们血清中miR-193a-5p的水平可作为食管癌预后的非侵入性诊断指标。
[Abstract]:1 background and objective esophageal cancer (esophageal cancer) is one of the most prevalent digestive tract malignant tumors with high morbidity and mortality. At present, the incidence of cancer is ranked ninth in the world, and the total mortality is sixth. In developed countries, the incidence of esophageal cancer ranks twentieth, but the death row is eleventh. In developing countries, the incidence of esophageal cancer is in row. The mortality rate is fifth. Esophageal cancer has risen from the seventh largest cancer in 1990 to the sixth major cancer in 2013. Aging and growing population are the driving factors for the increase of esophageal cancer. In esophageal cancer, squamous cell carcinoma and adenocarcinoma are the main pathological types, and squamous cell carcinoma accounts for more than 90% in our country. The world is about eighth in the world. 400 thousand people died of esophageal cancer, and China is one of the most frequent areas of esophageal cancer in the world. The average death toll of the disease is more than 150 thousand per year. Because of the early symptoms of esophageal cancer and the lack of early diagnosis, most patients are in the advanced stage of the first diagnosis. The 5 year survival rate after esophagectomy is 9.5-45%, and the tumor invades the tumor. Attack and metastasis are the main causes of death in patients with esophageal cancer. Invasion and metastasis are mainly dependent on the activation of proto oncogenes and the inactivation of tumor suppressor genes, and the cooperation of cell proliferation and apoptosis. If we can study the occurrence and development of esophageal cancer from the perspective of genetics and epigenetics, it is possible to study the occurrence and development of esophageal cancer. It will provide new targets and new strategies for the diagnosis and treatment of esophageal cancer. Human epidermal growth factor receptor 2 (human epidermal growth factor receptor 2, ERBB2) is one of the members of the human epidermal growth factor receptor family, located in the chromosome 17q12-21.32, encoding the relative sub mass of the 185000 transmembrane receptor like protein, belonging to the transmembrane cheese. Tyrosine kinase receptor protein, which has tyrosine kinase activity, is associated with adverse consequences of breast cancer and prostate cancer. It has been shown that ERBB2 amplification has been shown to play an important role in the progression of invasive breast cancer, and ERBB2 egg white has become an important biomarker for 30% breast cancer patients. Target.ERBB2 plays an important role in cell growth, differentiation and migration, and its overexpression promotes tumor growth, metastasis and tumor angiogenesis. In the esophageal squamous cell carcinoma (ESCC), ERBB2 overexpression has been reported. ERBB2 is considered as a potential therapeutic target for esophageal cancer. However, ERBB2 is to be adjusted. The molecular mechanism of the occurrence of ESCC is still not clear that.Micro RNA (miRNA) is a small non coding RNA molecule found in recent years, containing about 22 nucleotides, widely distributed in eukaryotes. Although most miRNA is in the cell, it has also found some miRNA in the extracellular environment, known as circulating miRNA or extracellular miRNA. at present most. The regulation of target genes is that miRNA combines with some proteins to form miRNA induced silencing complex, and then leads to complete or incomplete pairing of 3'non coding region (3'-UTR) with messenger RNA (Messenger RNA, m RNA), to identify specific m RNA and to degrade or deter its transtranscriptional translation after the transcription of gene expression, that is, the gene expression after the target gene is suppressed. Expression of.MiRNA by regulating the expression of target genes plays an important role in cell proliferation, apoptosis and differentiation, and plays an important role in maintaining the normal function of cells. Researchers have found that miRNA disorders are related to many diseases, such as cancer, which are closely related to tumors. The expression of miR-193a-5p in squamous cell carcinoma and its relationship with the clinicopathological features; to elucidate the molecular mechanism of inhibiting ERBB2 translation to reveal the new regulation of ERBB2, to further detect the expression level of miR-193a-5p in the sera of the esophageal cancer and the patients, and to evaluate the value of miR-193a-5p as a prognostic indicator of esophageal cancer and to evaluate the value of miR-193a-5p. The expression and significance of ERBB2 in the detection of ERBB2 in squamous cell carcinoma of the esophagus by immunohistochemistry. We used immunohistochemistry to investigate the expression of ERBB2 in esophageal squamous cell carcinoma and its relationship with the clinicopathological features. The expression rate of ERBB2 (35/68,51.5%) in esophageal squamous cell carcinoma (35/68,51.5%) is higher than that of normal esophageal tissue (6/68) (x 2=33.03 P0.01).ERBB2 Tumor lymph node metastasis and tumor staging were correlated with statistical significance (P0.01). There was no statistical significance (P0.01) with age and sex (P0.05). The expression of.2.2 miR-193a-5p at the translation level inhibited ERBB2 expression. We detected ERBB2 in normal esophageal mucosa cells Het-1A and esophageal cancer cell strain KYSE150, KYSE410, and Western miR-193a-5p at the translation level. 09, the expression of mRNA and protein in TE-2, TE-13. It is found that the mRNA level and protein expression level of ERBB2 are different from normal cells in esophageal cancer cell lines. We infer that there is a post transcriptional regulation mechanism for ERBB2 in esophageal cancer to inhibit its expression by using the three main micro RNA prediction software, Targetscan, miRanda, and Micro. Inspector, predicting the potential of combining with the ERBB2 3'URT region, it is found that there is a binding site in the 3'URT region of miR-193a-5p and ERBB2, and the 41nt. in the downstream of the transcriptional termination codon up-regulated the expression level of miR-193a-5p by transfection of miR-193a-5p mimics, and then detects the level of ERBB2 protein and the level of M expression. The level of ERBB2 protein has no significant effect on the level of RNA. The results suggest that miR-193a-5p can inhibit the expression of.2.3 miR-193a-5p in the tissue and serum by binding to the ERBB2 3'URT region and the expression of the expression of.2.3 miR-193a-5p in the tissue and serum and the relationship between the expression level of miR-193a-5p in 35 cases by QRT-PCR, by paired T test ratio. The expression level of miR-193a-5p in the carcinoma tissue and adjacent tissue showed that the expression of miR-193a-5p in the squamous cell carcinoma of the esophagus was significantly lower than that of the normal tissue adjacent to the carcinoma. According to the clinical data, we divided the specimens into lymph node metastasis group and non lymph node metastasis group. By the Wilcoxon rank sum test, we found the miR-19 in the lymph node metastasis group. The expression level of 3a-5p was significantly lower than that in the non metastasis group (P0.05). The results showed that miR-193a-5p might be involved in inhibiting the metastasis of esophageal cancer. The expression level of ERBB2 was classified into ERBB2 high expression group and ERBB2 low expression group. The expression level of miR-193a-5p in the high expression group of ERBB2 was significantly lower than that of the ERBB2 low expression group (P0) (P0). .05) showed a negative correlation between the expression level of ERBB2 in the squamous cell carcinoma of the esophagus and the expression level of miR-193a-5p. The survival of the miR-193a-5p high expression group and the low expression group was compared by the logarithmic rank test. The results showed that the prognosis of the high expression group of miR-193a-5p was significantly higher than that of the miR-193a-5p low expression group (P0.05). 35 cases of frozen esophageal squamous cells were compared. MiRNA was extracted from the tissue and serum of the cell carcinoma, and the expression level of miR-193a-5p was detected by QRT-PCR. The correlation between the serum miR-193a-5p and the miR-193a-5p in the tissues was analyzed by Pearson correlation. The results showed that the expression level of miR-193a-5p in serum was positively correlated with the level of miR-193a-5p expression in the tissue and the.3 conclusion 3.1 ERBB2 exempts. The higher expression level of ERBB2 indicates that the prognosis of esophageal cancer is poor, the related reports of reference scholars in breast cancer, ERBB2 may be involved in the related mechanisms of tumor invasion and metastasis, and the up-regulation of ERBB2 is one of the risk factors for the poor prognosis of esophageal cancer,.3.2 in the post transcriptional level of ERBB2 in the squamous cell carcinoma of the esophagus Expression was suppressed, and the possible regulation of miRNA, miR-193a-5b combined with ERBB2 3'URT region and inhibition of ERBB2 translation and silence expression, the new regulatory mechanism of ERBB2 revealed the negative correlation between the expression level of ERBB2 in the squamous cell carcinoma of.3.3 and the expression level of miR-193a-5p, and the expression level of miR-193a-5p and the metastasis and prognosis of the patients. The level of miR-193a-5p expression in the serum can reflect the level of miR-193a-5p expression in the tissue, suggesting that the level of miR-193a-5p in the serum can be used as a noninvasive diagnostic index for the prognosis of esophageal cancer.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.1

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