MiRNA-153-5p对食管鳞状细胞癌临床特征评估的应用价值及其机制研究

发布时间：2018-04-27 11:14

本文选题：食管鳞状细胞癌 + miR-153-5p　；参考：《南京医科大学》2017年博士论文

【摘要】：背景和目的:食管癌是常见的恶性肿瘤,发病率和病死率在中国的恶性肿瘤中位列第五和第四。中国每年发病病例占全球食管癌一半以上,主要组织学类型是食管鳞状细胞癌。尽管手术、放疗和化疗等针对于食管癌的多学科综合治疗不断进步,但是食管癌患者的预后仍然不乐观。因此,进一步认识该疾病的分子特征,推进临床生物标志物应用以及改善治疗模式有广泛的社会需求和重要的临床义。微小RNA(microRNA,miRNA)是一类内源性短链(长度约约22个核苷酸)的非编码单链RNA,主要通过绑定3'非翻译区(UTR)调节目标基因的表达,导致翻译抑制。越来越多的证据表明miRNA可以调节肿瘤发生、发展影响肿瘤细胞浸润和转移,miRNAs的致癌或抑癌的功效取决于miRNA作用的靶基因。近年来,miR-153-5p被发现是一种人类癌症的抑癌因子,调节肿瘤抑癌基因、参与肿瘤的生长、转移和侵润,miR-153-5p在ESCC的作用机制仍不清楚。我们在miRDB检索miR-153-5p直接靶向因子,其中将WT1(肾母细胞瘤抑癌基因1)作为研究对象。WT1位于人染色体11P13,有报道在多种恶性肿瘤中发现WT1起抑癌因子作用。因此,本研究旨在探索miR153-5p在ESCC中的作用,评估临床资料,探讨其在食管鳞状细胞癌中的生物功能。探索ESCC细胞中miR-153-5p如何调控WT1表达。本课题包括以下三部分:第一部分:食管鳞状细胞癌组织中异常表达miRNA筛选及血清miRNA-153-5p与临床病理特征相关性分析;第二部分:体外调控miRNA-153-5p表达对食管癌细胞系TE-1增殖、侵袭的影响;第三部分:miRNA-153-5p靶基因的初步研究。第一部分食管鳞状细胞癌组织中异常表达miRNA筛选及血清miRNA-153-5p与临床病理特征相关性分析方法:1.收集标本,包括47例食管鳞状上皮细胞癌患者和50名健康人选血清标本,以及上述47例食管鳞状上皮细胞癌组织与其相应配对的癌旁正常组织标本。2.随机抽取4例食管癌病例和4例健康人选的血清,运用Agilent miRNA芯片检测miRNA表达情况,并对差异表达基因进行初步功能分析,筛选出显著异常表达的十二个miRNA;同样在这4例食管鳞状上皮细胞癌组织与其相应配对的癌旁组织标本进行对照分析。3.进一步运用qRT-PCR检测上述12种miRNA中的7种miRNA表达情况水平,包括5个食管癌中表达上调的miRNA(miR-25、miR-155、miR-214、miR-200c、miR-92-c)和2个食管癌中表达下调的miRNA基因(miR-518b 和 miR-153-5p)。4.在50例健康人选和47例ESCC病例依据血清miRNA-153-5p表达水平,运用ROC曲线分析其与食管鳞状细胞癌病人血清miRNA-153-5p表达水平与健康人群血清表达水平以及与食管鳞状细胞癌患者淋巴结转移、肿瘤长度、肿瘤分期、肿瘤分化程度等之间的相关性。5.统计学分析:采用统计软件SPSS 22.0对实验数据进行统计分析。血清miRNA-153-5p表达与临床诊断相关性分析进行聚类分析,ROC曲线,所有结果均以means ± SE表示,student t-test进行数据比较分析,P0.05为有统计学意义。结果:1.Agilent miRNA芯片检测分析得到研究发现有25个miRNA表达水平存在显著差异(P0.05和差异倍数≥2),包括13个高表达miRNA和12个低表达miRNA。组织miRNA表达水平与血清的研究结果一致。2.已被基因芯片检测的七个血清miRNA包括miR-25、miR-518b、miR-155、miR-214、miR-200c、miR-92-c 和 miR-153-5p,被 qRT-PCR 再次检测,总体qRT-PCR表达趋势与芯片结果一致;7个miRNA在两种组织样本qRT-PCR的表达趋势与血清结果一至。选择差异最为显著的具有负向调控作用的miRNA-153-5p作为研究对象。3.与健康人相比ESCC患者的血清miR-153-5p表达水平较低,ESCC患者的血清miR-153-5p表达水平与食管鳞状细胞癌患者的淋巴结转移、肿瘤长度和肿瘤TNM分期相关,与肿瘤分化程度无显著相关性。结论:1.本研究在食管癌组织中一共筛选分析得到25个差异表达的miRNA,其中有13个为上调miRNA基因,12个为下调miRNA基因。2.miR-153-5p在食管鳞状细胞癌患者血清和食管鳞癌组织中显著低表达,选择差异最为显著的具有负向调控作用的miRNA-153-5p作为食管鳞状细胞癌研究对象。3.食管鳞状细胞癌患者血清中miR-153-5p的表达水平在诊断食管鳞状细胞癌有潜在价值;与食管鳞状细胞癌患者的淋巴结转移、肿瘤长度和肿瘤TNM分期相关,与肿瘤分化程度无显著相关性,在评估食管鳞状细胞癌的临床特征具有良好的潜在应用价值。第二部分体外miR-153-5p表达对食管癌细胞系生物学行为的影响方法:1.运用 qRT-PCR 检测 HEEC、EC190、EC9706、SKGT-5 和 TE-1 等细胞系中miR-153-5p表达。2.实验分组为 Control 阴性组、miRNA-153-5pNC 组和 miRNA-153-5p mimics组,利用LipofectamineTM2000转染各组后TE-1细胞中miR-153-5p mRNA表达水平检测。3.MTT增殖实验检测各组TE-1细胞增殖能力影响。4.Transwell侵袭实验法检测各组TE-1细胞的侵袭转移能力。5.Western blot和qRT-PCR检测各实验组细胞中与肿瘤增殖和侵袭相关蛋白Vimentin和E-cadherin水平表达。6.统计学分析:采用SPSS20.0分析数据,各实验组独立实验计量资料据采集使用均数±标准差,组间均数比较采用非配对双侧t检验。qRT-PCR结果及肿瘤质量组间比较采用方差分析。P0.05为有统计学意义。结果:1.在HEEC(正常食管上皮细胞)miR-153-5p的表达明显高于人类食管癌细胞系 EC190、EC9706、SKGT-5 和 TE-1 等(P0.01),对比对照组,表达水平显著上调(P0.01)。2.MiR-153-5p mimics和相应的负控制成功转染到TE-1细胞,与NC组织相比,miR-153-5p水平在TE-1细胞中水平显著提高(P0.01)。3.MTT测定miR-153-5p过度表达抑制了 TE-1细胞的增殖(P0.01)4.Transwell侵袭试验表明,在miR-153-5p mimics组,TE-1细胞侵袭明显被抑制(P0.01)。miR-153-5pmimics组细胞的生长在转染2d后出现显著抑制(P0.05),并且随时间的延长而日益显著。5.Westernblot、qRT-PCR显示扩散和侵袭相关蛋白表达改变:与肿瘤发生、转移密切相关的vimentin表达减弱,与维持正常细胞结构、功能相关的E-cadherin表达增强。结论:1.miR-153-5p在体外食管癌细胞中水平显著降低。2.上调食管癌TE-1细胞中miR-153-5p表达可有效抑制食管癌TE-1细胞的增殖和侵袭能力,并且影响食管癌扩散和侵袭相关蛋白vimentin、E-cadherin 的表达。第三部分miRNA-153-5p靶基因的初步研究方法:1.开放应用程序miRDB、miRBase、starBase和TargetScan等用来分析潜在miR-153-5p的靶向目标。2.采用Western blot、qRT-PCR方法用来检测ESCC组织样本中WT1和miR-153-5p mRNA水平并建立相关性。3.Western blot 和 qRT-PCR 检测 WT1 在 miR-153-5p mimics 组、miR-NC组、对照组中的表达。4.构建野生型重组载体pmirGLO-WT1(WT),通过定向位点突变获得绑定位点的突变体结构(MUT)。采用双荧光素酶报告实验验证miR-153-5p的靶基因。结果:1.通过生物信息学分析推测WT1为miR-153-5p靶基因,miR-153-5p的互补序列被预测在WT1mRNA的3'-UTR。2.ESCC组织样本中miR-153-5p水平在ESCC组织中低于相应的正常组织,WT1mRNA表达明显增加,与miR-153-5p水平负相关(P0.01)。3.WT1在miR-153-5p mimics组、miR-NC组、对照组中的表达结果是与其他两组相比,miR-153-5p mimics组WT1蛋白表达和WT1 mRNA水平较低(P0.01)。4.食管鳞状癌细胞TE-1中,共转染miR-153-5pmimics和野生型3'UTR区的pmirGLO-WT1重组载体(WT)时,WT组中WT1荧光素酶活性被miR-153-5pmimics抑制,提示miR-153-5p可以通过作用于WT1的3'UTR区负向调控其表达,但在MUT中并没有(P0.01)。结论:1.WT1预测为miR-153-5p的靶基因,2.在食管鳞状细胞癌组织中WT1 mRNA表达水平明显增加,与miR-153-5p表达水平呈负相关。3.在TE-1细胞系中,miR-153-5pmimics作用于WT1,并抑制了 WT1的表达。4.MiR-153-5p通过作用于靶基因WT1的3'UTR,抑制WT1的表达参与食管癌TE-1细胞生长调控。
[Abstract]:Background and purpose: esophageal cancer is a common malignant tumor. The incidence and fatality rate are fifth and fourth in China's malignant tumors. The incidence of cancer in China is more than half of the global esophageal cancer in China every year. The main histological type is squamous cell carcinoma of the esophagus. Although surgery, radiotherapy and chemical therapy are not used for the multidisciplinary treatment of esophageal cancer However, the prognosis of patients with esophageal cancer is still not optimistic. Therefore, further understanding of the molecular characteristics of the disease, the application of clinical biomarkers and the improvement of treatment patterns have extensive social needs and important clinical meanings. Small RNA (microRNA, miRNA) is a class of non coded endogenetic short chains (about 22 nucleotides in length). Chain RNA, which regulates the expression of target genes by binding 3'untranslated region (UTR), leads to translation inhibition. More and more evidence suggests that miRNA can regulate the occurrence of tumor and the development of tumor cell infiltration and metastasis. The effect of miRNAs on carcinogenesis or tumor suppressor depends on the target gene of miRNA. In recent years, miR-153-5p has been found to be a kind of human being. Cancer suppressor factor, regulating tumor suppressor gene, participating in tumor growth, metastasis and invasion, the mechanism of miR-153-5p's action in ESCC is still unclear. We retrieved the direct target factor of miR-153-5p in miRDB, in which WT1 (nephroblastoma tumor suppressor gene 1) was used as the research object.WT1 in the human chromosome 11P13, and there were reports in a variety of malignant tumors. The purpose of this study is to explore the role of WT1 as a tumor suppressor factor. Therefore, the purpose of this study is to explore the role of miR153-5p in ESCC, to evaluate the clinical data and to explore its biological functions in the squamous cell carcinoma of the esophagus. To explore how miR-153-5p regulates the expression of WT1 in ESCC cells. This topic includes the following three parts: Part 1: abnormal expression in the tissues of squamous cell carcinoma of the esophagus MiRNA screening and the correlation analysis of serum miRNA-153-5p and clinicopathological features; the second part: the effect of miRNA-153-5p expression on the proliferation and invasion of the esophageal cancer cell line TE-1 in vitro; the third part: the preliminary study of the miRNA-153-5p target gene. The first part of the esophageal squamous cell carcinoma tissue abnormal expression of miRNA and the serum miRNA-153-5p and miRNA-153-5p The method of correlation analysis of clinicopathological features: 1. collection of specimens, including 47 cases of squamous cell carcinoma of the esophagus and 50 healthy persons, and 47 cases of squamous cell carcinoma of the esophagus and the corresponding normal tissue specimens of the paracancerous tissue adjacent to.2., the serum of 4 cases of tube cancer and 4 healthy persons were randomly selected, and Agi was used. Lent miRNA chips were used to detect the expression of miRNA, and a preliminary functional analysis of differentially expressed genes was used to screen out twelve miRNA with significant abnormal expression. The same 4 cases of esophageal squamous cell carcinoma and corresponding paired para cancerous tissue specimens were compared and analyzed by.3., and qRT-PCR was used to detect 7 mi of the 12 kinds of miRNA. The level of RNA expression, including the up regulated miRNA (miR-25, miR-155, miR-214, miR-200c, miR-92-c) and the down regulated miRNA gene (miR-518b and miR-153-5p) in 2 esophageal cancers in 5 esophageal cancers (miR-518b and miR-153-5p).4. in 50 healthy persons and 47 cases of ESCC cases on the basis of serum levels, and using the curve to analyze the squamous cells of the esophagus The correlation of serum miRNA-153-5p expression level with serum expression level in healthy people and the correlation of.5. with lymph node metastasis, tumor length, tumor stage and tumor differentiation in patients with squamous cell carcinoma of esophagus: statistical analysis of the experimental data by statistical software SPSS 22. The expression of serum miRNA-153-5p and the presence of miRNA-153-5p The correlation analysis of bed diagnosis was analyzed by clustering analysis, ROC curve, all the results were expressed in means + SE, student t-test was compared and analyzed, and P0.05 was statistically significant. The results of 1.Agilent miRNA chip detection analysis showed that there were 25 miRNA expression levels (P0.05 and difference multiple more than 2), including 13 high levels. The expression level of the expression of miRNA and 12 low expression miRNA. tissues was consistent with the results of the study in the serum. The seven serum miRNA, including miR-25, miR-518b, miR-155, miR-214, miR-200c, miR-92-c and miR-153-5p, were detected by the gene chip, and the overall expression trend was consistent with the results of the chip; 7 groups were in two groups. The expression trend of qRT-PCR and the result of sera were 1. The most significant difference was the negative regulation of miRNA-153-5p as the object of study. The level of miR-153-5p expression in the serum of ESCC patients was lower than that of the healthy people, and the level of miR-153-5p expression in the serum of ESCC patients and the lymph node metastasis of patients with squamous cell carcinoma of the tube was swollen. The length of the tumor was correlated with the TNM staging of the tumor, and there was no significant correlation with the degree of tumor differentiation. Conclusion: 1. a total of 25 differentially expressed miRNA were screened and analyzed in the esophageal carcinoma tissue, of which 13 were up to up miRNA gene, and 12 for miRNA gene.2.miR-153-5p in the sera and esophageal squamous cell carcinoma tissues of the patients with esophageal squamous cell carcinoma. The expression level of miRNA-153-5p, which has the most significant difference in the negative regulation, is the target of esophageal squamous cell carcinoma. The expression level of miR-153-5p in the sera of.3. esophageal squamous cell carcinoma is of potential value in the diagnosis of squamous cell carcinoma of the esophagus; the lymph node metastasis, the length of tumor and the T of the tumor in the patients with esophageal squamous cell carcinoma NM staging correlation has no significant correlation with the degree of tumor differentiation, and has good potential application value in evaluating the clinical characteristics of esophageal squamous cell carcinoma. Second the effect of miR-153-5p expression on the biological behavior of esophageal cancer cell lines in vitro: 1. using qRT-PCR to detect miR-15 in HEEC, EC190, EC9706, SKGT-5 and TE-1 3-5p expression of.2. was divided into Control negative group, miRNA-153-5pNC group and miRNA-153-5p mimics group, miR-153-5p mRNA expression in TE-1 cells was detected by LipofectamineTM2000 transfection, and.3.MTT proliferation test was used to detect the effect of.3.MTT proliferation test on the proliferation of TE-1 cells. .5.Western blot and qRT-PCR were used to detect the expression of.6. in the cells with tumor proliferation and invasion related protein Vimentin and E-cadherin in the experimental groups. The SPSS20.0 analysis was used to analyze the data of the experimental groups. The independent experimental data of the experimental groups were collected using the mean standard deviation, and the average number between the groups was compared with the non paired bilateral t test.QRT-PCR. The results and the contrast analysis of.P0.05 were statistically significant. Results: 1. the expression of miR-153-5p in HEEC (normal esophageal epithelial cells) was significantly higher than that of human esophageal carcinoma cell line EC190, EC9706, SKGT-5 and TE-1 (P0.01). Compared with the control group, the expression level was significantly up (P0.01).2.MiR-153-5p mimics and the corresponding negative. The control was successfully transfected into TE-1 cells. Compared with NC tissue, the level of miR-153-5p in TE-1 cells increased significantly (P0.01).3.MTT determination of miR-153-5p overexpression inhibited the proliferation of TE-1 cells (P0.01) 4.Transwell invasion test showed that the invasion of cells in miR-153-5p mimics group was obviously inhibited. The growth was significantly inhibited after transfection of 2D (P0.05), and the expression of proliferation and invasion related protein expression changed with the prolongation of time. QRT-PCR showed changes in the expression of diffusion and invasion related proteins: the expression of vimentin, which was closely related to tumor occurrence and metastasis, was weakened, and the expression of E-cadherin was enhanced with the maintenance of normal cell structure and function related. Conclusion: 1.miR-153-5p in 1.miR-153-5p is enhanced. The level of.2. in esophageal cancer cells significantly reduces the expression of miR-153-5p in esophageal cancer TE-1 cells, which can effectively inhibit the proliferation and invasion of esophageal cancer TE-1 cells, and affect the expression of vimentin and E-cadherin in the proliferation and invasion related proteins of esophageal cancer. The preliminary study method of the third part of the miRNA-153-5p target gene: 1. open application Sequential miRDB, miRBase, starBase, and TargetScan are used to analyze potential miR-153-5p target target.2. using Western blot. QRT-PCR method is used to detect WT1 and miR-153-5p levels in ESCC organization samples. A wild type recombinant vector pmirGLO-WT1 (WT) was constructed to obtain the mutant structure (MUT) of the binding site by directional site mutation. Double luciferase reporter experiment was used to verify the target gene of miR-153-5p. Results: 1. by bioinformatics analysis, WT1 was a miR-153-5p target gene, and the complementary sequence of miR-153-5p was predicted in the 3'-UTR.2.ESCC group of WT1mRNA. The miR-153-5p level in the ESCC tissue was lower than that of the corresponding normal tissue, and the expression of WT1mRNA was significantly increased, and the negative correlation with the miR-153-5p level (P0.01).3.WT1 in the miR-153-5p mimics group, miR-NC group and the control group was compared with the other two groups, and the WT1 protein expression and the lower level of miR-153-5p mimics group were lower than those of the other groups. In the squamous cell carcinoma cell TE-1, when the pmirGLO-WT1 recombinant vector (WT) was co transfected with miR-153-5pmimics and wild type 3'UTR region, the activity of WT1 luciferase in group WT was inhibited by miR-153-5pmimics, suggesting that miR-153-5p could regulate its expression by negative direction acting on 3'UTR region of WT1. 2. in esophageal squamous cell carcinoma, the expression of WT1 mRNA increased significantly, and the expression level of miR-153-5p was negatively correlated with the expression level of.3. in the TE-1 cell line. MiR-153-5pmimics acted on WT1 and inhibited the expression of.4.MiR-153-5p through the 3'UTR of the target gene WT1, and inhibited the expression of WT1 to participate in the regulation of the growth of esophageal cancer cells.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.1

【参考文献】