长链非编码RNA LOC100130476在食管鳞癌及贲门腺癌中的表达及其甲基化状态

发布时间：2018-04-20 17:06

本文选题：食管鳞癌 + 贲门腺癌　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:食管癌(esophageal cancer,EC)是最常见的消化系统恶性肿瘤之一,在全球有较高死亡率。EC的发生有明显地区差异,中国食管癌的发病率明显高于其他国家。其中食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)是EC的主要病理类型。尽管近年来,ESCC在多学科综合治疗中取得了一定的效果,但5年生存率仍然很低。贲门腺癌(gastric cardia adenocarcinoma,GCA)是我国北方地区较为常见的恶性肿瘤之一。GCA起病隐匿且其发病率总体呈上升趋势,有症状来就诊时患者多数已到中晚期。然而迄今为止,食管鳞癌和贲门腺癌的发病机制尚未完全明确。因此,寻找ESCC及GCA发生的早期生物标志物,探索其发病机制对于临床中的预防及治疗显得尤为重要。长链非编码RNA(long non-coding RNA,lnc RNAs)是指长度在200~100000 nt之间,具有调控基因表达作用的非编码RNA。近年来研究者通过基因芯片技术筛选出的lnc RNAs在ESCC及GCA发病中的作用机制仍不清楚,lnc RNAs与ESCC、GCA相关性的研究报道较少。因此,本研究旨在通过检测lnc RNA LOC100130476在食管癌细胞系、食管鳞癌及贲门腺癌组织中的表达情况及甲基化状态,分析该lnc RNA表达与甲基化状态的相关性,并结合临床资料探讨其与患者年龄、性别、淋巴结转移、组织分化程度、TNM分期之间的关系,为lnc RNA LOC100130476在ESCC及GCA发生和发展中的作用提供实验依据。方法:1应用反转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)法分别检测DNA甲基转移酶抑制剂5-氮杂-2’-脱氧胞苷(5-aza-2’-deoxycitydine,5-Aza-d C)处理前后的食管癌细胞系(TE1、TE13、T.Tn、Eca109)中、72例食管鳞癌及相应癌旁正常组织中和76例贲门腺癌及相应癌旁正常组织中LOC100130476的表达情况。2应用甲基化特异性聚合酶链反应(methylation specific polymerasechain reaction,MSP)法分别检测应用5-Aza-d C处理前后的食管癌细胞系、72例食管鳞癌及相应癌旁正常组织中和76例贲门腺癌及相应癌旁正常组织中LOC100130476的甲基化状态。3运用统计学软件SPSS13.0对数据进行统计学分析。结果:1食管癌细胞系中lnc RNA LOC100130476的表达情况及甲基化状态1食管癌细胞系中lnc RNA LOC100130476的表达情况及甲基化状态1.1 5-Aza-d C处理前后食管癌细胞系中lnc RNA LOC100130476的表达未经5-Aza-dC处理的食管癌TE1、TE13和T.Tn细胞中LOC100130476的表达均为阴性,在Eca109细胞中为弱阳性表达;用5-Aza-d C处理后,TE1、TE13和T.Tn细胞中LOC100130476均转变为阳性表达,而在Eca109细胞中较未用5-Aza-d C处理的细胞表达上调。1.2 5-Aza-d C处理前后的食管癌细胞系中lnc RNA LOC100130476的甲基化状态TE1、TE13、T.Tn和Eca109细胞中,在5-Aza-d C未处理前LOC100130476均表现为高甲基化的状态,应用5-Aza-d C处理后,Eca109细胞系中LOC100130476甲基化程度明显降低,其余3种细胞中的LOC100130476则均表现为非甲基化状态。2食管鳞癌组织中lnc RNA LOC100130476的表达水平、甲基化状态以及与临床病理学资料之间的关系2.1食管鳞癌组织中lnc RNA LOC100130476的表达LOC100130476在食管鳞癌组织中的表达显著低于相应癌旁正常组织(0.49±0.09 vs 0.56±0.08),两者差异具有统计学意义(t=-5.893,P0.05)。在食管鳞癌患者中,LOC100130476的表达与组织分化程度和TNM分期密切相关(P0.05),而与患者的年龄、性别以及淋巴结转移无关(P0.05)。2.2食管鳞癌组织中lnc RNA LOC100130476的甲基化状态食管鳞癌组织中LOC100130476的第一外显子区甲基化率显著高于癌旁正常组织[69.4%(50/72)vs 26.4%(19/72)],两者差异具有统计学意义(χ2=26.741,P0.05),并与组织分化程度和TNM分期密切相关(P0.05),而与患者的年龄、性别以及淋巴结转移无关(P0.05)。发生LOC100130476甲基化的食管鳞癌组织中LOC100130476的表达显著低于未发生甲基化的食管鳞癌组织(0.47±0.08 vs 0.55±0.08),两者差异具有统计学意义(t=-4.099,P0.05)。3贲门腺癌组织中lnc RNA LOC100130476的表达水平、甲基化状态以及与临床病理学资料之间的关系3.1贲门腺癌组织中lnc RNA LOC100130476的表达LOC100130476在贲门腺癌组织中的表达显著低于相应癌旁正常组织(0.61±0.22 vs 0.79±0.23),两者差异具有统计学意义(t=-5.387,P0.05)。在贲门腺癌患者中,LOC100130476的表达与组织分化程度和淋巴结转移密切相关(P0.05),而与患者的年龄、性别以及TNM分期无关(P0.05)。3.2贲门腺癌组织中lnc RNA LOC100130476的甲基化状态贲门腺癌组织中LOC100130476的第一外显子区甲基化率显著高于癌旁正常组织[64.5%(49/76)vs 23.7%(18/76)],两者差异具有统计学意义(χ2=25.649,P0.05),并与组织分化程度和淋巴结转移密切相关(P0.05),而与患者的年龄、性别以及TNM分期无关(P0.05)。发生LOC100130476甲基化的贲门腺癌组织中LOC100130476的表达显著低于未发生甲基化的贲门腺癌组织(0.56±0.21 vs 0.71±0.21),两者差异具有统计学意义(t=-3.055,P0.05)。结论:1 LOC100130476与食管鳞癌、贲门腺癌的发生、发展密切相关,并可能起抑癌基因的作用。2 LOC100130476第一外显子区甲基化异常增高可能是导致其表达下调的机制之一。3 LOC100130476第一外显子区甲基化状态可能与食管鳞癌、贲门腺癌的恶性程度及进展相关。
[Abstract]:Objective: esophageal cancer (EC) is one of the most common malignant tumors of the digestive system. There is a significant regional difference in the incidence of.EC in the world. The incidence of esophageal cancer in China is obviously higher than that of other countries. The main pathological type of EC is esophageal squamous cell carcinoma (esophageal squamous cell carcinoma, ESCC). In recent years, ESCC has achieved a certain effect in the multidisciplinary integrated treatment, but the 5 year survival rate is still very low. Gastric cardia adenocarcinoma (GCA) is one of the most common malignant tumors in northern China..GCA is insidious and its incidence is generally on the rise. Most of the patients have come to the middle and late stages when they have symptoms. So far, the pathogenesis of esophageal squamous and cardiac adenocarcinoma is not completely clear. Therefore, it is particularly important to find the early biomarkers of ESCC and GCA and explore its pathogenesis for the prevention and treatment of clinical. Long chain non coded RNA (long non-coding RNA, LNC RNAs) refers to the length between 200~100000 nt and modulation. The mechanism of non coding RNA. for controlling gene expression in recent years is not clear in the pathogenesis of LNC RNAs screened by gene chip technology in the pathogenesis of ESCC and GCA. There are few reports on the correlation between LNC RNAs and ESCC and GCA. Therefore, this study aims to detect LNC RNA LOC100130476 in esophageal cancer cell lines, esophageal squamous cell carcinoma and cardiac glands. The expression and methylation status of the cancer tissue were analyzed, and the correlation between the expression of LNC RNA and methylation status was analyzed, and the relationship between the age, sex, lymph node metastasis, the degree of tissue differentiation, TNM staging and the effect of LNC RNA LOC100130476 on the occurrence and development of ESCC and GCA were provided. Method: 1 the reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect the esophageal cancer cell lines before and after the DNA methyltransferase inhibitor 5- aza -2 'deoxycytidine (5-Aza-2' -deoxycitydine, 5-Aza-d C), and 72 cases of esophageal squamous cell carcinoma and the corresponding normal paracancerous group. Expression of LOC100130476 in 76 cases of gastric cardia adenocarcinoma and corresponding normal tissue adjacent to cancer.2 using methylation specific polymerase chain reaction (methylation specific polymerasechain reaction, MSP) to detect esophageal cancer cell lines before and after 5-Aza-d C treatment, 72 cases of esophageal squamous cell carcinoma and 76 cases of corresponding normal tissue adjacent to cancer. Methylation status of LOC100130476 in gastric cardia adenocarcinoma and corresponding normal tissue.3 using statistical software SPSS13.0 to analyze the data. Results: the expression of LNC RNA LOC100130476 in 1 esophageal carcinoma cell lines and methylation status, the expression of LNC RNA LOC100130476 in 1 esophageal cancer cell lines and methylation status 1.1 5-Az The expression of LNC RNA LOC100130476 in esophageal cancer cell lines before and after A-D C treatment was negative in TE1, TE13 and T.Tn cells in TE1, TE13 and T.Tn cells, and was weakly positive in Eca109 cells. The methylation status of LNC RNA LOC100130476 in esophageal cancer cell lines before and after.1.2 5-Aza-d C treatment was up before and after.1.2 5-Aza-d C treatment. The methylation status of LNC RNA LOC100130476 in TE13, T.Tn and Eca109 cells The degree of LOC100130476 in the other 3 cells showed the expression level of LNC RNA LOC100130476 in the non methylation state of.2 esophageal squamous cell carcinoma, the methylation status and the relationship with the clinicopathological data. 2.1 the expression of LOC100130476 in esophageal squamous cell carcinoma tissue in the esophageal squamous cell carcinoma tissue The expression was significantly lower than that of the normal tissues adjacent to the carcinoma (0.49 + 0.09 vs 0.56 + 0.08). The difference was statistically significant (t=-5.893, P0.05). In the patients with esophageal squamous cell carcinoma, the expression of LOC100130476 was closely related to the degree of tissue differentiation and TNM staging (P0.05), but was not related to the age, sex, and lymph node metastasis of the patients (P0.05).2.2 esophageal squamous cell carcinoma group. The methylation status of LNC RNA LOC100130476 in the tissue of the tissue was significantly higher than that of [69.4% (50/72) vs 26.4% (19/72) in the normal tissue adjacent to the normal tissue of the carcinoma of the esophagus. The difference was statistically significant (x 2=26.741, P0.05), and was closely related to the degree of tissue differentiation and TNM (P0.05), and the age of the patients. Sex and lymph node metastasis were not related (P0.05). The expression of LOC100130476 in the tissue of LOC100130476 methylation of esophageal squamous cell carcinoma was significantly lower than that of non methylation esophageal squamous cell carcinoma (0.47 + 0.08 vs 0.55 + 0.08). The difference was statistically significant (t=-4.099, P0.05) the expression level of LNC RNA LOC100130476 in.3 cardiac adenocarcinoma tissue The relationship between methylation and clinicopathological data in 3.1 cardiac adenocarcinoma tissue, expression of LNC RNA LOC100130476 in gastric cardia adenocarcinoma was significantly lower than that of normal tissues adjacent to the carcinoma (0.61 + 0.22 vs 0.79 + 0.23), and the difference was statistically significant (t=-5.387, P0.05). In the patients with cardiac adenocarcinoma, LO The expression of C100130476 is closely related to the degree of tissue differentiation and lymph node metastasis (P0.05), but not with the age, sex, and TNM staging of the patients (P0.05) the methylation of LNC RNA LOC100130476 in the.3.2 cardiac adenocarcinoma tissue of.3.2, the methylation rate of the first exon of the LOC100130476 in the gastric cardia adenocarcinoma tissue is significantly higher than that of the normal tissue adjacent to the carcinoma [64.5%. (49/76) vs 23.7% (18/76)], the differences were statistically significant (x 2=25.649, P0.05), closely related to the degree of tissue differentiation and lymph node metastasis (P0.05), but not with age, sex, and TNM staging (P0.05). The expression of LOC100130476 in the cardiac adenocarcinoma tissue with LOC100130476 methylation was significantly lower than that of non methylation. The difference of cardiac adenocarcinoma (0.56 + 0.21 vs 0.71 + 0.21) was statistically significant (t=-3.055, P0.05). Conclusion: 1 LOC100130476 is closely related to the development of carcinoma of the esophagus, gastric cardia adenocarcinoma, and may play the role of tumor suppressor gene in.2 LOC100130476 first exon methylation The methylation status of the first exon region of.3 LOC100130476 may be related to the malignancy and progression of esophageal squamous cell carcinoma and cardiac adenocarcinoma.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735

【参考文献】