MiR-106b调控靶基因Smad7通过EMT促进食管鳞癌的侵袭和转移

发布时间：2018-08-27 08:37

【摘要】：目的:研究微小RNAl06b(mi R-106b)在食管癌浸润转移中的作用机制,及其对Smad7的调控影响,为临床治疗食管癌提供新的理论依据。方法:1.在组织水平,免疫组织化学方法检测90对食管鳞癌及其对应的正常食管组织中Smad7的表达情况,分析mi R-106b与Smad7的相关性,探讨mi R-106b、Smad7表达水平与临床病理参数的相关性。2.在细胞水平,分别转染慢病毒LV-has-mir-106b和阴性对照病毒CON238于食管癌细胞株KYSE150,转染慢病毒LV-has-mir-106b-inhibition和阴性对照病毒CON137于食管癌细胞株KYSE450后,实验分为上调组和下调组。上调组:control组(未转染mi R-106b的KYSE150细胞组)、NC组(转染随机序列组,阴性对照病毒CON238)和mi R-106b组(转染mi R-106b的KYSE150的细胞组);下调组:control组(未转染mi R-106b-inhibition的KYSE450细胞组)、NC-inhibitor组(转染随机序列组,阴性对照病毒CON137)和mi R-106b-inhibition组(转染mi R-106b-inhibitor的KYSE450细胞组),采用实时荧光定量聚合酶联反应(real-time quantitative PCR,q RT-PCR)和免疫印迹(Western Blot)分别检测mi R-106b、Smad7 m RNA的表达水平、Smad7的蛋白表达水平和EMT的相关标记物(上皮型钙黏蛋白(E-Cadherin)、神经型钙黏蛋白(N-Cadherin)的蛋白表达水平,四甲基偶氮唑盐微量酶反应比色法(MTT法)检测细胞的增殖能力,划痕实验检测细胞的迁移能力,Transwell方法检测细胞的侵袭能力,流式细胞术检测细胞凋亡情况,双荧光素酶报告基因实验验证mi R-106与Smad7的调控关系。结果:1.Smad7在食管鳞癌组织中的表达显著低于对应的正常食管组织(P0.05),其表达水平与肿瘤的淋巴结转移相关(P0.05),与年龄、性别、分化程度、大体分型无关(P㧐0.05)。2.慢病毒稳定转染mi R-106b后,Smad7m RNA表达水平和蛋白表达水平明显降低(P0.05)。食管癌细胞的增殖、侵袭和迁移能力均增强(P0.05);q RT-PCR与Western blot检测显示mi R-106b组E-Cadherin表达较NC组、control组、明显降低(P0.05);N-Cadherin表达明显升高(P0.05)。反之,慢病毒稳定转染mi R-106b-inhibition后,Smad7 m RNA表达水平和蛋白表达水平明显升高(P0.05),食管癌细胞的增殖、侵袭和迁移能力均降低(P0.05);q RT-PCR与Western blot检测显示mi R-106b-inhibition组E-Cadherin表达较NC-inhibitor组、control组明显升高(P0.05);N-Cadherin表达明显降低(P0.05)。双荧光素酶报告基因实验验证了mi R-106b与Smad7有直接调控关系。结论:1.Smad7参与了食管癌的发生,且与转移相关。2.在食管鳞癌中,mi R-106b直接调控了Smad7蛋白的表达。3.mi R-106b通过调控Smad7促进了食管癌细胞的增殖、迁移和侵袭。
[Abstract]:Objective: to study the mechanism of tiny RNAl06b (mi R-106b) in invasion and metastasis of esophageal carcinoma and its regulatory effect on Smad7, so as to provide a new theoretical basis for clinical treatment of esophageal carcinoma. Method 1: 1. At the tissue level, immunohistochemical method was used to detect the expression of Smad7 in esophageal squamous cell carcinoma and its corresponding normal esophageal tissues, to analyze the correlation between mi R-106b and Smad7, and to explore the correlation between mi R-106b Smad7 expression and clinicopathological parameters. At the cell level, lentivirus LV-has-mir-106b and negative control virus CON238 were transfected into esophageal carcinoma cell line KYSE150, and negative control virus CON137 were transfected into KYSE450 cell line respectively. The cells were divided into up-regulation group and down-regulation group. Upregulation group: control group (KYSE150 cell group not transfected with mi R-106b) NC group (transfected random sequence group, negative control virus CON238 group) and mi R-106b group (mi R-106b transfected KYSE150 cell group); down-regulation group: control group (KYSE450 cell group not transfected with mi R-106b-inhibition) and NC-inhibitor group (transfected random sequence group), The expression level of mi R-106b Smad7 m RNA and EMT were detected by real-time fluorescent quantitative polymerase chain reaction (real-time quantitative PCR,q RT-PCR) and Western blot (Western Blot), respectively, in the negative control virus (CON137) and mi R-106b-inhibition group (KYSE450 cells transfected with mi R-106b-inhibitor). The expression levels of epithelial cadherin (E-Cadherin) and neural cadherin (N-Cadherin), The cell proliferation was detected by MTT assay, the migration ability was detected by scratch assay, the invasion ability was detected by Transwell method, and apoptosis was detected by flow cytometry. The regulatory relationship between mi R-106 and Smad7 was verified by double luciferase reporter gene experiment. Results: 1. The expression of Smad7 in esophageal squamous cell carcinoma was significantly lower than that in normal esophageal tissues (P0.05). The expression level of Smad7 was correlated with lymph node metastasis (P0.05), but not related to age, sex, differentiation, gross classification (P0.05). The expression level of Smad7m RNA and protein of mi R-106b were significantly decreased after lentivirus transfection (P0.05). The expression of E-Cadherin in mi R-106b group was significantly lower than that in NC group (P0.05). On the contrary, the expression level of Smad7 m RNA and protein were significantly increased after lentivirus transfection into mi R-106b-inhibition (P0.05), and the proliferation of esophageal cancer cells was observed. The expression of E-Cadherin in mi R-106b-inhibition group was significantly higher than that in NC-inhibitor group (P0.05) and the expression of N-Cadherin in mi R-106b-inhibition group was significantly lower than that in NC-inhibitor group (P0.05). Double luciferase reporter gene experiment confirmed that mi R-106b has direct regulatory relationship with Smad7. Conclusion: 1. Smad7 is involved in the development of esophageal carcinoma and is related to metastasis. In esophageal squamous cell carcinoma (ESCC), the expression of Smad7 protein was regulated directly. 3.mi R-106b promoted the proliferation, migration and invasion of esophageal carcinoma cells by regulating Smad7.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.1

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