【摘要】：目的:研究微小RNAl06b(mi R-106b)在食管癌浸潤(rùn)轉(zhuǎn)移中的作用機(jī)制,及其對(duì)Smad7的調(diào)控影響,為臨床治療食管癌提供新的理論依據(jù)。方法:1.在組織水平,免疫組織化學(xué)方法檢測(cè)90對(duì)食管鱗癌及其對(duì)應(yīng)的正常食管組織中Smad7的表達(dá)情況,分析mi R-106b與Smad7的相關(guān)性,探討mi R-106b、Smad7表達(dá)水平與臨床病理參數(shù)的相關(guān)性。2.在細(xì)胞水平,分別轉(zhuǎn)染慢病毒LV-has-mir-106b和陰性對(duì)照病毒CON238于食管癌細(xì)胞株KYSE150,轉(zhuǎn)染慢病毒LV-has-mir-106b-inhibition和陰性對(duì)照病毒CON137于食管癌細(xì)胞株KYSE450后,實(shí)驗(yàn)分為上調(diào)組和下調(diào)組。上調(diào)組:control組(未轉(zhuǎn)染mi R-106b的KYSE150細(xì)胞組)、NC組(轉(zhuǎn)染隨機(jī)序列組,陰性對(duì)照病毒CON238)和mi R-106b組(轉(zhuǎn)染mi R-106b的KYSE150的細(xì)胞組);下調(diào)組:control組(未轉(zhuǎn)染mi R-106b-inhibition的KYSE450細(xì)胞組)、NC-inhibitor組(轉(zhuǎn)染隨機(jī)序列組,陰性對(duì)照病毒CON137)和mi R-106b-inhibition組(轉(zhuǎn)染mi R-106b-inhibitor的KYSE450細(xì)胞組),采用實(shí)時(shí)熒光定量聚合酶聯(lián)反應(yīng)(real-time quantitative PCR,q RT-PCR)和免疫印跡(Western Blot)分別檢測(cè)mi R-106b、Smad7 m RNA的表達(dá)水平、Smad7的蛋白表達(dá)水平和EMT的相關(guān)標(biāo)記物(上皮型鈣黏蛋白(E-Cadherin)、神經(jīng)型鈣黏蛋白(N-Cadherin)的蛋白表達(dá)水平,四甲基偶氮唑鹽微量酶反應(yīng)比色法(MTT法)檢測(cè)細(xì)胞的增殖能力,劃痕實(shí)驗(yàn)檢測(cè)細(xì)胞的遷移能力,Transwell方法檢測(cè)細(xì)胞的侵襲能力,流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡情況,雙熒光素酶報(bào)告基因?qū)嶒?yàn)驗(yàn)證mi R-106與Smad7的調(diào)控關(guān)系。結(jié)果:1.Smad7在食管鱗癌組織中的表達(dá)顯著低于對(duì)應(yīng)的正常食管組織(P0.05),其表達(dá)水平與腫瘤的淋巴結(jié)轉(zhuǎn)移相關(guān)(P0.05),與年齡、性別、分化程度、大體分型無(wú)關(guān)(P㧐0.05)。2.慢病毒穩(wěn)定轉(zhuǎn)染mi R-106b后,Smad7m RNA表達(dá)水平和蛋白表達(dá)水平明顯降低(P0.05)。食管癌細(xì)胞的增殖、侵襲和遷移能力均增強(qiáng)(P0.05);q RT-PCR與Western blot檢測(cè)顯示mi R-106b組E-Cadherin表達(dá)較NC組、control組、明顯降低(P0.05);N-Cadherin表達(dá)明顯升高(P0.05)。反之,慢病毒穩(wěn)定轉(zhuǎn)染mi R-106b-inhibition后,Smad7 m RNA表達(dá)水平和蛋白表達(dá)水平明顯升高(P0.05),食管癌細(xì)胞的增殖、侵襲和遷移能力均降低(P0.05);q RT-PCR與Western blot檢測(cè)顯示mi R-106b-inhibition組E-Cadherin表達(dá)較NC-inhibitor組、control組明顯升高(P0.05);N-Cadherin表達(dá)明顯降低(P0.05)。雙熒光素酶報(bào)告基因?qū)嶒?yàn)驗(yàn)證了mi R-106b與Smad7有直接調(diào)控關(guān)系。結(jié)論:1.Smad7參與了食管癌的發(fā)生,且與轉(zhuǎn)移相關(guān)。2.在食管鱗癌中,mi R-106b直接調(diào)控了Smad7蛋白的表達(dá)。3.mi R-106b通過(guò)調(diào)控Smad7促進(jìn)了食管癌細(xì)胞的增殖、遷移和侵襲。
[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.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R735.1

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