發(fā)布時間：2018-06-29 12:51

  本文選題：甲基化 + MicroRNA-335��； 參考：《南昌大學》2016年碩士論文

【摘要】：目的:探討胃癌轉移中表達下調MicroRNA-335是否存在表觀遺傳調控的機制,并通過干預實驗進行驗證。同時在胃癌中預測并驗證MicroRAN-335參與胃癌侵襲和轉移的靶向基因。方法:生物信息學平臺驗證理論的可能性,實時定量PCR檢測胃癌細胞株中和胃組織中MicroRNA-335表達水平;重亞硫酸鹽測序PCR(BSP)和甲基化特異性PCR(MSP)檢測二者MicroRNA-335啟動子區(qū)的甲基化狀態(tài);采用去甲基化方法(5-Azadeoxycytidine)處理胃癌細胞株,分別用實時定量PCR和BSP法檢測處理后的MicroRNA-335表達的變化及其甲基化的變化;利用多個生物靶向在線軟件(microRNA;Targetscan;Pic Tar;Rnahybrid)分析miR-335可能的靶基因及靶向3’-UTR的位點,實時定量PCR和Western Blot對預測出的靶基因做mRNA和蛋白水平的驗證,利用脂質體轉染法將hsa-miR-335模擬物及miR-335抑制劑及其對應的陰性對照、空白對照組分別轉入人胃癌SGC-7901和BGC-823細胞系,進一步驗證這種靶向關系。結果:(1)MicroRNA-335基因序列的啟動子區(qū)具有穩(wěn)定的CpG島:生物信息學網站(UCSC、NCBI、Mirbase)綜合對比分析MicroRNA-335基因上游5000bp范圍的序列,用確定的基因序列進行啟動子和CpG島搜索,Cpgislands發(fā)現(xiàn)2個甲基島,Methprimer和Cpgplot分別有4個和3個甲基島,三者重疊率為88.9%,對比分析甲基島和啟動子區(qū)域存在交匯。(2)人胃癌細胞系中MicroRNA-335表達降低。通過5-Azadeoxycytidine去甲基化預處理,人胃癌細胞系中miR-335表達升高。胃癌組織中miR-335表達降低,尤以有腹膜轉移的組織表達最低。(3)人胃癌細胞系中miR-335的啟動子區(qū)CpG島高甲基化,通過5-Azadeoxycytidine去甲基化預處理,miR-335啟動子區(qū)甲基化水平降低。胃癌中miR-335的啟動子區(qū)CpG島高甲基化與miR-335表達存在相關性。通過甲基化特異性PCR(MSP)檢測,胃癌組織中miR-335有更高的甲基化,且高甲基化信號與胃癌TNM分期、淋巴結轉移、腹膜轉移有明顯的相關性。胃癌組織中,高甲基化的組織呈現(xiàn)較低的miR-335表達水平。(4)生物信息學預測GTPase activating protein 1(RASA1)可能是miR-335作用于胃癌侵襲轉移的靶基因。實時定量PCR及Western Blot初步檢測胃癌中尤其是轉移組織中RASA1表達升高,外生高表達miR-335后RASA1表達顯著減低。結論:(1)胃癌轉移中表達下調MicroRNA-335受其啟動子CpG島高甲基化的調控。(2)胃癌組織中miR-335啟動子區(qū)高甲基化信號與胃癌侵襲轉移相關臨床病理因素有顯著相關性。(3)miR-335可能通過調節(jié)RASA1基因表達發(fā)揮抑制胃癌侵襲轉移作用。
[Abstract]:Aim: to investigate the mechanism of epigenetic regulation of down-regulation of microRNA-335 in gastric cancer metastasis and verify it by intervention experiment. At the same time, microRAN-335 gene involved in invasion and metastasis of gastric cancer was predicted and verified in gastric cancer. Methods: bioinformatics platform was used to test the possibility of microRNA-335 expression in gastric cancer cell lines and gastric tissues by real-time quantitative PCR, and the methylation status of microRNA-335 promoter region was detected by heavy sulfite sequencing PCR (BSP) and methylation specific PCR (MSP). Gastric cancer cell lines were treated with demethylation method (5-Azadeoxycytidine). The changes of microRNA-335 expression and methylation were detected by real-time quantitative PCR and BSP-PCR, respectively. The possible target genes of miR-335 and the sites targeting 3H-UTR were analyzed by microRNA-TargetscanPic tag Rnahybrid. The mRNA and protein levels of the predicted target genes were verified by real-time quantitative PCR and Western Blot. The hsa-miR-335 mimics, miR-335 inhibitors and their corresponding negative controls were transfected into human gastric cancer SGC-7901 and BGC-823 cell lines by liposome transfection. Results: (1) the promoter region of the microRNA-335 gene sequence had stable CpG island: a comprehensive comparative analysis of the 5000bp region upstream of the microRNA-335 gene. The promoter and CpG islands were used to search for two methyl islands, Methprimer and Cpgplot, which were found to have 4 and 3 methyl islands, respectively. The overlap rate among the three groups was 88.9, which was compared and analyzed. (2) the expression of microRNA-335 was decreased in human gastric cancer cell line. The expression of miR-335 in human gastric cancer cell line was increased by 5-Azadeoxycytidine demethylation preconditioning. The expression of miR-335 was decreased in gastric cancer tissues, especially in the tissues with peritoneal metastasis. (3) the promoter region of miR-335 was hypermethylated, and the methylation level of the promoter region of miR-335 was decreased by 5-Azadeoxycytidine demethylation preconditioning. The expression of miR-335 was correlated with CpG island hypermethylation in the promoter region of miR-335 in gastric cancer. The results of methylation specific PCR (MSP) showed that miR-335 had higher methylation in gastric cancer tissues, and the hypermethylation signal was significantly correlated with TNM stage, lymph node metastasis and peritoneal metastasis. In gastric cancer tissues, the hypermethylated tissues showed a low expression level of miR-335. (4) Bioinformatics predicted that GTPase activating protein 1 (RASA1) might be the target gene of miR-335 acting on invasion and metastasis of gastric cancer. The expression of RASA1 in gastric carcinoma, especially in metastatic tissues, was detected by real-time quantitative PCR and Western blot, but the expression of RASA1 was significantly decreased after miR-335 was overexpressed. Conclusion: (1) the down-regulation of microRNA-335 in gastric cancer metastasis is regulated by the hypermethylation of its promoter CpG island. (2) there is a significant correlation between the hypermethylation signal of miR-335 promoter and the clinicopathological factors associated with invasion and metastasis of gastric cancer. Regulation of RASA1 gene expression inhibits invasion and metastasis of gastric cancer.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R735.2

【相似文獻】

相關碩士學位論文 前1條

1 李道江;MicroRNA-335在胃癌中的表達及其表觀遺傳調控機制的研究[D];南昌大學;2016年

，

本文編號：2082171