Hsa-miR-29c调控人胆囊癌细胞增殖转移的机制研究

发布时间：2019-01-01 13:02

【摘要】：研究目的胆囊癌是胆道系统最常见的恶性肿瘤,其发病率占消化道肿瘤的7位。由于胆囊癌细胞能较快地通过直接浸润、淋巴转移、血运转移等方式在早期发生广泛扩散,大多数患者发现时已是中晚期,根治性手术切除率低,预后极差,中位生存期小于1年。Micro RNA(miRNA)是一类新发现的非编码小RNA分子,它们能够通过与靶基因的m RNA分子从而起到对靶基因的调控作用。已有大量报道发现这种小RNA分子几乎参与调控了细胞生命周期中所以的关键步骤,但在胆囊癌领域研究较少。方法1、通过高通量Agilent人类miRNA芯片(V.19.0)检测胆囊癌组织及其相对应的癌旁组织中miRNA的表达谱,选择表达量显著异常的miRNA作为研究对象。2、通过使用Real-Time PCR方法检测40例胆囊癌组织及其癌旁组织中miR-29c-5p的表达量发现,miR-29c-5p在肿瘤组织中异常低表达,同时发现miR-29c-5p与胆囊癌患者的预后密切相关。3、体内体外实验发现,miR-29c-5p调控胆囊癌细胞增殖及抑制其浸润转移的能力,这一现象可能是由miR-29c-5p抑制胆囊癌细胞上皮间质转化(EMT)所调控的。4、MiR-29c-5p的靶基因的确定:结合miRDB和miRanda等生物信息学预测与双荧光素酶报告基因方法检测miR-29c-5p对靶基因3‘UTR的结合作用,Western blot在蛋白水平检测其对靶基因的抑制作用,免疫组化检测SPOCK1基因和胆囊癌患者预后的关系。5、SPOCK1作为miR-29c-5p的靶基因在miR-29c-5p的生物学功能中的作用:SPOCK1在miR-29c-5p对胆囊癌细胞增殖和侵袭转移中的作用—SPOCK1过表达质粒和miR-29c-5p共转染后进行Transwell实验;Western blot验证EMT相关指标。结果1、通过胆囊癌差异性表达检测确定miR-29c-5p为核心的胆囊癌相关miRNA;2、体内体外实验证明miR-29c-5p过表达通过调控EMT抑制胆囊癌细胞的侵袭转移,并抑制胆囊癌细胞的增殖;3、SPOCK1被证实为miR-29c-5p的靶基因;4、SPOCK1在胆囊癌中起癌基因作用,并且在miR-29c-5p的抑癌作用中起关键作用。结论MiR-29c-5p通过靶向SPOCK1在胆囊癌的生长和转移中起关键作用,提示干预miR-29c-5p可能成为胆囊癌治疗的重要部分。
[Abstract]:Objective gallbladder carcinoma is the most common malignant tumor in the biliary system. Because gallbladder cancer cells can spread widely in the early stage through direct infiltration, lymphatic metastasis, blood metastasis and so on, most of the patients are found to be in the middle and late stage, the rate of radical resection is low, and the prognosis is extremely poor. . Micro RNA (miRNA) with median survival of less than one year is a new class of non-coding small RNA molecules, which can regulate the target gene by interacting with the m RNA molecule of the target gene. It has been widely reported that this small RNA molecule is almost involved in the key steps of regulating cell life cycle, but less research has been done in the field of gallbladder carcinoma. Methods 1. High throughput Agilent human miRNA microarray (V.19.0) was used to detect the miRNA expression profile in gallbladder carcinoma tissues and their corresponding adjacent tissues. By using Real-Time PCR method to detect the expression of miR-29c-5p in 40 cases of gallbladder carcinoma and its adjacent tissues, it was found that the expression of miR-29c-5p was abnormally low in tumor tissues. It was also found that miR-29c-5p was closely related to the prognosis of gallbladder cancer patients. In vivo and in vitro experiments showed that miR-29c-5p could regulate the proliferation and inhibit the invasion and metastasis of gallbladder cancer cells. This phenomenon may be regulated by miR-29c-5p inhibiting epithelial interstitial transformation (EMT) of gallbladder cancer cells. Identification of target genes of MiR-29c-5p: combined with bioinformatics prediction such as miRDB and miRanda and double luciferase reporter gene method, the binding of miR-29c-5p to target gene 3'UTR was detected. The inhibitory effect of Western blot on target gene was detected at protein level, and the relationship between SPOCK1 gene and prognosis of gallbladder carcinoma was detected by immunohistochemistry. The role of SPOCK1 as a target gene of miR-29c-5p in the biological function of miR-29c-5p: the role of SPOCK1 in the proliferation, invasion and metastasis of cholecyst carcinoma cells by miR-29c-5p-SPOCK1 overexpression plasmid and miR-29c-5p co-transfection were used for Transwell assay. Western blot was used to verify the EMT related indexes. Results 1. By the differential expression detection of gallbladder carcinoma, miRNA; related to gallbladder carcinoma with miR-29c-5p as the core was determined. 2. In vitro and in vivo experiments showed that the overexpression of miR-29c-5p inhibited the invasion and metastasis of gallbladder cancer cells by regulating EMT, and inhibited the proliferation of gallbladder cancer cells, 3SPOCK1 was confirmed as the target gene of miR-29c-5p. 4 SPOCK1 plays an oncogene role in gallbladder carcinoma and plays a key role in the inhibition of miR-29c-5p. Conclusion MiR-29c-5p plays a key role in the growth and metastasis of gallbladder carcinoma by targeting SPOCK1, suggesting that intervention of miR-29c-5p may be an important part in the treatment of gallbladder carcinoma.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.8

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