miR-126调控LIMK-1与卵巢癌细胞侵袭相关的研究

发布时间：2018-03-01 00:06

本文关键词： 卵巢癌侵袭 miR-126 LIMK-1　出处：《浙江大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究背景和目的：卵巢癌是女性生殖系统常见的恶性肿瘤之一,具有发展迅速、恶性程度高、极易发生局部侵袭和腹腔内转移的特点,其复发率和致死率高居妇科恶性肿瘤之首。LIMK-1的主要作用是组装含肌动蛋白的细胞骨架,在肿瘤细胞的增殖与侵袭中具有重要的作用。miRNA即microRNA,是一类新发现的内源性小分子非编码单链RNA,长约18～26个核苷酸,具体作用方式取决于靶mlRNA的3’末端非翻译区碱基互补配对与匹配的程度,最终致靶mRNA降解或转录后翻译受抑,广泛参与动植物生长发育、细胞分化、增殖与凋亡以及肿瘤的发生发展和侵袭转移等各种生理及病理过程。通过我们先前的研究发现miR-126通过调节VEGF的表达对肿瘤的增殖、侵袭有抑制的作用,然而作为血管内皮生长因子的VEGF如何抑制肿瘤细胞的侵袭的机制目前还不明了,为此,本研究拟构建miR-126的慢病毒表达载体LV-miR-126,并将其转染上皮性卵巢癌细胞株,明确miR-126对卵巢癌细胞LIMK-1的影响,从而为阐明卵巢癌侵袭的机制以及为抑制卵巢癌的转移提供一条新的途径。研究材料和方法培养人卵巢癌细胞株SKOV3,通过慢病毒包装miR-126转染卵巢癌SKOV3细胞系,将其分为三组：转染miR-126表达组(SKOV3/LV3-has-miR-126组)、转染阴性对照组(SKOV3/LV3NC组)和空白对照组(SKOV3组),通过Transwell侵袭实验观察各组细胞的侵袭能力,通过免疫荧光比较各组细胞LIMK-1表达量的差别,分析miR-126与LIMK-1表达的关系。结果： 1. Transwell侵袭实验结果显示SKOV3细胞侵过基质胶的穿膜细胞数在SKOV3组细胞中是261±8。与SKOV3组细胞相比较,转染阴性对照组SKOV3/LV3NC细胞侵过基质胶的穿膜细胞数没有明显改变(276±20,P0.05),转染LV3-has-miR-126组细胞侵过基质胶的穿膜细胞数明显减少(232±17,P0.05)。 2. SKOV3组与SKOV3/LV3NC组的细胞LIMK-1蛋白的表达量相近；与SKOV3组与SKOV3/LV3NC组的细胞LIMK-1蛋白的表达量相比,LV3-has-miR-126组细胞LIMK-1蛋白的表达量明显减少。结论： 1.miR-126转染的SKOV3卵巢癌细胞侵过基质胶的穿膜细胞数明显减少,提示miR-126的表达可抑制卵巢癌的侵袭。 2.miR-126转染的SKOV3卵巢癌细胞LIMK-1的表达受到抑制,提示miR-126可能通过VEGF/LMK-1来抑制卵巢癌细胞骨架的变形从而抑制其侵袭。
[Abstract]:Background and objectives of the study:. Ovarian cancer is one of the most common malignant tumors in the female reproductive system, which is characterized by rapid development, high degree of malignancy, local invasion and intraperitoneal metastasis. The main function of LIMK-1 is to assemble actin containing cytoskeleton. MiRNA is a new class of endogenous small molecule non-coding single-stranded RNAs, which is about 18 ~ 26 nucleotides long, which plays an important role in the proliferation and invasion of tumor cells. The specific mode of action depends on the degree of complementary pairing and matching of the 3 '-terminal untranslated regions of target mlRNA, resulting in the degradation of target mRNA or post-transcriptional suppression of translation, and its extensive participation in the growth and development of animals and plants and cell differentiation. Through our previous studies, we found that miR-126 can inhibit the proliferation and invasion of tumor by regulating the expression of VEGF. However, it is not clear how VEGF, as a vascular endothelial growth factor, inhibits the invasion of tumor cells. Therefore, the lentivirus expression vector LV-miR-126 of miR-126 was constructed and transfected into epithelial ovarian cancer cell line. To elucidate the effect of miR-126 on ovarian cancer cell line LIMK-1 and provide a new way to elucidate the mechanism of ovarian cancer invasion and inhibit the metastasis of ovarian cancer. Research materials and methods. Human ovarian cancer cell line SKOV3 was cultured and transfected into ovarian cancer SKOV3 cell line by lentivirus packaging miR-126. They were divided into three groups: transfected miR-126 expression group (SKOV3 / LV3-has-miR-126), negative control group (SKOV3 / LV3NC) and blank control group (SKOV3). The invasive ability of each group was observed by Transwell invasion experiment, and the difference of LIMK-1 expression in each group was compared by immunofluorescence. To analyze the relationship between miR-126 and LIMK-1 expression. Results:. 1. The results of Transwell invasion assay showed that the number of SKOV3 cells infiltrating matrix glue was 261 卤8 in SKOV3 group, which was significantly higher than that in SKOV3 group. There was no significant change in the number of transmembrane cells infiltrating matrix glue in SKOV3/LV3NC cells in negative control group, while in LV3-has-miR-126 group, the number of transmembrane cells infiltrated through matrix glue was significantly decreased by 232 卤17 (P 0.05). 2. The expression of LIMK-1 protein in SKOV3 group was similar to that in SKOV3/LV3NC group, and the expression of LIMK-1 protein in LV3-has-miR-126 group was significantly lower than that in SKOV3 group and SKOV3/LV3NC group. Conclusion:. 1. The number of transmembrane cells infiltrating matrix glue in SKOV3 ovarian cancer cells transfected with miR-126 was significantly decreased, suggesting that the expression of miR-126 could inhibit the invasion of ovarian cancer. 2. The expression of LIMK-1 in SKOV3 ovarian cancer cells transfected with miR-126 was inhibited, suggesting that miR-126 might inhibit the cytoskeleton deformation and invasion of ovarian cancer cells by VEGF/LMK-1.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

【参考文献】