本文關鍵詞： 卵巢癌 侵襲 miR-126 LIMK-1　出處：《浙江大學》2014年碩士論文　論文類型：學位論文

【摘要】：研究背景和目的： 卵巢癌是女性生殖系統(tǒng)常見的惡性腫瘤之一,具有發(fā)展迅速、惡性程度高、極易發(fā)生局部侵襲和腹腔內轉移的特點,其復發(fā)率和致死率高居婦科惡性腫瘤之首。LIMK-1的主要作用是組裝含肌動蛋白的細胞骨架,在腫瘤細胞的增殖與侵襲中具有重要的作用。miRNA即microRNA,是一類新發(fā)現(xiàn)的內源性小分子非編碼單鏈RNA,長約18～26個核苷酸,具體作用方式取決于靶mlRNA的3’末端非翻譯區(qū)堿基互補配對與匹配的程度,最終致靶mRNA降解或轉錄后翻譯受抑,廣泛參與動植物生長發(fā)育、細胞分化、增殖與凋亡以及腫瘤的發(fā)生發(fā)展和侵襲轉移等各種生理及病理過程。通過我們先前的研究發(fā)現(xiàn)miR-126通過調節(jié)VEGF的表達對腫瘤的增殖、侵襲有抑制的作用,然而作為血管內皮生長因子的VEGF如何抑制腫瘤細胞的侵襲的機制目前還不明了,為此,本研究擬構建miR-126的慢病毒表達載體LV-miR-126,并將其轉染上皮性卵巢癌細胞株,明確miR-126對卵巢癌細胞LIMK-1的影響,從而為闡明卵巢癌侵襲的機制以及為抑制卵巢癌的轉移提供一條新的途徑。 研究材料和方法 培養(yǎng)人卵巢癌細胞株SKOV3,通過慢病毒包裝miR-126轉染卵巢癌SKOV3細胞系,將其分為三組：轉染miR-126表達組(SKOV3/LV3-has-miR-126組)、轉染陰性對照組(SKOV3/LV3NC組)和空白對照組(SKOV3組),通過Transwell侵襲實驗觀察各組細胞的侵襲能力,通過免疫熒光比較各組細胞LIMK-1表達量的差別,分析miR-126與LIMK-1表達的關系。 結果： 1. Transwell侵襲實驗結果顯示SKOV3細胞侵過基質膠的穿膜細胞數(shù)在SKOV3組細胞中是261±8。與SKOV3組細胞相比較,轉染陰性對照組SKOV3/LV3NC細胞侵過基質膠的穿膜細胞數(shù)沒有明顯改變(276±20,P0.05),轉染LV3-has-miR-126組細胞侵過基質膠的穿膜細胞數(shù)明顯減少(232±17,P0.05)。 2. SKOV3組與SKOV3/LV3NC組的細胞LIMK-1蛋白的表達量相近；與SKOV3組與SKOV3/LV3NC組的細胞LIMK-1蛋白的表達量相比,LV3-has-miR-126組細胞LIMK-1蛋白的表達量明顯減少。 結論： 1.miR-126轉染的SKOV3卵巢癌細胞侵過基質膠的穿膜細胞數(shù)明顯減少,提示miR-126的表達可抑制卵巢癌的侵襲。 2.miR-126轉染的SKOV3卵巢癌細胞LIMK-1的表達受到抑制,提示miR-126可能通過VEGF/LMK-1來抑制卵巢癌細胞骨架的變形從而抑制其侵襲。
[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 鹵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 鹵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.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.31

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