【摘要】：目的： 驗(yàn)證miR-224在宮頸癌組織中表達(dá)上調(diào),并明確miR-224對(duì)宮頸癌細(xì)胞生物學(xué)行為的作用及調(diào)控機(jī)制。 方法： 采用Stem-loop RT-PCR法檢測(cè)148例宮頸鱗癌組織和64例宮頸正常上皮組織的miR-224的表達(dá)水平,收集整理臨床病理資料,分析miR-224的表達(dá)水平與宮頸癌臨床不良預(yù)后因素的相關(guān)性。 在宮頸癌細(xì)胞株Siha和CaSki中,利用細(xì)胞轉(zhuǎn)染技術(shù)過表達(dá)miR-224以及siRNA干擾RASSF8的表達(dá),利用MTT法檢測(cè)細(xì)胞增殖能力,流式細(xì)胞技術(shù)檢測(cè)細(xì)胞周期分布,劃痕試驗(yàn)和Transwell侵襲試驗(yàn)檢測(cè)細(xì)胞的遷移和侵襲能力,并利用Real time RT-PCR和Western Blot的方法檢測(cè)宮頸癌組織中IASSF8的mRNA和蛋白水平,最后利用雙熒光素酶報(bào)告基因的方法確認(rèn)miR-224與RASSF8靶向關(guān)系 結(jié)果： 1.miR-224在宮頸鱗癌組織的表達(dá)水平與宮頸正常上皮組織相比顯著下調(diào)。miR-224高表達(dá)組有更高的FIGO分期、更大的腫瘤體積,更易于發(fā)生盆腔淋巴結(jié)轉(zhuǎn)移、深間質(zhì)浸潤(rùn)、以及脈管浸潤(rùn)。 2.在宮頸癌細(xì)胞株中,過表達(dá)miR-224可增加Siha和CaSki細(xì)胞的增殖能力,促進(jìn)細(xì)胞劃痕的愈合和細(xì)胞的侵襲并且下調(diào)RASSF8的蛋白水平,但是對(duì)周期以及凋亡無明顯影響。 3.宮頸癌組織中IASSF8的表達(dá)量明顯低于正常宮頸組織。 4.miR-224可下調(diào)含RASSF83'UTR區(qū)報(bào)告質(zhì)粒熒光素酶的活性,而不影響3'UTR區(qū)突變報(bào)告質(zhì)粒的熒光素酶活性。 5.在宮頸癌細(xì)胞中,干擾RASSF8的表達(dá)可促進(jìn)Siha和CaSki細(xì)胞的遷移和侵襲能力但是對(duì)增值無影響。結(jié)論： 1.miR-224的表達(dá)水平在宮頸癌組織與正常組織相比顯著上調(diào)并且miR-224高表達(dá)與宮頸癌預(yù)后不良因素相關(guān). 2.miR-224表達(dá)上調(diào)參與了宮頸癌的進(jìn)展過程,miR-224通過靶向RASSF8調(diào)控宮頸癌細(xì)胞的生物學(xué)功能。
[Abstract]:Aim: to verify the up-regulation of miR-224 expression in cervical cancer tissues and to clarify the role of miR-224 in the biological behavior of cervical cancer cells and its regulatory mechanism. Methods: the expression of miR-224 was detected by Stem-loop RT-PCR in 148 cases of cervical squamous cell carcinoma and 64 cases of normal cervical epithelium, and the clinicopathological data were collected. To analyze the correlation between the expression of miR-224 and the prognostic factors of cervical cancer. In cervical cancer cell lines Siha and CaSki, the over-expression of miR-224 and siRNA interfered with the expression of RASSF8 in cervical cancer cell line Siha and CaSki, the proliferation ability of cells was detected by MTT method, and the cell cycle distribution was detected by flow cytometry. Scratch test and Transwell invasion test were used to detect the migration and invasion ability of cells, and the mRNA and protein levels of IASSF8 in cervical cancer tissues were detected by Real time RT-PCR and Western Blot methods. Finally, the double luciferase reporter gene method was used to confirm the target relationship between miR-224 and RASSF8. The expression level of 1.miR-224 in cervical squamous cell carcinoma was significantly down-regulated compared with that in normal cervical epithelium. 224 high expression group had higher FIGO stage. Larger tumor size, more prone to pelvic lymph node metastasis, deep interstitial infiltration, and vascular infiltration. 2. In cervical cancer cell lines, overexpression of miR-224 can increase the proliferation ability of Siha and CaSki cells, promote the healing of cell scratches and invasion of cells, and down-regulate the protein level of RASSF8, but have no obvious effect on cell cycle and apoptosis. 3. The expression of IASSF8 in cervical cancer tissues was significantly lower than that in normal cervical tissues. 4.miR-224 could down-regulate the luciferase activity of the reporter plasmid containing the RASSF83' UTR region, but did not affect the luciferase activity of the mutated reporter plasmid in the 3 'UTR region. 5. In cervical cancer cells, interference with the expression of RASSF8 can promote the migration and invasion of Siha and CaSki cells, but has no effect on proliferation. Conclusion: the expression of 1.miR-224 is significantly up-regulated in cervical cancer tissues and normal tissues, and the high expression of miR-224 is associated with poor prognosis of cervical cancer. The up-regulated expression of 2.miR-224 is involved in the progression of cervical cancer. MiR-224 regulates the biological function of cervical cancer cells by targeting RASSF8.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.33

【共引文獻(xiàn)】

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