本文選題：宮頸癌 + 淋巴結(jié)轉(zhuǎn)移　； 參考：《復(fù)旦大學(xué)》2014年博士論文

【摘要】：目的：淋巴結(jié)轉(zhuǎn)移是宮頸癌主要轉(zhuǎn)移途徑,也是影響宮頸癌患者預(yù)后的重要因素。假設(shè)miRNA能調(diào)節(jié)宮頸癌淋巴結(jié)轉(zhuǎn)移,研究不同淋巴結(jié)轉(zhuǎn)移能力的宮頸癌組織中的miRNA表達(dá)差異,篩選出淋巴結(jié)轉(zhuǎn)移相關(guān)的miRNA,并且研究其調(diào)節(jié)淋巴結(jié)轉(zhuǎn)移的機(jī)制。材料和方法：在宮頸癌新鮮標(biāo)本中,用miRNA芯片篩選出不同淋巴結(jié)轉(zhuǎn)移能力的宮頸癌的miRNA的差異表達(dá),Real-time PCR驗(yàn)證miRNA芯片篩選的結(jié)果。在SiHa細(xì)胞株中過(guò)表達(dá)或低表達(dá)候選miRNA含量,通過(guò)體外Transwell實(shí)驗(yàn)檢測(cè)細(xì)胞的遷移和侵襲能力。用慢病毒載體構(gòu)建穩(wěn)定轉(zhuǎn)染候選miRNA的穩(wěn)轉(zhuǎn)株,并建立裸鼠宮頸癌細(xì)胞轉(zhuǎn)移模型。通過(guò)生物信息軟件、靶基因mRNA real-time PCR和Western Blot蛋白檢測(cè)三種方法綜合推斷miR-652的靶基因。結(jié)果：niRNA芯片篩查顯示,對(duì)比無(wú)盆腔淋巴結(jié)轉(zhuǎn)移的宮頸癌組織,有盆腔淋巴結(jié)轉(zhuǎn)移的宮頸癌組織中2個(gè)miRNA高表達(dá)(miR-142-3p,miR-652) (P0.05),3個(gè) miRNA低表達(dá)(miR-196b, miR-320b和miR-424) (P0.05)。Real-time PCR證實(shí)篩選出的miRNA表達(dá)變化趨勢(shì)與芯片結(jié)果相似,但只有miR-196b及miR-652的差異表達(dá)具有統(tǒng)計(jì)學(xué)差異(P0.05)。Transwell實(shí)驗(yàn)顯示,低表達(dá)miR-196b和過(guò)表達(dá)miR-652可以增進(jìn)細(xì)胞遷移和侵襲能力,過(guò)表達(dá)miR-196b和低表達(dá)miR-652可以抑制細(xì)胞遷移和侵襲能力。未能發(fā)現(xiàn)改變niR-142-3p、miR-320b和miR-424表達(dá)量會(huì)影響細(xì)胞遷移或侵襲功能。綜合上述結(jié)論,miR-196b和miR-652可能與宮頸癌淋巴結(jié)轉(zhuǎn)移有關(guān)。從創(chuàng)新性考慮,暫選miR-652進(jìn)行后續(xù)研究。在體內(nèi)實(shí)驗(yàn)中,未有足夠證據(jù)說(shuō)明miR-652能增進(jìn)腫瘤細(xì)胞的轉(zhuǎn)移能力,但PET-CT顯示miR-652可能有促進(jìn)肝臟轉(zhuǎn)移病變的趨勢(shì)。綜合Targetscan、miRDB、miRSearch、miRTarBase 和 miRwalk共5個(gè)miRNA數(shù)據(jù)庫(kù)聯(lián)合預(yù)測(cè),并查閱靶基因資料文獻(xiàn),獲得6個(gè)miR-652的候選靶基因,分別為BCL11A、ISL1、LLGL1、NPTN、NR4A3、YWHAH。在對(duì)以上基因mRNA表達(dá)水平的PCR檢測(cè)中,發(fā)現(xiàn)miR-652過(guò)表達(dá)組ISL1、NR4A3和YWHAH基因的mRNA表達(dá)量下降(P0.05), miR-652低表達(dá)組ISL1、NR4A3和YWHAH基因的mRNA表達(dá)量上升(P0.05),變化趨勢(shì)符合miRNA-mRNA結(jié)合改變。其余3個(gè)靶基因mRNA表達(dá)量變化未發(fā)現(xiàn)統(tǒng)計(jì)學(xué)差異(P0.05)。miR-652過(guò)表達(dá)組YWHAH蛋白表達(dá)量減少,miR-652低表達(dá)組YWHAH蛋白表達(dá)量上升。未能發(fā)現(xiàn)miR-652的低表達(dá)和過(guò)表達(dá)影響ISL1和NR4A3蛋白表達(dá)量的改變。結(jié)論：miR-196b有抑制宮頸癌淋巴結(jié)轉(zhuǎn)移的功能,miR-652有促進(jìn)宮頸癌淋巴結(jié)轉(zhuǎn)移的功能。YWHAH可能是miR-652的靶基因。
[Abstract]:Objective: lymph node metastasis is the main metastasis pathway of cervical cancer and an important factor affecting the prognosis of cervical cancer patients. It is assumed that miRNA can regulate lymph node metastasis of cervical cancer. The difference of miRNA expression in cervical cancer tissues with different lymph node metastasis ability is studied. The miRNAs associated with lymph node metastasis are screened out and the mechanism of their regulation of lymph node metastasis is studied. Materials and methods: miRNA microarray was used to screen the differential expression of miRNA in cervical cancer with different lymph node metastasis ability. Real-time PCR was used to verify the results of miRNA microarray screening. Candidate miRNAs were overexpressed or low expressed in SIHA cell lines. The migration and invasion of SIHA cells were detected by Transwell assay in vitro. The stable transfer strain of candidate miRNA was constructed by using lentivirus vector and the metastasis model of cervical cancer cells in nude mice was established. The target gene of miR-652 was inferred by three methods: bioinformatics software, real-time PCR of target gene and Western Blot protein detection. Results compared with cervical cancer tissues without pelvic lymph node metastasis, the microarray screening showed that, In cervical carcinoma with pelvic lymph node metastasis, two miRNA overexpression (miR-142-3pnmiR-652), three miRNA low expression (miR-196b, miR-320b and miR-424) (P0.05). Real-time PCR confirmed that the change trend of miRNA expression was similar to that of microarray. However, only the difference of miR-196b and miR-652 expression was statistically significant (P0.05) .Transwell experiments showed that the low expression of miR-196b and over-expression of miR-652 could enhance the ability of cell migration and invasion, and over-expression of miR-196b and low-expression of miR-652 could inhibit the ability of cell migration and invasion. No change in the expression of niR-142-3pmmiR-320b and miR-424 was found to affect cell migration or invasion. These results suggest that miR-196b and miR-652 may be associated with lymph node metastasis of cervical cancer. From the innovative point of view, miR-652 was selected for follow-up study. In vivo, there was not enough evidence that miR-652 could enhance the metastasis ability of tumor cells, but PET-CT showed that miR-652 might promote liver metastasis. Combined prediction of 5 miRNA databases from TargetscanmiRDBmiRsearch chong miRTarBase and miRwalk, six candidate target genes of miR-652 were obtained, which were BCL11A, ISL1, LLGL1, NPNLGL1, NPNLGL1, NR4A3, YWHAH, respectively, and obtained six candidate target genes of miR-652 (BCL11A1, ISL1, LLGL1, NPNLGL1, NPNR4A3, YWHAH). In the PCR analysis of the mRNA expression level of the above genes, it was found that the mRNA expression of ISL1, NR4A3 and YWHAH gene decreased in the overexpression group of miR-652 (P0.05), and the mRNA expression of ISL1, NR4A3 and YWHAH gene increased in the low expression group of miR-652 (P0.05), and the change trend was consistent with the change of miRNA-mRNA binding. There was no significant difference in the mRNA expression of the other three target genes (P0.05). The expression of YWHAH protein decreased in the over-expression group of miR-652 and increased in the low expression group of miR-652. The low expression and overexpression of miR-652 had no effect on the expression of ISL1 and NR4A3. Conclusion: miR-196b can inhibit lymph node metastasis of cervical cancer. MiR-652 has the function of promoting lymph node metastasis of cervical cancer. YWHAH may be the target gene of miR-652.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.33

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相關(guān)期刊論文 前1條

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