本文選題：宮頸癌 + 淋巴結(jié)轉(zhuǎn)移　； 參考：《復(fù)旦大學》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)移的機制。材料和方法：在宮頸癌新鮮標本中,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,Real-time PCR驗證miRNA芯片篩選的結(jié)果。在SiHa細胞株中過表達或低表達候選miRNA含量,通過體外Transwell實驗檢測細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胞轉(zhuǎn)移模型。通過生物信息軟件、靶基因mRNA real-time PCR和Western Blot蛋白檢測三種方法綜合推斷miR-652的靶基因。結(jié)果：niRNA芯片篩查顯示,對比無盆腔淋巴結(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miRNA高表達(miR-142-3p,miR-652) (P0.05),3個 miRNA低表達(miR-196b, miR-320b和miR-424) (P0.05)。Real-time PCR證實篩選出的miRNA表達變化趨勢與芯片結(jié)果相似,但只有miR-196b及miR-652的差異表達具有統(tǒng)計學差異(P0.05)。Transwell實驗顯示,低表達miR-196b和過表達miR-652可以增進細胞遷移和侵襲能力,過表達miR-196b和低表達miR-652可以抑制細胞遷移和侵襲能力。未能發(fā)現(xiàn)改變niR-142-3p、miR-320b和miR-424表達量會影響細胞遷移或侵襲功能。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進行后續(xù)研究。在體內(nèi)實驗中,未有足夠證據(jù)說明miR-652能增進腫瘤細胞的轉(zhuǎn)移能力,但PET-CT顯示miR-652可能有促進肝臟轉(zhuǎn)移病變的趨勢。綜合Targetscan、miRDB、miRSearch、miRTarBase 和 miRwalk共5個miRNA數(shù)據(jù)庫聯(lián)合預(yù)測,并查閱靶基因資料文獻,獲得6個miR-652的候選靶基因,分別為BCL11A、ISL1、LLGL1、NPTN、NR4A3、YWHAH。在對以上基因mRNA表達水平的PCR檢測中,發(fā)現(xiàn)miR-652過表達組ISL1、NR4A3和YWHAH基因的mRNA表達量下降(P0.05), miR-652低表達組ISL1、NR4A3和YWHAH基因的mRNA表達量上升(P0.05),變化趨勢符合miRNA-mRNA結(jié)合改變。其余3個靶基因mRNA表達量變化未發(fā)現(xiàn)統(tǒng)計學差異(P0.05)。miR-652過表達組YWHAH蛋白表達量減少,miR-652低表達組YWHAH蛋白表達量上升。未能發(fā)現(xiàn)miR-652的低表達和過表達影響ISL1和NR4A3蛋白表達量的改變。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.
【學位授予單位】：復(fù)旦大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R737.33

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