【摘要】：背景惡性黑色素瘤(malignant melanoma,MM)是一種皮膚腫瘤,其是由來源于神經(jīng)脊的黑色素細(xì)胞異常增生而產(chǎn)生的,一旦產(chǎn)生并進(jìn)入快速生長期,則具有易侵襲、易轉(zhuǎn)移、預(yù)后差、死亡率高等特點(diǎn)。microRNA(mi RNA)是一類長度為21-25nt的內(nèi)源性非編碼單鏈RNA,它們通過與靶基因mRNA的3'UTRs(非編碼區(qū))或編碼區(qū)的特異性結(jié)合,從而調(diào)控靶基因的表達(dá)。本課題旨在探究mi RNA在惡性黑色素瘤B16F0和B16F10細(xì)胞中的差異表達(dá)情況及過表達(dá)miR-763對B16F10細(xì)胞的影響[1]。目的1.探討惡性黑色素瘤B16F0和B16F10細(xì)胞miRNA的差異表達(dá)情況;2.探討miR-763對惡性黑色素瘤B16F10細(xì)胞的影響及潛在分子機(jī)制;方法使用惡性黑色素瘤B16F0和B16F10細(xì)胞作為研究對象,構(gòu)建動(dòng)物模型。1.觀測在體內(nèi)和體外惡性黑色素瘤B16F0和B16F10細(xì)胞遷移、侵襲和增殖方面的差異;2.檢測惡性黑色素瘤B16F0和B16F10細(xì)胞mi RNA的表達(dá)情況,并利用生物信息學(xué)分析其差異性;3.利用Real-time PCR技術(shù)檢測小鼠黑色素瘤細(xì)胞miR-763、miR-431-5p、miR-205-5p、let-7C-2-3p、mi R-26a-1-3p、miR-28b、miR-29b-3p和miR-24-2-5p的表達(dá)水平,驗(yàn)證miRNA芯片結(jié)果的準(zhǔn)確性。4.在惡性黑色素瘤B16F10細(xì)胞過表達(dá)miR-763,觀測其對B16F10細(xì)胞增殖的影響;5.檢測生物信息學(xué)預(yù)測的miR-763靶基因的表達(dá)變化,利用激酶磷酸化特異性抗體檢測各個(gè)信號通路中的關(guān)鍵激酶的磷酸化水平變化,確定miR-763通過靶基因影響哪些信號通路進(jìn)而對B16F10細(xì)胞產(chǎn)生影響。結(jié)果1.惡性黑色素瘤B16F10細(xì)胞在體外的遷移、侵襲和增殖能力高于B16F0細(xì)胞。2.通過生物信息學(xué)分析發(fā)現(xiàn)惡性黑色素瘤B16F0和B16F10細(xì)胞在miRNA表達(dá)水平存在明顯的差異性。3.Real-time PCR檢測發(fā)現(xiàn)小鼠黑色素瘤細(xì)胞miR-763、miR-431-5p、miR-205-5p、let-7C-2-3p表達(dá)水平下調(diào),miR-26a-1-3p、miR-28b、miR-29b-3p和miR-24-2-5p表達(dá)水平上調(diào),結(jié)果與芯片結(jié)果相一致,miRNA芯片結(jié)果準(zhǔn)確可信。4.過表達(dá)miR-763可以抑制惡性黑色素瘤B16F10細(xì)胞的體外增殖。5.miR-763下調(diào)潛在靶基因Grb2的表達(dá),進(jìn)而影響下游分子Cyclin D1的變化。結(jié)論1.生物信息學(xué)分析發(fā)現(xiàn)兩株細(xì)胞之間在miRNA表達(dá)水平存在顯著差異,這可能是惡性黑色素瘤B16F10與B16F0細(xì)胞遷移、侵襲和增殖能力差異的重要原因。2.miR-763可能通過靶向調(diào)節(jié)Grb2的表達(dá),抑制惡性黑色素瘤B16F10細(xì)胞的增殖。
[Abstract]:Background malignant melanoma (malignant melanoma,MM) is a kind of skin tumor, which is produced by the abnormal proliferation of melanocytes from the spinal cord. Once it comes into rapid growth, it is prone to invasion, metastasis and poor prognosis. MicroRNAs are a class of endogenous non-coding single-stranded RNA, with length of 21-25nt which regulate the expression of target genes by binding specifically to the 3'UTRs (non-coding region) or coding region of the target gene mRNA. The purpose of this study was to investigate the differential expression of mi RNA in B16F0 and B16F10 cells of malignant melanoma and the effect of overexpression of miR-763 on B16F10 cells [1]. Objective 1. To investigate the differential expression of miRNA between B16F0 and B16F10 cells in malignant melanoma. To investigate the effect and potential molecular mechanism of miR-763 on B16F10 cells of malignant melanoma, the animal model was constructed by using B16F0 and B16F10 cells of malignant melanoma as research objects. To observe the difference in migration, invasion and proliferation of B16F0 and B16F10 cells in vivo and in vitro. To detect the expression of mi RNA in B16F0 and B16F10 cells of malignant melanoma and analyze its difference by bioinformatics. The expression levels of miR-763,miR-431-5p,miR-205-5p,let-7C-2-3p,mi R-26a-1-3pmmiR-28bmmiR-29b-3p and miR-24-2-5p in mouse melanoma cells were detected by Real-time PCR technique to verify the accuracy of miRNA microarray results. 4. Overexpression of miR-763, in B16F10 cells of malignant melanoma and its effect on the proliferation of B16F10 cells were observed. The expression of miR-763 target gene was detected by bioinformatics, and the phosphorylation level of key kinase in each signal pathway was detected by kinase phosphorylation specific antibody. Determine which signaling pathways miR-763 affect B16F10 cells through target genes. Result 1. The ability of migration, invasion and proliferation of malignant melanoma B16F10 cells was higher than that of B16F0 cells in vitro. By bioinformatics analysis, we found that there was significant difference between B16F0 and B16F10 cells in miRNA expression level. 3. Real-time PCR detection showed that miR-763,miR-431-5p,miR-205-5p,let-7C-2-3p expression level of murine melanoma cells down-regulated miR-26a-1-3pmmiR-28btimiR-29b-3p and miR-24-2-5p expression. The results were consistent with the results of microarray. Overexpression of miR-763 could inhibit the proliferation of malignant melanoma B16F10 cells in vitro. 5. 5. MiR-763 down-regulated the expression of potential target gene Grb2, and then affected the change of downstream molecule Cyclin D1. Conclusion 1. Bioinformatics analysis showed that there was a significant difference in miRNA expression between the two cells, which may be the important reason for the difference in migration, invasion and proliferation between B16F10 and B16F0 cells. 2. MiR-763 may regulate the expression of Grb2 by targeting. Inhibit the proliferation of malignant melanoma B16F10 cells.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R739.5

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