本文選題：MTA1 + miR-183��； 參考：《鄭州大學》2017年碩士論文

【摘要】：研究背景骨肉瘤(osteosarcoma)是一種侵襲性的惡性腫瘤,從間質(zhì)細胞系發(fā)展而來,具有成骨細胞分化的特點,且極易發(fā)生肺轉(zhuǎn)移,同時它也是兒童癌癥中生存率較低的腫瘤之一,患者的五年生存率僅為65%-70%,嚴重威脅著青少年的健康。近年來在基因分子的水平對疾病進行研究越來越受關注,因此從基因分子水平上研究骨肉瘤的發(fā)病機制及防治,對其未來的診斷和治療有更積極的作用。腫瘤轉(zhuǎn)移相關基因(metastasis associated gene 1,MTA1)是核小體重塑和組蛋白去乙酰化酶復合物(Nucleosome remodeling and histone deacetylase,NuRD)的重要組成部分。大量的研究發(fā)現(xiàn)MTA1在多種腫瘤中高表達,并促進腫瘤的惡化,參與腫瘤的增殖、侵襲、遷移、凋亡和細胞周期等過程。有研究報道m(xù)iR-183在骨肉瘤細胞系MG63、U2-OS、Saos-2和HOS中均異常低表達,并能靶向埃茲基因(Ezrin,其蛋白又稱細胞骨架結(jié)合蛋白)抑制MG63細胞的侵襲和遷移,多種生物信息學軟件預測顯示miR-183可能與MTA1存在靶向結(jié)合區(qū)。因此,探討miR-183靶向調(diào)控MTA1基因,對骨肉瘤增殖、侵襲、遷移及凋亡的影響,將為骨肉瘤發(fā)病機制研究及臨床防治提供新的思路和方向。miR-183靶向調(diào)控MTA1基因?qū)侨饬黾毎挠绊懠捌浞肿幼饔脵C制的研究尚未見報道。研究目的本研究將通過實驗驗證MTA1是miR-183的直接靶基因,轉(zhuǎn)染miR-183mimic至骨肉瘤細胞系MG63,觀察miR-183對MTA1的靶向調(diào)控作用,及對MG63細胞增殖、侵襲、遷移與凋亡的影響,由此尋找骨肉瘤研究及防治新的靶點和策略。研究方法1.收集標本:自2014年9月至2016年6月從鄭州大學第一附屬醫(yī)院收集了25例骨肉瘤患者的癌組織及與其配對的癌旁組織,并置于-80℃冰箱中長期保存。采用實時熒光定量PCR技術檢測已收集標本中miR-183及MTA1基因的mRNA表達水平,并通過免疫印跡Western blot檢測MTA1蛋白的表達并分析結(jié)果。2.采用TargetScan和miRanda等生物信息學軟件預測靶向MTA1的microRNA。3.構(gòu)建含有MTA1 3’端非翻譯區(qū)域(3’UTR)的野生型和突變型載體,運用雙熒光素酶報告基因?qū)嶒烌炞CMTA1與miR-183的靶向作用。4.RT-qPCR法檢測正常成骨細胞株hFOB1.19與人骨肉瘤細胞系MG63中miR-183與MTA1基因的mRNA表達水平。5.體外合成miR-183 mimic和miR-183 negative control,采用脂質(zhì)體將其分別轉(zhuǎn)染到各組人骨肉瘤細胞系MG63中。實驗分為3組,miR-183 mimic組:轉(zhuǎn)染miR-183 mimic;NC組:轉(zhuǎn)染miR-183 negative control;Control組:僅加脂質(zhì)體。6.CCK-8實驗檢測骨肉瘤細胞系MG63的增殖情況。7.細胞劃痕實驗檢測骨肉瘤細胞系MG63遷移能力改變情況。8.Transwell實驗檢測骨肉瘤細胞系MG63侵襲能力改變的情況。9.流式細胞術及TUNEL實驗檢測骨肉瘤細胞系MG63凋亡的情況。10.免疫印跡Western blot法檢測轉(zhuǎn)染后MTA1蛋白的表達。實驗結(jié)果1.RT-qPCR檢測結(jié)果顯示,骨肉瘤組織中miR-183的表達量顯著低于其在癌旁組織中的表達(P0.01),而MTA1基因的mRNA在骨肉瘤中的表達量明顯高于其在癌旁組織中的表達(P0.01)。Western blot的結(jié)果顯示,與癌旁組織比較,MTA1蛋白在癌組織中呈現(xiàn)高表達。2.雙熒光素酶報告基因?qū)嶒炞C明miR-183與MTA1基因的3'UTR區(qū)域結(jié)合,與生物信息學軟件預測結(jié)果一致,表明MTA1是miR-183的靶基因。3.RT-qPCR結(jié)果顯示,在骨肉瘤細胞系MG63中miR-183的表達顯著低于成骨細胞株hFOB1.19中的表達(P0.01),而MTA1基因的mRNA表達則明顯高于其在成骨細胞株hFOB1.19中的表達(P0.01)。4.轉(zhuǎn)染后,miR-183 mimic組的miR-183表達量明顯高于NC組與Control組,差異具有統(tǒng)計學意義(P0.01)。表明miR-183 mimic轉(zhuǎn)染骨肉瘤細胞成功。5.CCK-8結(jié)果顯示,與NC組和Control組比較,轉(zhuǎn)染miR-183 mimic組的骨肉瘤細胞在相同時間點的OD490吸光值有所下降,差異具有統(tǒng)計學意義(P0.05),而NC組與Control組比較則無顯著差異(P0.05)。6.劃痕實驗結(jié)果顯示,miR-183組細胞的遷移能力較NC組與Control組低,而NC組與Control組比較則遷移能力無明顯差異。7.Transwell實驗結(jié)果表明,miR-183組的骨肉瘤MG63細胞穿過基底膜的細胞數(shù)明顯低于NC組與Control組,差異具有統(tǒng)計學意義(P0.01),但NC組與Control組穿過基底膜的細胞數(shù)量無明顯差異(P0.05)。8.流式細胞術與TUNEL實驗結(jié)果顯示,與NC組和Control組比較,miR-183組的骨肉瘤MG63細胞的凋亡率明顯升高,差異具有統(tǒng)計學意義(P0.05),而NC組與Control組比較,MG63細胞的凋亡率無顯著差異(P0.05)。9.免疫印跡Western blot檢測結(jié)果顯示,轉(zhuǎn)染miR-183 mimic后,與NC組和Control組比較,MTA1蛋白表達下降,而NC組與Control組比較,MTA1表達無顯著差異。結(jié)論1.骨肉瘤組織與細胞系MG63中miR-183低表達,MTA1高表達。2.miR-183可以通過靶向作用MTA1基因的3’UTR區(qū)域降低其轉(zhuǎn)錄表達,并發(fā)揮調(diào)控作用。3.過表達miR-183可能通過降低靶基因MTA1表達而有效抑制骨肉瘤細胞系MG63的增殖、侵襲、遷移并促進其細胞凋亡。
[Abstract]:Background osteosarcoma (osteosarcoma) is an invasive malignant tumor, developed from interstitial cell lines, characterized by osteoblast differentiation, and highly susceptible to lung metastasis. At the same time, it is also one of the low survival rates in children's cancer. The five year survival rate of patients is only 65%-70%, which threatens the health of young people in recent years. It is becoming more and more concerned about the study of disease at the level of gene molecules, so it is more active to study the pathogenesis and prevention of osteosarcoma from the gene molecular level. The tumor metastasis related gene (metastasis associated gene 1, MTA1) is the recombination of nucleosome remodeling and histone deacetylase. An important part of Nucleosome remodeling and histone deacetylase (NuRD). A large number of studies have found that MTA1 is highly expressed in a variety of tumors and promotes the deterioration of the tumor and participates in the process of tumor proliferation, invasion, migration, apoptosis and cell cycle. It is reported that miR-183 is different in the osteosarcoma cell lines MG63, U2-OS, Saos-2, and HOS. It is often low expressed and can target Ezi gene (Ezrin, its protein also called cytoskeleton binding protein) to inhibit the invasion and migration of MG63 cells. A variety of bioinformatics software predicts that miR-183 may have a target binding zone with MTA1. Therefore, the effect of miR-183 targeting MTA1 gene on proliferation, invasion, migration and apoptosis of osteosarcoma will be discussed. The study of the pathogenesis and clinical prevention of osteosarcoma provides new ideas and directions for the study of the effect of.MiR-183 targeting MTA1 gene on osteosarcoma cells and its molecular mechanism. The purpose of this study is to verify that MTA1 is a direct target gene of miR-183 and transfected from miR-183mimic to osteosarcoma cell line MG63. The effects of miR-183 on the targeting of MTA1, and the effects on proliferation, invasion, migration and apoptosis of MG63 cells were observed, and the new targets and strategies for the study and prevention of osteosarcoma were sought. Methods 1. collection of specimens from the First Affiliated Hospital of Zhengzhou University from September 2014 to June 2016 were collected from the cancer tissues of 25 patients with osteosarcoma and their pairing. The para cancerous tissue was preserved in the -80 C refrigerator for a long time. The mRNA expression level of the miR-183 and MTA1 genes in the collected specimens was detected by real-time fluorescence quantitative PCR, and the expression of MTA1 protein was detected by immunoblotting Western blot and the results of.2. were predicted by the bioinformatics software such as TargetScan and miRanda. ORNA.3. constructs a wild type and mutant vector containing MTA1 3 'untranslated region (3' UTR), and uses double luciferase reporter gene test to verify the target action of MTA1 and miR-183 to detect the mRNA expression level.5. in normal osteoblast cell line hFOB1.19 and human osteosarcoma cell line MG63, miR-183 and MTA1 genes in vitro synthesis 3 mimic and miR-183 negative control were transfected into the human osteosarcoma cell line MG63 respectively with liposomes. The experiment was divided into 3 groups, and the miR-183 mimic group was transfected with miR-183 mimic; NC group: transfected to miR-183 negative. Changes in the migration ability of osteosarcoma cell line MG63.8.Transwell test detection of invasion ability of osteosarcoma cell line MG63.9. flow cytometry and TUNEL test detection of apoptosis in osteosarcoma cell line MG63.10. immunoblotting Western blot method to detect the expression of MTA1 protein after transfection. Experimental results 1.RT-qPCR detection junction The results showed that the expression of miR-183 in osteosarcoma was significantly lower than that in paracancerous tissue (P0.01), while the expression of mRNA in osteosarcoma was significantly higher than that of its expression in the paracancerous tissues (P0.01).Western blot, which showed that MTA1 protein expressed a high expression of.2. double fluorescein in the tissue of the cancer tissue compared with that of the para cancerous tissue. The enzyme reporter gene experiment showed that miR-183 was associated with the 3'UTR region of MTA1 gene, which was consistent with the prediction results of bioinformatics software, indicating that MTA1 was the target gene of miR-183 and that the expression of miR-183 in the osteosarcoma cell line MG63 was significantly lower than the expression of the osteoblast strain hFOB1.19 (P0.01), while mRNA expression of the MTA1 gene was clear. The expression of miR-183 in the miR-183 mimic group was significantly higher than that in the osteoblast cell line hFOB1.19 (P0.01).4.. The difference was statistically significant (P0.01). The results showed that the miR-183 mimic transfected osteosarcoma cells were successfully transfected with the osteosarcoma cells. The OD490 absorption value of the tumor cells decreased at the same time point, the difference was statistically significant (P0.05), but there was no significant difference between the NC group and the Control group (P0.05).6. scratch test results showed that the migration ability of the miR-183 group was lower than that of the NC group and the Control group, while the NC and Control group had no significant difference in the migration capacity. The experimental results showed that the number of osteosarcoma MG63 cells passing through the basement membrane in the miR-183 group was significantly lower than that in the NC group and the Control group. The difference was statistically significant (P0.01), but there was no significant difference in the number of cells passing through the basement membrane in the NC group and Control group (P0.05).8. flow cytometry and TUNEL experimental results, compared with NC and Control groups. The apoptosis rate of osteosarcoma MG63 cells in the group was significantly higher, the difference was statistically significant (P0.05), but there was no significant difference in the apoptosis rate of MG63 cells in the group NC and the Control group (P0.05).9. immunoblotting Western blot detection results showed that after the miR-183 mimic, the expression of the protein decreased with the NC group and the group. Conclusion there is no significant difference in MTA1 expression. Conclusion 1. osteosarcoma tissue and cell line MG63 are low expression of miR-183, MTA1 high expression.2.miR-183 can reduce its transcriptional expression through the 3 'UTR region targeting the target action of MTA1 gene, and play the regulatory role of.3. overexpression miR-183 may effectively inhibit the osteosarcoma cell line MG by reducing the target gene MTA1 expression. 63 of the proliferation, invasion, migration, and promotion of cell apoptosis.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R738.1

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