本文選題：骨肉瘤　切入點(diǎn)：miR-143　出處：《山東大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景骨肉瘤是最常見的起源于骨骼系統(tǒng)的原發(fā)性惡性骨腫瘤,特點(diǎn)是高發(fā)生率,高度惡性,高轉(zhuǎn)移率,因此預(yù)后較差,腫瘤的發(fā)生給患者家庭和社會(huì)帶來了很大的花費(fèi)和負(fù)擔(dān)。近20年以來,盡管國際諸多研究機(jī)構(gòu)進(jìn)行了大量的努力,但骨肉瘤患者生存率依然停滯不前,仿佛進(jìn)入了骨肉瘤治療的瓶頸期。同時(shí)化療耐藥的出現(xiàn)也讓化療方案的推廣舉步維艱,激進(jìn)的化療方案的副作用讓很多骨肉瘤患者難以堅(jiān)持完成治療。為此,對骨肉瘤的深入研究迫在眉睫。近年來有關(guān)微小RNA(microRNA,miRNA)的研究成為腫瘤領(lǐng)域研究的熱點(diǎn)。在人類多種癌細(xì)胞中都發(fā)現(xiàn)了miRNA的表達(dá)異常,miRNA既可以促進(jìn)腫瘤生長而起到癌基因的作用,也可抑制腫瘤生長而起到抑癌基因的作用。最近的研究提示miR-143參與了腫瘤細(xì)胞的發(fā)生、增殖、分化、遷移侵襲、凋亡等生理過程,但總體而言miR-143的作用主要起到類似抑癌基因的功能。然而,針對miR-143的研究大多集中于上皮組織來源的癌細(xì)胞,對于間葉源性的惡性腫瘤細(xì)胞尤其是骨肉瘤中miR-143的作用報(bào)道較少。我們在本研究通過檢測骨肉瘤組織和鄰近正常組織中miR-143的表達(dá),并通過生物信息學(xué)技術(shù)預(yù)測靶基因后檢測靶基因的表達(dá)情況。通過細(xì)胞轉(zhuǎn)染上調(diào)miR-143及特異性抑制序列來敲除miR-143的作用,觀察體外培養(yǎng)的骨肉瘤細(xì)胞系中靶基因的表達(dá)情況,并檢測miR-143對骨肉瘤細(xì)胞凋亡的影響,以期闡明miR-143在骨肉瘤中的作用,為靶向治療骨肉瘤提供可能的思路。研究目的1.觀察miR-143在骨肉瘤組織中的表達(dá)水平。2.觀察miR-143對骨肉瘤細(xì)胞的影響。3.尋找miR-143的靶基因,闡明miR-143對靶基因的調(diào)節(jié)作用。研究方法1.收集骨肉瘤病人的標(biāo)本,應(yīng)用RT-PCR技術(shù)檢測骨肉瘤標(biāo)本及周圍正常組織中miR-143的表達(dá)水平,比較miR-143在骨肉瘤和正常組織中的表達(dá)差異。2.通過生物信息學(xué)方法及軟件預(yù)測miR-143的靶基因?yàn)锽cl-2,RT-PCR技術(shù)檢測骨肉瘤標(biāo)本及周圍正常組織中Bcl-2的mRNA表達(dá)水平,應(yīng)用免疫組織化學(xué)技術(shù)和Western Blot方法檢測骨肉瘤標(biāo)本及周圍正常組織中Bcl-2的蛋白表達(dá)水平。3.利用脂質(zhì)體2000將miR-143模擬物及陰性對照序列瞬時(shí)轉(zhuǎn)染MG63骨肉瘤細(xì)胞系,建立過表達(dá)miR-143的骨肉瘤細(xì)胞系。4.利用CCK-8檢測不同時(shí)間段過表達(dá)miR-143的骨肉瘤細(xì)胞系及陰性對照組細(xì)胞的細(xì)胞活性,觀察miR-143對骨肉瘤細(xì)胞增殖能力的影響。5.利用Transwell方法檢測不同時(shí)間段過表達(dá)miR-143的骨肉瘤細(xì)胞系及陰性對照組細(xì)胞的細(xì)胞穿膜數(shù)量,觀察miR-143對骨肉瘤細(xì)胞遷移能力的影響。6.預(yù)測miR-143的靶基因,并構(gòu)建相應(yīng)靶基因的野生型和突變型熒光素酶報(bào)告基因質(zhì)粒,與miR-143模擬物及陰性對照共轉(zhuǎn)染MG63骨肉瘤細(xì)胞,檢測熒光素酶活性,判斷miR-143的靶基因。7.轉(zhuǎn)染miR-143模擬物及抑制物后通過RT-PCR和Western Blot技術(shù)檢測靶基因的mRNA水平和蛋白表達(dá)水平的變化,分析miR-143對靶基因的調(diào)節(jié)作用。8.將miR-143模擬物及陰性對照序列轉(zhuǎn)染MG63骨肉瘤細(xì)胞系后,應(yīng)用Annexin V/PI雙染色后通過流式細(xì)胞術(shù)檢測細(xì)胞凋亡比率的變化,來觀察miR-143對骨肉瘤細(xì)胞凋亡的影響。實(shí)驗(yàn)結(jié)果1.12例骨肉瘤標(biāo)本中的miR-143表達(dá)水平同鄰近正常組織中相比明顯下降,只有鄰近正常組織的56%。2.通過TargetScan軟件預(yù)測miR-143的靶基因可能是Bcl-2。免疫組織化學(xué)方法發(fā)現(xiàn)骨肉瘤標(biāo)本中的Bcl-2表達(dá)水平較正常組織相比明顯升高。RT-PCR檢測發(fā)現(xiàn)Bcl-2的mRNA表達(dá)水平較正常組織明顯升高,Western Blot結(jié)果顯示骨肉瘤組織中Bcl-2的蛋白表達(dá)水平明顯增加,約為鄰近正常組織的1.9倍。3.通過細(xì)胞轉(zhuǎn)染的方法成功建立miR-143高表達(dá)的骨肉瘤細(xì)胞系。4.轉(zhuǎn)染miR-143 48h及72h時(shí),骨肉瘤細(xì)胞活性明顯下降,分別為對照組的78.6%和65.4%。miR-143能夠抑制骨肉瘤細(xì)胞的增殖。5.轉(zhuǎn)染miR-143 72h時(shí),骨肉瘤細(xì)胞的穿出Transwell小室的細(xì)胞數(shù)量明顯減少。miR-143能夠抑制骨肉瘤細(xì)胞的遷移能力。6.成功構(gòu)建候選靶基因Bcl-2的熒光素酶報(bào)告基因質(zhì)粒,共轉(zhuǎn)染miR-143和Bcl-2報(bào)告基因后熒光素酶失活,證實(shí)Bcl-2是miR-143的靶基因。7.過表達(dá)miR-143的骨肉瘤細(xì)胞中,Bcl-2的mRNA明顯下調(diào),蛋白表達(dá)水平明顯降低,而應(yīng)用miR-143抑制物可上調(diào)Bcl-2的蛋白表達(dá)。miR-143通過抑制Bcl-2表達(dá)發(fā)揮作用。8.過表達(dá)miR-143明顯增加骨肉瘤細(xì)胞凋亡比率,其機(jī)制可能與miR-143介導(dǎo)的Bcl-2下調(diào)有關(guān)。結(jié)論1.骨肉瘤組織中miR-143的表達(dá)降低,而Bcl-2的表達(dá)水平增高。2.過表達(dá)miR-143能夠抑制骨肉瘤細(xì)胞的增殖與遷移。3.Bcl-2可能是miR-143的靶基因之一,miR-143可介導(dǎo)Bcl-2的表達(dá)水平。4.miR-143可誘導(dǎo)骨肉瘤細(xì)胞凋亡,其機(jī)制可能與miR-143介導(dǎo)的Bcl-2下調(diào)有關(guān)。
[Abstract]:Background osteosarcoma is the most common origin in the skeletal system of primary malignant bone tumor, characterized by high incidence, high malignancy, high transfer rate, so the prognosis is poor, cancer has brought great cost burden for patients and families and the society. Over the past 20 years, as many international research institutions a great deal of effort, but patients with osteosarcomasurvival remains stagnant, as if into the bottleneck period of treatment of osteosarcoma. At the same time the chemotherapy resistant also make chemotherapy promotion difficult, side effects of chemotherapy of radical so many patients with osteosarcoma is difficult to adhere to the completion of treatment. Therefore, in-depth study of osteosarcoma imminent. In recent years on the micro RNA (microRNA, miRNA) research has become a hot research field of tumor. In many human cancer cells are found in the abnormal expression of miRNA, miRNA can promote tumor The growth and play the role of oncogenes can also inhibit tumor growth and play the role of tumor suppressor gene. Recent studies suggest that miR-143 is involved in tumor cell proliferation, differentiation, migration, invasion, apoptosis and other physiological processes, but the overall effect of miR-143 mainly plays like the function of tumor suppressor gene. However, Research on miR-143 mostly concentrated in epithelial cancer cells for malignant mesenchymal tumor cells especially miR-143 in osteosarcoma effect reported less. In this study we through the detection of the expression of miR-143 in osteosarcoma tissues and adjacent normal tissues, and through bioinformatics prediction of target gene to detect the expression of target gene. The cells transfected with the up regulation of miR-143 and sequence specific inhibition to knock out the role of miR-143, the target gene of human osteosarcoma cell line in vitro expression of the situation, and To study the effect of miR-143 on apoptosis of osteosarcoma cells, to elucidate the role of miR-143 in osteosarcoma, as the target may provide ideas to the treatment of osteosarcoma. The purpose of the study is to observe 1. miR-143 in osteosarcoma and the expression level of.2. effect of miR-143 on osteosarcoma cell.3. targeting gene for miR-143, regulation clarify miR-143 on target gene. The method of collecting 1. patients with osteosarcoma specimens, the expression level of miR-143 RT-PCR was used to detect osteosarcoma specimens and adjacent normal tissues, miR-143 in osteosarcoma and normal tissues in the expression of the target base difference of.2. by bioinformatics methods and software for predicting miR-143 because of Bcl-2, Bcl-2 RT-PCR detection of osteosarcoma specimens and surrounding normal tissues, the expression level of mRNA, immunohistochemistry and Western Blot method for detection of osteosarcoma specimens and normal group The expression of Bcl-2 in protein level of.3. fabric by Lipofectamine 2000 analogue of miR-143 and negative control sequence transient transfection of human osteosarcoma MG63 cells, the establishment of the over expression of miR-143 osteosarcoma cell line CCK-8 was detected by.4. in different time after expression of cell activity in osteosarcoma cell line miR-143 and negative control group cells, observe the effect of miR-143 on osteosarcoma cell proliferation of.5. was detected by Transwell in different time after expression of osteosarcoma cell line miR-143 and negative control group cells penetrating quantity, the effect of miR-143 on osteosarcoma cell migration effect of.6. can force prediction of miR-143 target gene, and construct the corresponding wild-type and mutant luciferase gene reporter plasmid were co transfected into MG63 osteosarcoma cells with miR-143 mimics and negative, detection of luciferase activity,.7. gene was transfected into miR miR-143 The level of mRNA and expression of target gene RT-PCR and Western Blot technology, the expression of -143 mimics and inhibitors, miR-143 analysis on target gene regulation of.8. analogue of miR-143 and negative control sequence were transfected into MG63 osteosarcoma cell lines, to detect the changes of cell apoptosis rate by flow cytometry by Annexin V/ PI staining to observe the effect of miR-143 on apoptosis of osteosarcoma cells. Results expression in 1.12 cases of osteosarcoma specimens miR-143 levels in adjacent normal tissues compared with adjacent normal tissue decreased significantly, only 56%.2. by TargetScan software to predict the target gene miR-143 may be Bcl-2. immunohistochemistry found in osteosarcoma specimens the expression level of Bcl-2 was significantly higher than normal tissue.RT-PCR detected Bcl-2 mRNA expression level increased significantly compared with the normal tissue, Western Bl Ot results showed that the expression level of Bcl-2 protein in osteosarcoma was significantly increased, about 1.9 times the.3. of the adjacent normal tissue by transfected cells successfully established the high expression of miR-143 in osteosarcoma cell line.4. transfected with miR-143 48h and 72h, osteosarcoma cell activity decreased significantly, the control group respectively 78.6% and 65.4%.miR-143 can inhibit the growth of osteosarcoma cells.5. transfected with miR-143 luciferase reporter gene plasmid 72h, the number of cells through the Transwell cell of osteosarcoma cells significantly reduced.MiR-143 can inhibit osteosarcoma cell migration.6. successfully constructed candidate target genes of Bcl-2, miR-143 and Bcl-2 were co transfected with reporter gene luciferase inactivation, confirmed that Bcl-2 is the target gene of.7. miR-143 the expression of miR-143 in osteosarcoma cells, Bcl-2 mRNA down-regulation of protein expression level decreased significantly, and the inhibition effect of miR-143. Can up regulate the protein expression of Bcl-2.MiR-143 through inhibiting the expression of Bcl-2 play a role in.8. over expression of miR-143 significantly increased the apoptosis rate of osteosarcoma cells, and its mechanism may be mediated by down-regulation of miR-143 Bcl-2. Conclusion the expression of miR-143 decreased 1. in osteosarcoma, and the expression level of Bcl-2 increased in.2. overexpression of miR-143 could inhibit the proliferation and migration of.3.Bcl-2 osteosarcoma cells may be one of the target genes of miR-143, miR-143 expression level of.4.miR-143 mediated Bcl-2 can induce apoptosis of osteosarcoma cells, and its mechanism may be mediated by down-regulation of miR-143 Bcl-2.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R738.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 周云飛;龍新華;張志宏;陳宣銀;劉家明;黃山虎;劉志禮;;miR-129-5p靶向調(diào)控VCP抑制骨肉瘤細(xì)胞體外遷徙侵襲[J];臨床與實(shí)驗(yàn)病理學(xué)雜志;2014年10期

2 Xiao-Li Wu;Bin Cheng;Pei-Yuan Li;Huan-Jun Huang;Qiu Zhao;Zi-Li Dan;Dean Tian;Peng Zhang;;MicroRNA-143 suppresses gastric cancer cell growth and induces apoptosis by targeting COX-2[J];World Journal of Gastroenterology;2013年43期

3 劉兵;;Bcl-2和Caspase-3在骨肉瘤中的表達(dá)及其臨床意義[J];國際檢驗(yàn)醫(yī)學(xué)雜志;2009年06期

，

本文編號：1627765