一种调控脑胶质瘤表达B7-H3的新MicroRNA-Mir-339-5p的发现及其对脑胶质瘤细胞侵袭作用的研究
发布时间：2018-07-21 20:29
【摘要】：脑胶质瘤是中枢脑系统最常见的恶性肿瘤，约占人颅内肿瘤的45%-60%，侵袭性生长是脑胶质瘤的一个重要特征。肿瘤细胞这种侵袭性生长的行为限制了脑胶质瘤局部治疗如手术切割和放疗等手段的可行性和有效性。因此探索脑胶质瘤病理进程的分子机理和基于此的新靶点业已成为诊断和治疗脑胶质瘤的研究热点。B7-H3是参与肿瘤免疫逃逸的重要负性B7家族分子，已有研究表明，B7-H3分子在诸多肿瘤组织包括脑胶质瘤中存在异常表达，并与肿瘤生物学行为直接相关。但是B7-H3在脑胶质瘤异常表达的调控机制以及其确切的临床意义尚不清楚。MicroRNAs，一种非编码蛋白质的单链小分子RNA(大约22nt)，可在转录后水平调控基因翻译，在脑胶质瘤细胞的生物学特性调控包括细胞增殖、转移侵袭、分化及肿瘤干细胞等方面起重要的作用。有研究发现miR-29a可以靶定B7-H33’-UTR序列，并调控成神经细胞瘤细胞B7-H3的表达。但是否存在其它调控脑胶质瘤B7-H3的microRNA，并且microRNA对B7-H3的调控在脑胶质瘤细胞侵袭中的作用及机制还有待进一步的研究。 鉴此，本课题通过生物学信息方法预测和双荧光素酶报告系统检测相结合，发现miR-339-5p具有调控B7-H3表达的作用，在此基础上分析其在脑胶质瘤组织的表达及临床意义。并且通过体外转染miR-339-5p到脑胶质瘤U87细胞株，探讨miR-339-5p调控B7-H3和脑胶质细胞侵袭的可能作用机制，为进一步探索脑胶质瘤侵袭的分子调控机制及为临床靶向治疗提供理论依据。 本论文分为两个部分： 一miR-339-5p对B7-H3表达的调控和其在脑胶质瘤中的表达及临床意义 【目的】：分析调控B7-H3在脑胶质瘤表达的mircroRNAs，并检测其对脑胶质瘤表达B7-H3的调控机制及临床意义。 【方法】：通过生物信息学的方法预测与B7-H33’-UTR序列存在结合位点的mircroRNAs，并分析脑胶质瘤原位瘤模型的microRNA芯片数据，获得可能调控B7-H3并在脑胶质瘤侵袭中起调控作用microRNAs，进而采用双荧光素酶报告系统验证对B7-H3表达调节作用的miRNAs，实时荧光定量（RT-PCR）的方法分析调控B7-H3的miRNAs在脑胶质瘤不同病理组织中的表达及临床意义。 【结果】：（1）microRNA在线预测（网址http://www.microrna.org）结果显示，在B7-H33’-UTR序列中存在20个不同的microRNA结合位点，通过脑胶质瘤原位瘤模型的microRNA芯片数据分析，得出3条较为特异性的microRNAs：miR-339-5p，miR-185-5p和miR-539-5p。经双荧光素酶报告系统验证，发现miR-339-5p可以直接靶定B7-H33’-UTR序列并下调B7-H3基因的表达，下调效果与已报导的miR-29a对B7-H3调控作用相似。（2）实时定量PCR结果显示，脑胶质瘤组织中miR-339-5p的表达与临床病理分级呈负相关，与B7-H3基因的表达也呈负相关。 【结论】：发现一种调控脑胶质瘤表达B7-H3的新的miRNA—miR-339-5p。初步结果显示，B7-H3在高级别脑胶质瘤组织中呈高表达，而miR-339-5p的表达呈下调，两者呈显著的负相关，由此表明，miR-339-5p可能在脑胶质瘤中参与B7-H3异常表达的调控。 二miR-339-5p调控脑胶质瘤异常表达B7-H3和对肿瘤细胞侵袭性的作用探讨 【目的】：分析miR-339-5p在脑胶质瘤中对B7-H3分子的调节作用；观察miR-339-5p下调B7-H3对脑胶质瘤生物学特性的影响及可能的作用机制。 【方法】：采用基因转染技术将miR-339-5p转入脑胶质瘤细胞株U87细胞，通过流式细胞术和real-time PCR方法检测转染后B7-H3分子的蛋白和mRNA表达水平；通过细胞侵袭实验和CCK-8法检测miR-339-5p对脑胶质瘤细胞侵袭和增殖的影响；同时通过流式细胞术检测转染后U87中CXC趋化因子受体(CXCchemokine receptor)CXCR1、CXCR2、CXCR3、CXCR4和CXCR7的表达。 【结果】：（1）流式细胞术和real-time PCR结果显示，转染miR-339-5p能够抑制脑胶质瘤细胞中B7-H3分子和B7-H3mRNA的表达；（2）细胞侵袭实验结果表明，转染miR-339-5p能够降低脑胶质瘤细胞的侵袭和转移能力，miR-339-5p的抑制剂能够逆转其作用；而对脑胶质瘤细胞的增值并没有影响；（3）转染miR-339-5p的脑胶质瘤细胞对CXCR4的表达有明显的下降，而其它趋化因子受体的表达没有明显改变。 【结论】：miR-339-5p可以下调脑胶质瘤中B7-H3的表达，并在体外实验证实其对脑胶质瘤细胞侵袭有抑制作用，但并不影响其增殖。同时转染miR-339-5p可以下调脑胶质瘤上CXCR4的表达，而CXCR4经microRNA在线软件预测并不存在miR-339-5p的结合位点，由此表明miR-339-5p抑制肿瘤细胞侵袭可能通过下调B7-H3表达来下调CXCR4信号途径，但确切机制还有待进一步探讨。 小结 本论文在研究小组之前的脑胶质瘤原位瘤模型的microRNA芯片数据基础之上，通过生物信息学预测的方法，分析出有3个可能调控B7-H3的microRNAs，经双荧光素酶报告系统验证发现miR-339-5p能够靶定B7-H3基因序列并下调其表达，下调效果与已报导miR-29a相似。进而分析B7-H3和miR-339-5p在脑胶质瘤组织和细胞中的表达发现，，B7-H3的表达与miR-339-5p的表达呈负相关，与脑胶质瘤病理进程呈正相关，由此推测，B7-H3在脑胶质瘤中可能受到miR-339-5p的调控。因此，我们进一步通过体外转染实验发现，miR-339-5p下调了脑胶质瘤细胞株U87上B7-H3的表达，并抑制脑胶质瘤细胞的侵袭，但不影响肿瘤细胞的增殖。同时转染miR-339-5p还显著下调了化学趋化因子受体CXCR4的表达，表明miR-339-5p可能通过B7-H3和CXCR4相互作用影响肿瘤细胞的侵袭。
[Abstract]:Glioma is the most common malignant tumor of the central brain system, which accounts for the 45%-60% of the human intracranial tumor. Invasive growth is an important feature of glioma. The invasive growth behavior of tumor cells restricts the feasibility and effectiveness of local treatment of glioma, such as surgical incision and radiotherapy. The molecular mechanism of the process and the new targets based on this have become a hot spot in the diagnosis and treatment of gliomas..B7-H3 is an important negative B7 family involved in tumor immune escape. It has been shown that B7-H3 molecules have abnormal expression in a number of tumor tissues including gliomas, and are directly related to the biological behavior of tumors. It is the regulatory mechanism of abnormal expression of B7-H3 in glioma and its exact clinical significance is not clear.MicroRNAs, a single strand small molecule RNA (about 22nt), a non coding protein, which can regulate gene translation at post transcriptional level. The regulation of biological characteristics of glioma cells includes cell proliferation, metastasis and invasion, differentiation and tumor stem. It has been found that miR-29a can target B7-H33 '-UTR sequence and regulate the expression of B7-H3 in neurocytoma cells. However, there are other microRNA to regulate the B7-H3 of brain glioma, and the role and mechanism of microRNA in the regulation of B7-H3 in the invasion of glioma cells remains to be further studied.
Therefore, by combining the biological information method prediction and the double Luciferase Report System detection, we found that miR-339-5p has the role of regulating the expression of B7-H3. On this basis, the expression and clinical significance of B7-H3 in brain glioma tissue are analyzed, and through transfection of miR-339-5p into glioma U87 cell lines in vitro, miR-339-5p is used to investigate the regulation of B. The possible mechanism of 7-H3 and glial invasion is to further explore the molecular regulation mechanism of glioma invasion and provide a theoretical basis for clinical targeted therapy.
This paper is divided into two parts:
Regulation of B7-H3 expression by miR-339-5p and its expression in glioma and its clinical significance
[Objective] to analyze and control the expression of mircroRNAs in B7-H3 glioma and detect its regulatory mechanism and clinical significance in the expression of B7-H3 in glioma.
[method]: using the Bioinformatics Method to predict the mircroRNAs of the binding site of the B7-H33 '-UTR sequence, and analyze the microRNA chip data of the tumor in situ tumor model of brain glioma, obtain the possible regulation of B7-H3 and play a regulatory role in the invasion of brain glioma, and then use the dual luciferase reporter system to verify the expression of B7-H3. MiRNAs and real-time fluorescence quantitative (RT-PCR) analysis were used to analyze the expression and clinical significance of miRNAs regulated by B7-H3 in different pathological tissues of glioma.
[results]: (1) the microRNA online prediction (http://www.microrna.org) results showed that there were 20 different microRNA binding sites in the B7-H33 '-UTR sequence and 3 specific microRNAs:miR-339-5p, miR-185-5p and miR-539-5p. via double fluorescence were obtained by the microRNA chip data analysis of the brain glioma in situ tumor model. It was found that miR-339-5p could directly target the B7-H33 '-UTR sequence and downregulate the expression of B7-H3 gene. The downregulation effect was similar to that of the reported miR-29a on B7-H3. (2) real-time quantitative PCR results showed that the expression of miR-339-5p in glioma tissues was negatively correlated with the clinicopathological classification and the expression of the B7-H3 gene. There is also a negative correlation.
[Conclusion]: a new miRNA - miR-339-5p. preliminary result that regulates the expression of B7-H3 in glioma shows that B7-H3 is highly expressed in the high level glioma tissue, and the expression of miR-339-5p is down, and there is a significant negative correlation. Thus, miR-339-5p may be involved in the regulation of abnormal expression of B7-H3 in glioma.
Regulation of B7-H3 expression by two miR-339-5p in glioma and its effect on tumor cell invasiveness
Objective: to analyze the role of miR-339-5p in the regulation of B7-H3 in glioma, and to observe the effect of B7-H3 on the biological characteristics of glioma and the possible mechanism of action of miR-339-5p.
[method]: gene transfection was used to transfer miR-339-5p into glioma cell line U87 cells. The protein and mRNA expression levels of B7-H3 molecules after transfection were detected by flow cytometry and real-time PCR, and the effects of miR-339-5p on the invasion and proliferation of glioma cells were detected by cell invasion test and CCK-8 method. The expression of CXC chemokine receptor (CXCchemokine receptor) CXCR1, CXCR2, CXCR3, CXCR4 and CXCR7 in transfected U87 was detected by flow cytometry.
[results]: (1) the results of flow cytometry and real-time PCR showed that the transfection of miR-339-5p could inhibit the expression of B7-H3 and B7-H3mRNA in glioma cells. (2) the results of cell invasion experiment showed that the transfection of miR-339-5p could reduce the invasion and transfer ability of glioma cells, and the inhibitor of miR-339-5p could reverse its effect. There was no effect on the proliferation of glioma cells. (3) the expression of CXCR4 in the glioma cells transfected with miR-339-5p was significantly decreased, but the expression of other chemokine receptors did not change significantly.
[Conclusion]: miR-339-5p can downregulate the expression of B7-H3 in glioma, and in vitro experiments have confirmed that it can inhibit the invasion of glioma cells, but does not affect the proliferation of glioma cells. At the same time, transfection of miR-339-5p can reduce the expression of CXCR4 on glioma, and CXCR4 does not predict the binding site of miR-339-5p in the line software of microRNA. This indicates that miR-339-5p inhibits tumor cell invasion by down regulating B7-H3 expression and down regulating CXCR4 signaling pathway, but the exact mechanism remains to be further explored.
Summary
In this paper, on the basis of microRNA chip data of the brain glioma in situ tumor model before the team, 3 microRNAs regulating B7-H3 are analyzed by bioinformatics prediction method. The results show that miR-339-5p can target the B7-H3 gene sequence and downregulate its expression by double luciferase reporter system. It was reported that miR-29a was similar. Then the expression of B7-H3 and miR-339-5p in brain glioma tissues and cells showed that the expression of B7-H3 was negatively correlated with the expression of miR-339-5p and was positively related to the pathological process of glioma. Therefore, it is suggested that B7-H3 may be regulated by miR-339-5p in glioma. Therefore, we further transfect in vitro by transfection. It was found that miR-339-5p downregulated the expression of B7-H3 on the glioma cell line U87 and inhibited the invasion of glioma cells, but did not affect the proliferation of tumor cells. At the same time, transfection of miR-339-5p also significantly lowered the expression of chemokine receptor CXCR4, indicating that miR-339-5p may affect the invasion of tumor cells through the interaction of B7-H3 and CXCR4. Attack.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41
本文编号：2136764
[Abstract]:Glioma is the most common malignant tumor of the central brain system, which accounts for the 45%-60% of the human intracranial tumor. Invasive growth is an important feature of glioma. The invasive growth behavior of tumor cells restricts the feasibility and effectiveness of local treatment of glioma, such as surgical incision and radiotherapy. The molecular mechanism of the process and the new targets based on this have become a hot spot in the diagnosis and treatment of gliomas..B7-H3 is an important negative B7 family involved in tumor immune escape. It has been shown that B7-H3 molecules have abnormal expression in a number of tumor tissues including gliomas, and are directly related to the biological behavior of tumors. It is the regulatory mechanism of abnormal expression of B7-H3 in glioma and its exact clinical significance is not clear.MicroRNAs, a single strand small molecule RNA (about 22nt), a non coding protein, which can regulate gene translation at post transcriptional level. The regulation of biological characteristics of glioma cells includes cell proliferation, metastasis and invasion, differentiation and tumor stem. It has been found that miR-29a can target B7-H33 '-UTR sequence and regulate the expression of B7-H3 in neurocytoma cells. However, there are other microRNA to regulate the B7-H3 of brain glioma, and the role and mechanism of microRNA in the regulation of B7-H3 in the invasion of glioma cells remains to be further studied.
Therefore, by combining the biological information method prediction and the double Luciferase Report System detection, we found that miR-339-5p has the role of regulating the expression of B7-H3. On this basis, the expression and clinical significance of B7-H3 in brain glioma tissue are analyzed, and through transfection of miR-339-5p into glioma U87 cell lines in vitro, miR-339-5p is used to investigate the regulation of B. The possible mechanism of 7-H3 and glial invasion is to further explore the molecular regulation mechanism of glioma invasion and provide a theoretical basis for clinical targeted therapy.
This paper is divided into two parts:
Regulation of B7-H3 expression by miR-339-5p and its expression in glioma and its clinical significance
[Objective] to analyze and control the expression of mircroRNAs in B7-H3 glioma and detect its regulatory mechanism and clinical significance in the expression of B7-H3 in glioma.
[method]: using the Bioinformatics Method to predict the mircroRNAs of the binding site of the B7-H33 '-UTR sequence, and analyze the microRNA chip data of the tumor in situ tumor model of brain glioma, obtain the possible regulation of B7-H3 and play a regulatory role in the invasion of brain glioma, and then use the dual luciferase reporter system to verify the expression of B7-H3. MiRNAs and real-time fluorescence quantitative (RT-PCR) analysis were used to analyze the expression and clinical significance of miRNAs regulated by B7-H3 in different pathological tissues of glioma.
[results]: (1) the microRNA online prediction (http://www.microrna.org) results showed that there were 20 different microRNA binding sites in the B7-H33 '-UTR sequence and 3 specific microRNAs:miR-339-5p, miR-185-5p and miR-539-5p. via double fluorescence were obtained by the microRNA chip data analysis of the brain glioma in situ tumor model. It was found that miR-339-5p could directly target the B7-H33 '-UTR sequence and downregulate the expression of B7-H3 gene. The downregulation effect was similar to that of the reported miR-29a on B7-H3. (2) real-time quantitative PCR results showed that the expression of miR-339-5p in glioma tissues was negatively correlated with the clinicopathological classification and the expression of the B7-H3 gene. There is also a negative correlation.
[Conclusion]: a new miRNA - miR-339-5p. preliminary result that regulates the expression of B7-H3 in glioma shows that B7-H3 is highly expressed in the high level glioma tissue, and the expression of miR-339-5p is down, and there is a significant negative correlation. Thus, miR-339-5p may be involved in the regulation of abnormal expression of B7-H3 in glioma.
Regulation of B7-H3 expression by two miR-339-5p in glioma and its effect on tumor cell invasiveness
Objective: to analyze the role of miR-339-5p in the regulation of B7-H3 in glioma, and to observe the effect of B7-H3 on the biological characteristics of glioma and the possible mechanism of action of miR-339-5p.
[method]: gene transfection was used to transfer miR-339-5p into glioma cell line U87 cells. The protein and mRNA expression levels of B7-H3 molecules after transfection were detected by flow cytometry and real-time PCR, and the effects of miR-339-5p on the invasion and proliferation of glioma cells were detected by cell invasion test and CCK-8 method. The expression of CXC chemokine receptor (CXCchemokine receptor) CXCR1, CXCR2, CXCR3, CXCR4 and CXCR7 in transfected U87 was detected by flow cytometry.
[results]: (1) the results of flow cytometry and real-time PCR showed that the transfection of miR-339-5p could inhibit the expression of B7-H3 and B7-H3mRNA in glioma cells. (2) the results of cell invasion experiment showed that the transfection of miR-339-5p could reduce the invasion and transfer ability of glioma cells, and the inhibitor of miR-339-5p could reverse its effect. There was no effect on the proliferation of glioma cells. (3) the expression of CXCR4 in the glioma cells transfected with miR-339-5p was significantly decreased, but the expression of other chemokine receptors did not change significantly.
[Conclusion]: miR-339-5p can downregulate the expression of B7-H3 in glioma, and in vitro experiments have confirmed that it can inhibit the invasion of glioma cells, but does not affect the proliferation of glioma cells. At the same time, transfection of miR-339-5p can reduce the expression of CXCR4 on glioma, and CXCR4 does not predict the binding site of miR-339-5p in the line software of microRNA. This indicates that miR-339-5p inhibits tumor cell invasion by down regulating B7-H3 expression and down regulating CXCR4 signaling pathway, but the exact mechanism remains to be further explored.
Summary
In this paper, on the basis of microRNA chip data of the brain glioma in situ tumor model before the team, 3 microRNAs regulating B7-H3 are analyzed by bioinformatics prediction method. The results show that miR-339-5p can target the B7-H3 gene sequence and downregulate its expression by double luciferase reporter system. It was reported that miR-29a was similar. Then the expression of B7-H3 and miR-339-5p in brain glioma tissues and cells showed that the expression of B7-H3 was negatively correlated with the expression of miR-339-5p and was positively related to the pathological process of glioma. Therefore, it is suggested that B7-H3 may be regulated by miR-339-5p in glioma. Therefore, we further transfect in vitro by transfection. It was found that miR-339-5p downregulated the expression of B7-H3 on the glioma cell line U87 and inhibited the invasion of glioma cells, but did not affect the proliferation of tumor cells. At the same time, transfection of miR-339-5p also significantly lowered the expression of chemokine receptor CXCR4, indicating that miR-339-5p may affect the invasion of tumor cells through the interaction of B7-H3 and CXCR4. Attack.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41
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