一種調(diào)控腦膠質(zhì)瘤表達B7-H3的新MicroRNA-Mir-339-5p的發(fā)現(xiàn)及其對腦膠質(zhì)瘤細胞侵襲作用的研究
發(fā)布時間：2018-07-21 20:29
【摘要】：腦膠質(zhì)瘤是中樞腦系統(tǒng)最常見的惡性腫瘤，約占人顱內(nèi)腫瘤的45%-60%，侵襲性生長是腦膠質(zhì)瘤的一個重要特征。腫瘤細胞這種侵襲性生長的行為限制了腦膠質(zhì)瘤局部治療如手術切割和放療等手段的可行性和有效性。因此探索腦膠質(zhì)瘤病理進程的分子機理和基于此的新靶點業(yè)已成為診斷和治療腦膠質(zhì)瘤的研究熱點。B7-H3是參與腫瘤免疫逃逸的重要負性B7家族分子，已有研究表明，B7-H3分子在諸多腫瘤組織包括腦膠質(zhì)瘤中存在異常表達，并與腫瘤生物學行為直接相關。但是B7-H3在腦膠質(zhì)瘤異常表達的調(diào)控機制以及其確切的臨床意義尚不清楚。MicroRNAs，一種非編碼蛋白質(zhì)的單鏈小分子RNA(大約22nt)，可在轉(zhuǎn)錄后水平調(diào)控基因翻譯，在腦膠質(zhì)瘤細胞的生物學特性調(diào)控包括細胞增殖、轉(zhuǎn)移侵襲、分化及腫瘤干細胞等方面起重要的作用。有研究發(fā)現(xiàn)miR-29a可以靶定B7-H33’-UTR序列，并調(diào)控成神經(jīng)細胞瘤細胞B7-H3的表達。但是否存在其它調(diào)控腦膠質(zhì)瘤B7-H3的microRNA，并且microRNA對B7-H3的調(diào)控在腦膠質(zhì)瘤細胞侵襲中的作用及機制還有待進一步的研究。 鑒此，本課題通過生物學信息方法預測和雙熒光素酶報告系統(tǒng)檢測相結合，發(fā)現(xiàn)miR-339-5p具有調(diào)控B7-H3表達的作用，在此基礎上分析其在腦膠質(zhì)瘤組織的表達及臨床意義。并且通過體外轉(zhuǎn)染miR-339-5p到腦膠質(zhì)瘤U87細胞株，探討miR-339-5p調(diào)控B7-H3和腦膠質(zhì)細胞侵襲的可能作用機制，為進一步探索腦膠質(zhì)瘤侵襲的分子調(diào)控機制及為臨床靶向治療提供理論依據(jù)。 本論文分為兩個部分： 一miR-339-5p對B7-H3表達的調(diào)控和其在腦膠質(zhì)瘤中的表達及臨床意義 【目的】：分析調(diào)控B7-H3在腦膠質(zhì)瘤表達的mircroRNAs，并檢測其對腦膠質(zhì)瘤表達B7-H3的調(diào)控機制及臨床意義。 【方法】：通過生物信息學的方法預測與B7-H33’-UTR序列存在結合位點的mircroRNAs，并分析腦膠質(zhì)瘤原位瘤模型的microRNA芯片數(shù)據(jù)，獲得可能調(diào)控B7-H3并在腦膠質(zhì)瘤侵襲中起調(diào)控作用microRNAs，進而采用雙熒光素酶報告系統(tǒng)驗證對B7-H3表達調(diào)節(jié)作用的miRNAs，實時熒光定量（RT-PCR）的方法分析調(diào)控B7-H3的miRNAs在腦膠質(zhì)瘤不同病理組織中的表達及臨床意義。 【結果】：（1）microRNA在線預測（網(wǎng)址http://www.microrna.org）結果顯示，在B7-H33’-UTR序列中存在20個不同的microRNA結合位點，通過腦膠質(zhì)瘤原位瘤模型的microRNA芯片數(shù)據(jù)分析，得出3條較為特異性的microRNAs：miR-339-5p，miR-185-5p和miR-539-5p。經(jīng)雙熒光素酶報告系統(tǒng)驗證，發(fā)現(xiàn)miR-339-5p可以直接靶定B7-H33’-UTR序列并下調(diào)B7-H3基因的表達，下調(diào)效果與已報導的miR-29a對B7-H3調(diào)控作用相似。（2）實時定量PCR結果顯示，腦膠質(zhì)瘤組織中miR-339-5p的表達與臨床病理分級呈負相關，與B7-H3基因的表達也呈負相關。 【結論】：發(fā)現(xiàn)一種調(diào)控腦膠質(zhì)瘤表達B7-H3的新的miRNA—miR-339-5p。初步結果顯示，B7-H3在高級別腦膠質(zhì)瘤組織中呈高表達，而miR-339-5p的表達呈下調(diào)，兩者呈顯著的負相關，由此表明，miR-339-5p可能在腦膠質(zhì)瘤中參與B7-H3異常表達的調(diào)控。 二miR-339-5p調(diào)控腦膠質(zhì)瘤異常表達B7-H3和對腫瘤細胞侵襲性的作用探討 【目的】：分析miR-339-5p在腦膠質(zhì)瘤中對B7-H3分子的調(diào)節(jié)作用；觀察miR-339-5p下調(diào)B7-H3對腦膠質(zhì)瘤生物學特性的影響及可能的作用機制。 【方法】：采用基因轉(zhuǎn)染技術將miR-339-5p轉(zhuǎn)入腦膠質(zhì)瘤細胞株U87細胞，通過流式細胞術和real-time PCR方法檢測轉(zhuǎn)染后B7-H3分子的蛋白和mRNA表達水平；通過細胞侵襲實驗和CCK-8法檢測miR-339-5p對腦膠質(zhì)瘤細胞侵襲和增殖的影響；同時通過流式細胞術檢測轉(zhuǎn)染后U87中CXC趨化因子受體(CXCchemokine receptor)CXCR1、CXCR2、CXCR3、CXCR4和CXCR7的表達。 【結果】：（1）流式細胞術和real-time PCR結果顯示，轉(zhuǎn)染miR-339-5p能夠抑制腦膠質(zhì)瘤細胞中B7-H3分子和B7-H3mRNA的表達；（2）細胞侵襲實驗結果表明，轉(zhuǎn)染miR-339-5p能夠降低腦膠質(zhì)瘤細胞的侵襲和轉(zhuǎn)移能力，miR-339-5p的抑制劑能夠逆轉(zhuǎn)其作用；而對腦膠質(zhì)瘤細胞的增值并沒有影響；（3）轉(zhuǎn)染miR-339-5p的腦膠質(zhì)瘤細胞對CXCR4的表達有明顯的下降，而其它趨化因子受體的表達沒有明顯改變。 【結論】：miR-339-5p可以下調(diào)腦膠質(zhì)瘤中B7-H3的表達，并在體外實驗證實其對腦膠質(zhì)瘤細胞侵襲有抑制作用，但并不影響其增殖。同時轉(zhuǎn)染miR-339-5p可以下調(diào)腦膠質(zhì)瘤上CXCR4的表達，而CXCR4經(jīng)microRNA在線軟件預測并不存在miR-339-5p的結合位點，由此表明miR-339-5p抑制腫瘤細胞侵襲可能通過下調(diào)B7-H3表達來下調(diào)CXCR4信號途徑，但確切機制還有待進一步探討。 小結 本論文在研究小組之前的腦膠質(zhì)瘤原位瘤模型的microRNA芯片數(shù)據(jù)基礎之上，通過生物信息學預測的方法，分析出有3個可能調(diào)控B7-H3的microRNAs，經(jīng)雙熒光素酶報告系統(tǒng)驗證發(fā)現(xiàn)miR-339-5p能夠靶定B7-H3基因序列并下調(diào)其表達，下調(diào)效果與已報導miR-29a相似。進而分析B7-H3和miR-339-5p在腦膠質(zhì)瘤組織和細胞中的表達發(fā)現(xiàn)，，B7-H3的表達與miR-339-5p的表達呈負相關，與腦膠質(zhì)瘤病理進程呈正相關，由此推測，B7-H3在腦膠質(zhì)瘤中可能受到miR-339-5p的調(diào)控。因此，我們進一步通過體外轉(zhuǎn)染實驗發(fā)現(xiàn)，miR-339-5p下調(diào)了腦膠質(zhì)瘤細胞株U87上B7-H3的表達，并抑制腦膠質(zhì)瘤細胞的侵襲，但不影響腫瘤細胞的增殖。同時轉(zhuǎn)染miR-339-5p還顯著下調(diào)了化學趨化因子受體CXCR4的表達，表明miR-339-5p可能通過B7-H3和CXCR4相互作用影響腫瘤細胞的侵襲。
[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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R739.41
本文編號：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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R739.41
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