口腔鳞状细胞癌中MALAT-1与CCR7及其相关基因相互作用的分子通路
发布时间：2018-09-13 13:39
【摘要】：口腔鳞状细胞癌(Oral squamous cell carcinoma,OSCC)是口腔恶性肿瘤中最常见的一类,且较易早期发生淋巴结转移;发病率每年仍在不断增长。癌症虽然成因复杂,不能一概而论,但是多年来,学者们一直致力于从遗传,环境,基因等多个层面研究恶性肿瘤形成机制,以期可以从根源上预防恶性肿瘤的发生,并找到更好的诊断、治疗恶性肿瘤的方法。本文着重于研究口腔鳞癌淋巴结转移的分子通路,细胞信号通路并对关键环节加以阻断,试图寻找诊治口腔鳞状细胞癌的新靶点。细胞癌变和转移是一个多因素、多基因、多环节参与的复杂过程,其中有着复杂的基因调控因素。近几年研究发现长链非编码RNA(long non-coding RNA,lnc RNAs)在细胞发育和代谢以及肿瘤的发生发展过程中发挥着重要的作用。某些肿瘤根本不存在蛋白质编码基因的变异,而只有lnc RNAs的表达异常,几个lnc RNAs就可以使转录过程发生改变。肺腺癌转移相关转录本1(metastasis-associated lung adenocarcinoma transcript1,MALAT-1)是第一个被发现与肿瘤转移能力可能相关的lnc RNAs。MALAT-1在RNA聚合酶Ⅱ作用下合成,其表达产物定位于核散斑中,而参与m RNA前体加工的丝氨酸/精氨酸富集蛋白家族(serine/arginine richedprotein,SR protein,SR蛋白家族)也定位于核散斑中,基于两者的共定位关系,有学者通过一系列研究发现MALAT-1与SR蛋白磷酸化直接相关。MALAT-1在核散斑的定位是调控SR蛋白家族成员表达,定位和活化的结构基础,有研究证实,MALAT-1与miRNA-320s关系密切,MALAT-1可调控下游hsa-miR-320a(hsa-miRNA-320s家族的成员之一)的表达。Y盒结合蛋白(Y-box binding protein 1,YB-1)由编码蛋白质基因YB-1编码,是一种多功能冷休克蛋白,具有结合核酸的功能,被认为是一种肿瘤蛋白,涉及肿瘤细胞增殖与凋亡过程的许多方面;有研究发现YB-1可以结合至hsa-miRNA-320s之后参与调节miRNA的表达和调控。晚近宏基因组学生物信息分析数据显示,趋化因子受体CCR7的靶基因之一可能是hsa-miR-320a,目前尚无该方面实证研究文献支持。肿瘤细胞迁移具有靶向组织特异性。向特定组织迁移的分子调控过程类似于炎症反应中免疫细胞的浸润与迁移,趋化因子及其受体在肿瘤的发生、浸润与转移及预后均有密切关系。趋化因子及其受体在肿瘤细胞选择性转移向特定组织器官产生作用已得到众多文献研究支持,研究表明趋化因子及其受体调控肿瘤细胞淋巴结转移过程,类似趋化因子在炎症过程中调节免疫细胞的迁移。本课题组前期研究及有文献研究均表明趋化因子受体CCR7在口腔鳞癌淋巴结转移中具有重要作用。综上所述,提出以下假设:MALAT-1通过活化SR蛋白或YB-1蛋白,调控趋化因子受体m RNA前体miRNA-320s的加工,影响趋化因子受体CCR7在口腔鳞癌中的表达,从而可能对口腔鳞癌淋巴结转移行为造成影响。课题研究分三个部分:1、实时荧光定量检测口腔鳞癌组织标本,定性观察MALAT-1、miRNA-320s、SRSF1、YB-1、CCR7等分子表达情况;2、人舌癌细胞株SCC-9、SCC-25,在细胞水平,通过免疫共沉淀、免疫印迹法检验相关RNA(MALAT-1,miRNA-320s)与SRSF1蛋白或YB-1的相关性;3、通过基因调控技术转染miRNA inhibitor沉默miRNA-320s后进行免疫共沉淀、免疫印迹及实时荧光定量检测趋化因子受体CCR7的表达,并分析可能存在的相互关系。创新:本课题的创新之处在于首次发现了在口腔鳞癌细胞株中看到长链非编码RNA MALAT-1与趋化因子受体CCR7间的相互作用。在口腔鳞癌组织标本中看到了MALAT-1与CCR7呈正相关关系;在人舌癌细胞株中看到了MALAT-1、SRSF1、miRNA-320d间可以相互结合,且miRNA-320d可以影响CCR7的表达。以上MALAT-1影响CCR7表达的可能分子通路尚未见文献报道。【研究方法】第一部分:实时荧光定量检测定性观察MALAT-1、miRNA-320s、SRSF1、YB-1、CCR7等分子在口腔鳞癌组织及癌旁组织中的表达1.切取、保存口腔鳞癌及癌旁组织标本术中切取少量T3/T4期口腔癌组织及癌旁组织6组置于冻存管,由病理科鉴定为口腔鳞癌组织,以有无淋巴结转移分为两组,-80℃冰箱保存备用。2.实时荧光定量PCR检测口腔鳞癌及癌旁组织中MALAT1,SRSF1,miRNA-320s,YB-1及CCR7的表达差异。2.1 TRIZOL法抽提总RNA。2.2 RNA逆转录为c DNA。2.3 Real-time q PCR检测目标分子表达量。3.统计分析利用SPSS19.0及Graphpad prism 7.0对得到的实时荧光定量数据进行统计分析,对计量资料两组间采用配对t检验,以P≤0.05为标准视为差异具有统计学意义。第二部分:舌癌细胞株的相关RNA(MALAT-1,miRNA-320s)与SRSF1蛋白或YB-1的相关性研究1.细胞培养人舌鳞癌细胞株(SCC9、SCC25),在含有10%胎牛血清的DMEM高糖完全培养基中传代培养,37℃,5%CO_2,95%饱和湿度,传至第六代细胞供本实验使用。2.RNA与蛋白质结合免疫共沉淀2.1实验分组(1)对照组:加入RIP试剂盒内提供的阴性对照兔抗;(2)实验组1:加入抗SF2抗体的RIP复合物;(3)实验组2:加入抗YB-1抗体的RIP复合物;2.2实验方法使用Sigma公司的免疫共沉淀试剂盒进行RNA结合蛋白免疫共沉淀,检测有无RNA-抗体-蛋白质复合物的存在。3.免疫印迹实验检测细胞裂解产物的表达及RIP复合物的表达。将细胞裂解产物用免疫印迹实验检测裂解效率即目的分子的显色条带,并检测免疫共沉淀实验的复合物表达情况。4.实时荧光定量PCR4.1免疫共沉淀实验中的RNA-抗体-蛋白质复合物浓度、纯度检验。4.2 RNA逆转录为c DNA。4.3 Real-time q PCR。5.统计分析:Graphpad prism 7.0对得到的实时荧光定量数据进行统计分析,对计量资料两组间采用配对t检验,以P≤0.05为标准视为差异具有统计学意义。第三部分:基因调控技术沉默miRNA-320d,观察CCR7的表达根据第二部分实验结果:hsa-miRNA-320d(hsa-miRNA-320s家族成员之一,筛选了a、b、d、e)与MALAT-1具有相关关系。1.细胞miRNA-320d inhibitor转染1.1分组(1)对照组:miRNA Inhibitor Negative Control#1(4464058);(2)实验组:miRNA-320d inhibitor(4464066);1.2方法使用Lipofectamine公司的RNAi MAX转染试剂盒进行细胞miRNA inhibitor转染,沉默细胞内miRNA-320d。2.免疫印迹实验检测转染后细胞裂解产物的表达及RIP复合物的表达。将细胞裂解产物用免疫印迹实验检测裂解效率即目的分子的表达情况,并检测免疫共沉淀实验的复合物表达情况。3.实时荧光定量PCR3.1转染后的RIP复合物纯度、浓度检测;3.2 RNA逆转录为c DNA;3.3 Real-time q PCR。4.统计分析:Graphpad prism 7.0对得到的实时荧光定量数据进行统计分析,对计量资料两组间采用配对t检验,以P≤0.05为标准视为差异具有统计学意义。【结果】第一部分:实时荧光定量检测定性观察MALAT-1、miRNA-320s、SRSF1、YB-1、CCR7等分子在口腔鳞癌组织及癌旁组织中的表达1.MALAT-1、SRSF1、YB-1、CCR7在淋巴结转移组的口腔鳞癌组织中的表达高于无淋巴结转移组且高于癌旁组织。(P0.01)2.miRNA-320d在淋巴结转移组的口腔鳞癌组织中的表达低于无淋巴结转移组。(P≤0.05)第二部分:舌癌细胞株的相关RNA(MALAT-1,miRNA-320s)与SRSF1蛋白或YB-1的相关性研究1.MALAT-1可以与SRSF1相互结合。(见WB条带及实时荧光定量统计分析)2.miRNA-320d(miRNA-320s家族系列中的一员)与SRSF1可以相互结合(见WB条带及实时荧光定量统计分析)3.MALAT-1、miRNA-320d与YB-1未见结合。(见WB条带)结果表明:MALAT-1,miRNA-320d通过SRSF1有相互结合。第三部分:基因调控技术沉默miRNA-320d,观察CCR7的表达转染沉默miRNA-320d后抑制了基因的表达,同时观察到CCR7表达量升高,证明miRNA-320d与CCR7之间存在相互作用。【结论】1.通过实时荧光定量检测定性观察发现淋巴结转移组lnc RNA MALAT-1、SRSF1、CCR7相对于无淋巴结转移组均有不同程度高表达;2.通过免疫共沉淀、免疫印迹法检验发现相关RNA(MALAT-1,miRNA-320d)通过SRSF1蛋白可以互相结合;3.实时荧光法定量RNA-蛋白质复合物中MALAT-1,miRNA-320s的含量,MALAT-1,miRNA-320d通过SRSF1有相互结合;4.基因调控技术沉默miRNA-320s,免疫共沉淀、免疫印迹及实时荧光定量实验发现趋化因子受体CCR7的表达增强,可以推论MALAT-1、SRSF1、miRNA-320d、CCR7间存在相互作用关系,从分子层面揭示MALAT-1对趋化因子受体CCR7的影响,为以MALAT-1为分子靶向的口腔鳞癌淋巴结转移相关基础与应用的进一步研究提供一定理论基础。
[Abstract]:Oral squamous cell carcinoma (OSCC) is the most common type of oral malignancies, and is prone to early lymph node metastasis; the incidence is still growing every year. This paper focuses on the molecular pathway of lymph node metastasis in oral squamous cell carcinoma, the cell signaling pathway and the blockade of the key links, trying to find a new target for the diagnosis and treatment of oral squamous cell carcinoma. Carcinogenesis and metastasis is a complex process involving many factors, genes and links, among which there are complex gene regulatory factors. In recent years, studies have found that long non-coding RNA (lnc RNAs) plays an important role in cell development and metabolism, as well as in the occurrence and development of tumors. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) was the first to be found to be associated with tumor metastasis. Synthesized under the action of II, the expressed products are localized in nuclear speckles, and the serine/arginine rich protein family (SR protein, SR protein family) involved in the processing of M RNA precursors is also localized in nuclear speckles. Based on the co-localization relationship between the two proteins, a series of studies have found that MALAT-1 and SR protein phosphorylation. The localization of MALAT-1 in nuclear speckles is the structural basis for regulating the expression, localization and activation of SR protein family members. Studies have confirmed that MALAT-1 is closely related to microRNAs-320s. MALAT-1 can regulate the expression of downstream hsa-microRNAs-320a (a member of the hsa-microRNAs-320s family). Y-box binding protein 1 (YB-1) is a coding protein. YB-1 is a multifunctional cold shock protein that binds nucleic acids and is considered to be a tumor protein involved in many aspects of the proliferation and apoptosis of tumor cells. The data show that one of the target genes of chemokine receptor CCR7 may be hsa-microRNA-320a, and there is no empirical literature to support this aspect.Tumor cell migration has tissue-specific targeting.The molecular regulation of migration to specific tissues is similar to the infiltration and migration of immune cells in inflammatory reaction, and chemokines and chemokine receptors in tumors. Chemokines and chemokine receptors have been supported by many literatures. Chemokines and chemokine receptors have been shown to regulate lymph node metastasis of tumor cells, and chemokines like chemokines regulate immunity during inflammation. In summary, the following hypothesis is proposed: MALAT-1 regulates chemokine receptor m RNA precursor microRNA-320s processing by activating SR protein or YB-1 protein, affecting chemokine receptor CCR7. The research is divided into three parts: 1. Real-time fluorescence quantitative detection of oral squamous cell carcinoma tissue samples, qualitative observation of MALAT-1, microRNA-320s, SRSF1, YB-1, CCR7 and other molecular expression; 2, human tongue cancer cell lines SCC-9, SCC-25, at the cellular level, through immunization. Co-precipitation, Western blot assay was used to detect the correlation between RNA (MALAT-1, microRNAs-320s) and SRSF1 protein or YB-1; 3. MiNA inhibitor silenced microRNAs-320s was transfected by gene regulation technology, then co-precipitated by immunoblotting and real-time fluorescence quantitative detection of chemokine receptor CCR7 expression, and to analyze the possible correlation. The innovation of this study is that the interaction between long-stranded non-coding RNA MALAT-1 and chemokine receptor CCR7 was found in oral squamous cell carcinoma cell lines for the first time. The possible molecular pathways of MALAT-1 affecting CCR7 expression have not been reported in literature. [Methods] Part 1: Qualitative observation of the expression of MALAT-1, microRNA320s, SRSF1, YB-1, CCR7 in oral squamous cell carcinoma and adjacent tissues by real-time fluorescence quantitative detection. Six groups of oral squamous cell carcinoma (OSCC) tissues and adjacent tissues (T3/T4 stage) were harvested and placed in cryopreserved tubes. The specimens were divided into two groups with or without lymph node metastasis. 2. The expressions of MALAT1, SRSF1, microRNA-320s, YB-1 and CCR7 in OSCC and adjacent tissues were detected by real-time quantitative PCR. Difference.2.1 Total RNA.2.2 RNA was retrieved by TRIZOL and the expression of target molecule was detected by C DNA.2.3 Real-time Q PCR. Part 2: Correlation between the expression of related RNA (MALAT-1, microRNA-320s) and SRSF1 protein or YB-1 in tongue cancer cell lines 1. Human tongue squamous cell carcinoma cell lines (SCC9, SCC25) were subcultured in DMEM high glucose complete medium containing 10% fetal bovine serum at 37 C, 5% CO_2, 95% saturated humidity and passed to the sixth generation cells for use in this experiment. Protein-binding immunoprecipitation 2.1 experimental group (1) control group: adding negative control rabbit anti-RIP kit; (2) experimental group 1: adding anti-SF2 antibody RIP complex; (3) experimental group 2: adding anti-YB-1 antibody RIP complex; 2.2 experimental method using Sigma company's immunoprecipitation kit for RNA-binding protein immunoprecipitation 3. Immunoblotting assay was used to detect the expression of cell lysate products and RIP complex. Immunoblotting assay was used to detect the color bands of the target molecule whose lysis efficiency was the target molecule. Immunocoprecipitation assay was used to detect the complex expression. 4. Real-time fluorescence quantitative analysis. The concentration and purity of RNA-antibody-protein complex in PCR 4.1 immunoprecipitation assay were tested. Part 3: Gene regulation technique silenced microRNAs-320d. CCR7 expression was observed according to the second part of the experiment: hsa-microRNAs-320d (hsa-microRNAs-320s family members, screened a, b, d, e) and MALAT-1 has a correlation. 1. Cell microRNAs-320d inhibitor transfection 1.1 grouping (1) control group: MiRNA Inhibitor Negative Control #1 (control group: MiRNA Inhibitor Negative Control #1) 4464058; (2) Experimental group: MiRNA-320d inhibitor (4464066); 1. Real-time fluorescence quantitative PCR 3.1 transfected RIP complex purity, concentration detection; 3.2 RNA reverse transcription into C DNA; 3.3 Real-time Q PCR.4. Statistical analysis: Graphpad prism 7.0 on the obtained real-time fluorescence quantitative data Statistical analysis showed that there was significant difference between the two groups by paired t test and P < 0.05 as the standard. [Results] Part 1: Qualitative observation of MALAT-1, microRNA-320s, SRSF1, YB-1, CCR7 in oral squamous cell carcinoma and adjacent tissues by real-time fluorescence quantitative detection. The expression of microRNA-320d in the lymph node metastasis group was lower than that in the non-lymph node metastasis group. (P < 0.05) Part 2: Correlation between the expression of related RNA (MALAT-1, microNA-320s) and SRSF1 protein or YB-1 in tongue cancer cell lines. Study 1. MALAT-1 can bind to SRSF1. (See WB banding and real-time fluorescence quantitative analysis) 2. MiNA-320d (a member of the family of microNA-320s) can bind to SRSF1 (see WB banding and real-time fluorescence quantitative analysis) 3. MALAT-1, microNA-320d and YB-1 do not bind to each other. (See WB banding) The results showed that MALAT-1, microNA-320d through S-1. RSF1 was combined with each other. Part 3: Gene regulation technique silenced microRNAs-320d, observed CCR7 expression transfected silenced microRNAs-320d inhibited gene expression, and observed CCR7 expression increased, indicating that there was interaction between microRNAs-320d and CCR7. [Conclusion] 1. Qualitative observation of lymph node metastasis by real-time fluorescence quantitative detection. Histological LNC RNA MALAT-1, SRSF1, CCR7 were highly expressed in different degrees compared with the non-lymph node metastasis group. 2. Through immunoprecipitation, Western blotting test showed that the related RNA (MALAT-1, microRNA320d) could bind to each other through SRSF1 protein. 3. Real-time fluorescence assay was used to quantify the contents of MALAT-1, microRNA320s, MALAT-1 and microRNA320d in RNA-protein complexes. Through SRSF1 binding to each other; 4. Gene regulation technology silencing of microRNAs-320s, immunoprecipitation, Western blot and real-time fluorescence quantitative assay found that the expression of chemokine receptor CCR7 increased, we can infer that there is interaction between MALAT-1, SRSF1, microRNAs-320d, CCR7, reveal the effect of MALAT-1 on chemokine receptor CCR7 from the molecular level. MALAT-1 as a molecular targeted lymph node metastasis of oral squamous cell carcinoma related to the basis and application of further research to provide a theoretical basis.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.8
本文编号：2241332
[Abstract]:Oral squamous cell carcinoma (OSCC) is the most common type of oral malignancies, and is prone to early lymph node metastasis; the incidence is still growing every year. This paper focuses on the molecular pathway of lymph node metastasis in oral squamous cell carcinoma, the cell signaling pathway and the blockade of the key links, trying to find a new target for the diagnosis and treatment of oral squamous cell carcinoma. Carcinogenesis and metastasis is a complex process involving many factors, genes and links, among which there are complex gene regulatory factors. In recent years, studies have found that long non-coding RNA (lnc RNAs) plays an important role in cell development and metabolism, as well as in the occurrence and development of tumors. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) was the first to be found to be associated with tumor metastasis. Synthesized under the action of II, the expressed products are localized in nuclear speckles, and the serine/arginine rich protein family (SR protein, SR protein family) involved in the processing of M RNA precursors is also localized in nuclear speckles. Based on the co-localization relationship between the two proteins, a series of studies have found that MALAT-1 and SR protein phosphorylation. The localization of MALAT-1 in nuclear speckles is the structural basis for regulating the expression, localization and activation of SR protein family members. Studies have confirmed that MALAT-1 is closely related to microRNAs-320s. MALAT-1 can regulate the expression of downstream hsa-microRNAs-320a (a member of the hsa-microRNAs-320s family). Y-box binding protein 1 (YB-1) is a coding protein. YB-1 is a multifunctional cold shock protein that binds nucleic acids and is considered to be a tumor protein involved in many aspects of the proliferation and apoptosis of tumor cells. The data show that one of the target genes of chemokine receptor CCR7 may be hsa-microRNA-320a, and there is no empirical literature to support this aspect.Tumor cell migration has tissue-specific targeting.The molecular regulation of migration to specific tissues is similar to the infiltration and migration of immune cells in inflammatory reaction, and chemokines and chemokine receptors in tumors. Chemokines and chemokine receptors have been supported by many literatures. Chemokines and chemokine receptors have been shown to regulate lymph node metastasis of tumor cells, and chemokines like chemokines regulate immunity during inflammation. In summary, the following hypothesis is proposed: MALAT-1 regulates chemokine receptor m RNA precursor microRNA-320s processing by activating SR protein or YB-1 protein, affecting chemokine receptor CCR7. The research is divided into three parts: 1. Real-time fluorescence quantitative detection of oral squamous cell carcinoma tissue samples, qualitative observation of MALAT-1, microRNA-320s, SRSF1, YB-1, CCR7 and other molecular expression; 2, human tongue cancer cell lines SCC-9, SCC-25, at the cellular level, through immunization. Co-precipitation, Western blot assay was used to detect the correlation between RNA (MALAT-1, microRNAs-320s) and SRSF1 protein or YB-1; 3. MiNA inhibitor silenced microRNAs-320s was transfected by gene regulation technology, then co-precipitated by immunoblotting and real-time fluorescence quantitative detection of chemokine receptor CCR7 expression, and to analyze the possible correlation. The innovation of this study is that the interaction between long-stranded non-coding RNA MALAT-1 and chemokine receptor CCR7 was found in oral squamous cell carcinoma cell lines for the first time. The possible molecular pathways of MALAT-1 affecting CCR7 expression have not been reported in literature. [Methods] Part 1: Qualitative observation of the expression of MALAT-1, microRNA320s, SRSF1, YB-1, CCR7 in oral squamous cell carcinoma and adjacent tissues by real-time fluorescence quantitative detection. Six groups of oral squamous cell carcinoma (OSCC) tissues and adjacent tissues (T3/T4 stage) were harvested and placed in cryopreserved tubes. The specimens were divided into two groups with or without lymph node metastasis. 2. The expressions of MALAT1, SRSF1, microRNA-320s, YB-1 and CCR7 in OSCC and adjacent tissues were detected by real-time quantitative PCR. Difference.2.1 Total RNA.2.2 RNA was retrieved by TRIZOL and the expression of target molecule was detected by C DNA.2.3 Real-time Q PCR. Part 2: Correlation between the expression of related RNA (MALAT-1, microRNA-320s) and SRSF1 protein or YB-1 in tongue cancer cell lines 1. Human tongue squamous cell carcinoma cell lines (SCC9, SCC25) were subcultured in DMEM high glucose complete medium containing 10% fetal bovine serum at 37 C, 5% CO_2, 95% saturated humidity and passed to the sixth generation cells for use in this experiment. Protein-binding immunoprecipitation 2.1 experimental group (1) control group: adding negative control rabbit anti-RIP kit; (2) experimental group 1: adding anti-SF2 antibody RIP complex; (3) experimental group 2: adding anti-YB-1 antibody RIP complex; 2.2 experimental method using Sigma company's immunoprecipitation kit for RNA-binding protein immunoprecipitation 3. Immunoblotting assay was used to detect the expression of cell lysate products and RIP complex. Immunoblotting assay was used to detect the color bands of the target molecule whose lysis efficiency was the target molecule. Immunocoprecipitation assay was used to detect the complex expression. 4. Real-time fluorescence quantitative analysis. The concentration and purity of RNA-antibody-protein complex in PCR 4.1 immunoprecipitation assay were tested. Part 3: Gene regulation technique silenced microRNAs-320d. CCR7 expression was observed according to the second part of the experiment: hsa-microRNAs-320d (hsa-microRNAs-320s family members, screened a, b, d, e) and MALAT-1 has a correlation. 1. Cell microRNAs-320d inhibitor transfection 1.1 grouping (1) control group: MiRNA Inhibitor Negative Control #1 (control group: MiRNA Inhibitor Negative Control #1) 4464058; (2) Experimental group: MiRNA-320d inhibitor (4464066); 1. Real-time fluorescence quantitative PCR 3.1 transfected RIP complex purity, concentration detection; 3.2 RNA reverse transcription into C DNA; 3.3 Real-time Q PCR.4. Statistical analysis: Graphpad prism 7.0 on the obtained real-time fluorescence quantitative data Statistical analysis showed that there was significant difference between the two groups by paired t test and P < 0.05 as the standard. [Results] Part 1: Qualitative observation of MALAT-1, microRNA-320s, SRSF1, YB-1, CCR7 in oral squamous cell carcinoma and adjacent tissues by real-time fluorescence quantitative detection. The expression of microRNA-320d in the lymph node metastasis group was lower than that in the non-lymph node metastasis group. (P < 0.05) Part 2: Correlation between the expression of related RNA (MALAT-1, microNA-320s) and SRSF1 protein or YB-1 in tongue cancer cell lines. Study 1. MALAT-1 can bind to SRSF1. (See WB banding and real-time fluorescence quantitative analysis) 2. MiNA-320d (a member of the family of microNA-320s) can bind to SRSF1 (see WB banding and real-time fluorescence quantitative analysis) 3. MALAT-1, microNA-320d and YB-1 do not bind to each other. (See WB banding) The results showed that MALAT-1, microNA-320d through S-1. RSF1 was combined with each other. Part 3: Gene regulation technique silenced microRNAs-320d, observed CCR7 expression transfected silenced microRNAs-320d inhibited gene expression, and observed CCR7 expression increased, indicating that there was interaction between microRNAs-320d and CCR7. [Conclusion] 1. Qualitative observation of lymph node metastasis by real-time fluorescence quantitative detection. Histological LNC RNA MALAT-1, SRSF1, CCR7 were highly expressed in different degrees compared with the non-lymph node metastasis group. 2. Through immunoprecipitation, Western blotting test showed that the related RNA (MALAT-1, microRNA320d) could bind to each other through SRSF1 protein. 3. Real-time fluorescence assay was used to quantify the contents of MALAT-1, microRNA320s, MALAT-1 and microRNA320d in RNA-protein complexes. Through SRSF1 binding to each other; 4. Gene regulation technology silencing of microRNAs-320s, immunoprecipitation, Western blot and real-time fluorescence quantitative assay found that the expression of chemokine receptor CCR7 increased, we can infer that there is interaction between MALAT-1, SRSF1, microRNAs-320d, CCR7, reveal the effect of MALAT-1 on chemokine receptor CCR7 from the molecular level. MALAT-1 as a molecular targeted lymph node metastasis of oral squamous cell carcinoma related to the basis and application of further research to provide a theoretical basis.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.8
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