miR-301b-3p对M-CSF诱导单核细胞自噬的调控

发布时间：2018-06-05 04:31

本文选题：微小核糖核酸 + 单核细胞　；参考：《第二军医大学》2017年硕士论文

【摘要】：一、背景与目的动脉粥样硬化被认为有炎症因素参与其病理过程,而其炎症过程与单核细胞和单核细胞来源巨噬细胞的招募、活化以及单核细胞的分化相关,且与动脉粥样硬化斑块稳定性及动脉粥样硬化性疾病的预后相关。然而,由于单核细胞半衰期较短,如果缺少自噬,循环血中的单核细胞将会自然凋亡。目前已知主要的可诱导单核细胞自噬的刺激物主要有粒细胞巨噬细胞刺激因子(GM-CSF)和巨噬细胞刺激因子(M-CSF)。目前认为自噬是一个普遍存在于真核细胞中、可通过双层膜结构的囊泡降解或回收不需要或无功能的细胞组分以维持细胞稳态、抗衰老以及细胞发育的过程。其过程中,微管相关蛋白轻链3 (LC3)以及泛素样结合蛋白p62/SQSTM1均为重要标志蛋白,故常作为检测自噬活性的常用指标之一。短链非编码RNA中微小核糖核酸(microRNA, miRNA, miR-)可对广泛细胞各种生物学活动进行转录后调控。故我们设计本研究旨在探讨miRNA在单核细胞自噬中的表达情况及其调控作用。我们通过将THP-1人单核细胞置于M-CSF刺激环境下,诱导单核细胞发生自噬,检测miRNA表达情况,筛查表达变化最为显著的miRNA,并进一步将其在单核细胞中过表达来观察细胞自噬活性的变化。进而探讨该miRNA调控M-CSF诱导的THP-1人单核细胞自噬的潜在靶基因。二、研究方法(一)THP-1人单核细胞的培养THP-1人单核细胞培养条件为完全1640培养基(90%RPMI-1640+10%胎牛血清+1%P/S)。诱导自噬条件为在完全1640培养液中加入M-CSF至100ng/ml。(二)流式细胞检测THP-1人单核细胞按上述方法培养,CYTO-ID自噬检测试剂盒中绿色染剂染色后用流式细胞仪的绿(FL1)通道分析样本。(三)蛋白免疫印迹(Western blot)单核细胞样品采用HelixGenRIPA裂解液提取蛋白,对LC3-Ⅰ、LC3-Ⅱ、p62/SQSTMI以及GAPDH等蛋白表达情况进行检测。一抗浓度如下:anti-LC3(1:1000)、anti-p62 (1:1000)、anti-GAPDH (1:1000)。二抗浓度如下:anti-rabbit(1:10000)、anti-mouse (1:5000)。蛋白条带应用 BIORADFlour-SMuiltilmager 成像系统检测。(四)透射电子显微镜单核细胞经M-CSF处理后使用多聚甲醛固定液4℃固定6h以上后送检,电镜下观察到双层膜结构囊泡状的自噬体作为自噬定性检测标准。(五)Agilent miRNA 芯片应用mirVana RNA提取试剂盒提取total RNA,芯片杂交后应用Aglient G2505C扫描仪中选择相应的参数扫描。(六)实时定量聚合酶链反应(qRT-PCR)应用miRcute miRNA提取分离试剂盒提取THP-1人单核细胞的miRNA,应用miRcute增强型miRNAcDNA第一链合成试剂盒进行反转录,miRcute增强型miRNA荧光定量检测试剂盒进行qRT-PCR,使用Roche LightCycler分析仪进行检测分析,以U6作为内参计算ACt值,以2-△△Ct法计算目标miRNA相对表达水平。(七)腺病毒构建及细胞转染构建重组腺病毒并扩增、测定滴度,继而通过腺病毒将miR-301b-3p转染入单核细胞。采用qRT-PCR方法检测转染效率。(八)双荧光素酶报告基因分别将MYB和CYLD的mRNA 3'UTR融合至荧光素酶质粒中,再将质粒分别与miR-301b-3p类似物共转染至293T细胞中,24 h后裂解细胞,应用双荧光素酶报告系统试剂盒测定荧光素酶活性并定量分析。(九)统计学分析计量资料以平均数±标准差表示,每组的实验数据重复至少3次。两组间比较采用两独立样本的t检验,多个组间比较采用单因素ANOVA分析。以P0.05作为显著性差异标准。三、结果(一)M-CSF诱导THP-1人单核细胞自噬M-CSF诱导处理后,应用CYTO-ID自噬检测试剂盒对THP-1人单核细胞进行流式细胞检测显示处理6 h时细胞自噬水平显著升高;Western blot显示LC3II/1比值显著高于对照组、p62/SQSTM1显著低于对照组,应用巴弗洛霉素A1抑制溶酶体活性后,相同处理条件下LC3Ⅱ/Ⅰ比值进一步增高、p62/SQSTM1则高于对照组;透射电子显微镜观察到M-CSF处理6 h时单核细胞内形成双侧膜结构自噬体,而对照组则未观察到该现象。上述结果均证实在M-CSF处理6 h后单核细胞自噬被显著激活。(二)miRNA表达情况Agilent miRNA芯片检测结果提示与对照组相比,M-CSF处理6 h时单核细胞表达 miRNA 中 miR-1249-3p、miR-301b-3p、miR-4653-3p、miR-513a-5p、miR-6734-5p存在显著差异,进一步应用qRT-PCR检测验证miR-301b-3p表达显著增高。(三)过表达miR-301b-3p促进M-CSF诱导的单核细胞自噬用miR-301b-3p转染单核细胞,48 h时qRT-PCR检测显示miR-301b-3p表达量与对照组相比显著增高,提示转染有效。M-CSF诱导单核细胞发生自噬,继而应用Western blot检测显示miR-301b-3p过表达后LC311/I1比值显著增高、p62/SQSTM1显著降低,提示过表达miR-301b-3p进一步促进M-CSF诱导的单核细胞自噬。(四)靶基因预测及验证应用TargetScan、microRNAorg等数据库检索预测筛选显示MYB、CYLD可能为miR-301b-3p的潜在靶基因,其3'UTR含有miR-301b-3p可能的结合位点且保守性较好,故本研究应用荧光素酶报告基因方法验证MYB与CYLD是否为miR-301b-3p的靶基因。结果显示过表达miR-301b-3p可显著抑制MYB-3'UTR与CYLD-3'URT的荧光素酶表达,提示MYB与CYLD为miR-301b-3p的直接靶基因。四、结论(一)M-CSF能够诱导THP-1人单核细胞发生自噬,在诱导6 h后自噬活性水平显著增高。(二)在M-CSF能够诱导THP-1人单核细胞自噬时,miR-301b-3p表达显著增高。(三)过表达miR-301b-3p能促进M-CSF诱导的单核细胞自噬。(四)miR-301b-3p靶向作用于MYB与CYLD,可能通过该途径实现促进M-CSF诱导的单核细胞自噬。
[Abstract]:Background and objective atherosclerosis is considered to be involved in the pathological process of inflammation, which is associated with the recruitment and activation of monocyte and monocyte derived macrophages, and the differentiation of monocyte, and is related to the stability of atherosclerotic plaque and the prognosis of atherosclerotic disease. Monocyte half-life is short. If autophagy is short of autophagy, mononuclear cells in circulating blood will be spontaneously apoptotic. At present, the main inducible stimulants for autophagy are granulocyte macrophage stimulating factor (GM-CSF) and macrophage stimulating factor (M-CSF). It is often used as one of the commonly used indicators for the detection of autophagic activity in the process of maintaining cell homeostasis, aging and cell development by degradation or recovery of non functional or non functional cell components through the bilayer membrane vesicles. In the process, the light chain 3 (LC3) and the ubiquitin like binding protein p62/SQSTM1 are all important markers. MicroRNA, miRNA (miRNA, miR-) in short chain non coding RNA can be regulated after transcriptional regulation of various biological activities in a wide range of cells. Therefore, we designed this study to explore the expression and regulation of miRNA in autophagy in monocyte. We induce mononuclear cells to induce mononuclear cells by placing THP-1 human mononuclear cells in a M-CSF stimulus environment. Autophagy, detection of miRNA expression, screening the most significant changes in the expression of miRNA, and further overexpression in mononuclear cells to observe the changes in cell autophagy. Further explore the potential target gene for the regulation of M-CSF induced autophagy in THP-1 human mononuclear cells. Two, the study method (1) THP-1 human mononuclear cell culture THP -1 human monocyte culture condition was a complete 1640 culture medium (90%RPMI-1640+10% fetal bovine serum +1%P/S). The induction of autophagy was cultured in complete 1640 culture medium by adding M-CSF to 100ng/ml. (two) flow cytometry to detect THP-1 human mononuclear cells, and the green dye in the CYTO-ID autophagy test kit was green with a flow cytometer. FL1) channel analysis samples. (three) protein immunoblotting (Western blot) mononuclear cells were used to extract protein from HelixGenRIPA lysate and detect the expression of LC3- I, LC3- II, p62/SQSTMI and GAPDH. The concentration of anti LC3- was as follows: anti-LC3 (1:1000), anti-p62 (1:1000), and two 10000), anti-mouse (1:5000). The protein strip was detected by the BIORADFlour-SMuiltilmager imaging system. (four) the mononuclear cells of the transmission electron microscope were treated with polyoxymethylene fixed solution at 4 degrees centigrade for more than 6h after M-CSF treatment, and the vesicular autophagy was observed as a qualitative test standard for autophagy under the electron microscope. (five) Agilent MiRNA chip is used to extract total RNA by mirVana RNA extraction kit. After chip hybridization, the corresponding parameter scanning is selected in Aglient G2505C scanner. (six) real-time quantitative polymerase chain reaction (qRT-PCR) is used to extract miRNA of THP-1 mononuclear cells by miRcute miRNA extraction and separation kit. Reagents were reverse transcribed, miRcute enhanced miRNA fluorescence quantitative detection kit was qRT-PCR, and Roche LightCycler analyzer was used for detection and analysis. U6 was used as the internal parameter to calculate ACt value. 2- Delta Delta Ct method was used to calculate the relative expression level of target miRNA. (seven) adenovirus construction and cell transfection construction of recombinant adenovirus and amplification, determination of drops And then transfection of miR-301b-3p into mononuclear cells by adenovirus. The transfection efficiency was detected by qRT-PCR method. (eight) double luciferase reporter gene fused mRNA 3'UTR of MYB and CYLD into luciferase plasmid, and then plasmids were co transfected with miR-301b-3p analogues to 293T cells, after 24 h, lysate cells were used, and double fluorescein was applied. The enzyme report system kits were used to determine the luciferase activity and quantitative analysis. (nine) the statistical data were expressed with mean standard deviation, and the experimental data in each group were repeated at least 3 times. The two groups were compared with two independent samples of t test, and a single factor ANOVA analysis was used in multiple groups. P0.05 was used as a significant difference standard. Three, knot Fruit (1) M-CSF induced THP-1 human mononuclear autophagic M-CSF induction treatment, the use of CYTO-ID autophagy detection kit for THP-1 human mononuclear cell flow cytometry showed that the level of autophagy increased significantly at 6 h; Western blot showed that the LC3II/1 ratio was significantly higher than the control group, p62/SQSTM1 was significantly lower than the control group, using buffalamycin. After the inhibition of lysosome activity by A1, the ratio of LC3 II / I increased further under the same treatment condition, and p62/SQSTM1 was higher than that of the control group; transmission electron microscopy observed that the autophagosome was formed in the mononuclear cells when M-CSF treatment was 6 h, while the control group did not observe the phenomenon. The results all showed that the mononuclear cells from the M-CSF treatment after 6 h self were from the mononuclear cells. (two) the Agilent miRNA chip detection results of miRNA expression showed that there were significant differences in the expression of miR-1249-3p, miR-301b-3p, miR-4653-3p, miR-513a-5p, miR-6734-5p in the mononuclear cells miRNA at 6 h when compared with the control group. (three) over expression (three) overexpression. MiR-301b-3p promoted monocyte autophagy induced by M-CSF to transfect mononuclear cells with miR-301b-3p. At 48 h, qRT-PCR detection showed that the expression of miR-301b-3p was significantly higher than that of the control group, suggesting that the transfection of effective.M-CSF induced autophagy induced mononuclear cells, and then the LC311/I1 ratio was significantly increased after Western blot detection showed that miR-301b-3p overexpressed. P62/SQSTM1 was significantly reduced, suggesting that over expression of miR-301b-3p further promoted autophagy induced by M-CSF. (four) target gene prediction and verification application of TargetScan, microRNAorg and other database retrieval and prediction screening showed MYB, CYLD might be a potential target gene for miR-301b-3p, and 3'UTR contained miR-301b-3p possible binding sites and conservatism. Well, this study uses luciferase reporter gene method to verify whether MYB and CYLD are the target genes of miR-301b-3p. The results show that overexpression of miR-301b-3p can significantly inhibit the luciferase expression of MYB-3'UTR and CYLD-3'URT, suggesting that MYB and CYLD are the direct target genes of miR-301b-3p. Four, nodal (1) M-CSF can induce THP-1 mononuclear cells. Autophagy increased significantly after induction of 6 h. (two) the expression of miR-301b-3p increased significantly when M-CSF could induce autophagy in THP-1 mononuclear cells. (three) overexpression of miR-301b-3p could promote M-CSF induced autophagy in monocytes. (four) miR-301b-3p targeted MYB and CYLD, possibly through this pathway to promote M-CSF induction. Nuclear cells are autophagy.
【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】：R543.5

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