miR-378对骨髓增生异常综合征细胞增殖的作用及其机制研究

发布时间：2018-02-14 11:51

本文关键词： 骨髓增生异常综合征 microRNA-378 细胞凋亡 BCL2L2 CDC40　出处：《重庆医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的检测骨髓增生异常综合征(myelodysplastic syndrome,MDS)患者骨髓标本中miR-378的表达水平,构建mi R-378过表达重组慢病毒载体,通过体内外实验去探讨miR-378过表达对人MDS细胞株SKM-1增殖和凋亡的影响,并进一步探索miR-378在MDS细胞中发挥作用所调控的靶基因及其机制。方法1、收集20例MDS患者新鲜骨髓标本,其中RA 5例,RARS 3例,RCMD 2例,RAEB 5例,5q-综合征1例,sAML 1例,MDS-U 3例。此外还收集13例健康人的骨髓标本作为对照。梯度离心分离出骨髓标本中的单个核细胞,提取总RNA,再利用qRT-PCR检测两组骨髓标本中miR-378的表达。2、构建miR-378过表达重组慢病毒载体(LV-miR-378)及阴性对照病毒载体(LV-control),分别用LV-miR-378和LV-control去感染SKM-1细胞,流式细胞术检测感染效率,qRT-PCR明确各组细胞中miR-378的表达。用CCK-8法研究miR-378对MDS细胞增殖的作用,Annexin V/7-AAD双染法检测各组细胞的凋亡率,流式细胞术观察细胞周期情况。最后利用Western blot检测内外源凋亡通路相关凋亡因子的表达。3、分别将感染了lv-mir-378的skm-1细胞、lv-control感染的skm-1细胞及未经任何处理的skm-1细胞接种于nod/scid小鼠皮下,以建立mds荷瘤小鼠模型。观察各组小鼠的荷瘤生长情况,测量移植瘤的体积和重量,并用tunel法检测瘤细胞的原位凋亡情况。4、应用生物信息学软件预测mir-378潜在的靶基因,构建候选靶基因mrna3'utr野生型和突变型的双荧光素酶载体,分别与mir-378过表达质粒及阴性对照质粒共转染293t细胞,并用双荧光素酶报告基因试剂盒检测并计算相对荧光素酶活性。最后用pcr和westernblot技术研究mir-378表达上调对靶基因表达的影响。结果1、实时荧光定量pcr结果显示:mir-378在mds患者中的表达水平低于正常对照组(p0.05)。成功地构建了高表达mir-378的mds细胞模型,流式细胞术检测慢病毒感染效率大于70%,qrt-pcr检测到lv-mir-378慢病毒转染组的mir-378表达水平明显高于阴性对照病毒转染组和未经处理的空白对照组(p0.05)。2、cck-8结果显示,上调mir-378的表达可以明显抑制skm-1细胞的增殖活性,mir-378过表达组的od值与阴性对照组和未感染组相比明显降低(p0.05)。过表达mir-378慢病毒感染组的细胞凋亡率为[(12.90±3.72)%],明显高于阴性对照病毒感染组[(3.21±1.92)%]和未感染组[(2.78±1.04)%]。此外,上调skm-1细胞中mir-378的表达使g1/g0期细胞比例增加,s期细胞比例减少(p0.05)。我们还发现mir-378过表达可以同时激活内外源凋亡通路,使cleaved-caspase-3、cleaved-caspase-8、cleaved-caspase-9及Bax蛋白的表达水平增高。3、miR-378过表达组的移植瘤体积和重量均小于阴性对照组和空白对照组(P0.05),TUNEL法结果显示miR-378过表达组瘤组织的原位细胞凋亡率[(37.08±7.65)%]明显高于阴性对照组[(17.49±3.13)%,P=0.001]和空白对照组[(16.93±2.95)%,P=0.001]。4、两个靶基因预测网站都提示抗凋亡蛋白基因BCL2L2是miR-378的潜在靶基因。双荧光素酶实验结果提示,与对照组相比,miR-378质粒与BCL2L2 3'UTR野生型质粒共转染组的荧光素酶活性明显下降(P0.05)。上调MDS细胞中miR-378的表达后,BCL2L2mRNA水平无明显变化(P0.05),而BCL2L2蛋白表达明显降低。此外,我们还发现miR-378可以降低miR-378的另一个靶基因CDC40蛋白的表达。结论miR-378在MDS中呈低表达,上调mi R-378的表达可以抑制MDS细胞的生长,促进MDS细胞凋亡及造成细胞周期阻滞。其发挥作用的机制可能与靶向作用于BCL2L2和CDC40有关。
[Abstract]:Objective to detect the myelodysplastic syndrome (myelodysplastic, syndrome, MDS) the expression level of miR-378 in patients with bone marrow specimens, construction of MI over expression of R-378 recombinant lentiviral vectors in vitro and in vivo to investigate the effect of overexpression of miR-378 on proliferation and apoptosis of human MDS cell line SKM-1, and to further explore the target genes regulated by miR-378 play a role and its mechanism in MDS cells. Methods 1, 20 MDS patients were collected fresh bone marrow specimens, including 5 cases of RA, 3 cases RARS, 2 cases RCMD, 5 cases of RAEB, 1 cases of 5q- syndrome, 1 cases of sAML, 3 cases of MDS-U. In addition, bone marrow samples were collected from 13 healthy people as control. The gradient isolated mononuclear cells in bone marrow samples, extraction of total RNA, using qRT-PCR to detect miR-378 expression of.2 in bone marrow samples of two groups, the construction of miR-378 over expression of recombinant lentiviral vector (LV-miR-378) and negative control vector (LV-control), LV-miR-378 and LV-control were used to infect SKM-1 cells. The infection efficiency was assessed by flow cytometry, the expression of miR-378 qRT-PCR in the cells were clear. To study the effect of miR-378 CCK-8 on MDS cell proliferation and apoptosis of Annexin V/7-AAD double staining method to detect cell rate, cell cycle was measured by flow cytometry. The expression of.3 Western blot detection of apoptosis related protein locus, were infected with lv-mir-378 skm-1 cells, lv-control infected skm-1 cells and skm-1 cells without any treatment inoculation in nod/scid mice skin, in order to establish MDS mice model. The growth of tumor bearing mice were observed, measured the tumor volume and weight the TUNEL method was used to detect tumor cells in situ apoptosis of.4, using bioinformatics software predicted the potential target gene of mir-378, construct the candidate target gene mrna3'utr in the wild Dual luciferase vector type and mutant, and overexpression of mir-378 plasmid and negative control plasmid were transfected into 293T cells, and dual luciferase reporter kit to detect and calculate the relative luciferase activity. Finally, using PCR and Westernblot technology research on the influence of mir-378 expression on target gene expression. Results 1, real-time fluorescence quantitative the result of PCR showed that the expression level of mir-378 in patients with MDS than those of the normal control group (P0.05). The successful construction of the model of MDS cells with high expression of mir-378 is greater than 70%, the efficiency of flow cytometry to detect slow virus infection, qRT-PCR detected lv-mir-378 lentiviral transfection group mir-378 expression levels were significantly higher than that in the negative control group and transfection the untreated control group (P0.05).2, CCK-8 results showed that the upregulation of the expression of mir-378 can significantly inhibit the proliferation of skm-1 cells, overexpression of mir-378 group The OD value decreased obviously compared with the negative control group and non infection group (P0.05). Expression of apoptosis mir-378 lentivirus infection group was [(12.90 + 3.72) detected in the negative control group was significantly higher than that of virus infection [(3.21 + 1.92) detected and uninfected Group [(2.78 + 1.04) in addition. The expression of mir-378, skm-1 in the cells increased the percentage of g1/g0 phase, decreased the proportion of cells in S phase (P0.05). We also found that overexpression of mir-378 can also activate exogenous apoptosis pathway, cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9 and Bax protein expression increased.3, overexpression of miR-378 group transplanted tumor volume and weight are less than the negative control group and blank control group (P0.05), TUNEL assay showed that cell apoptosis in miR-378 overexpression group tumor rate [(37.08 + 7.65) when it was significantly higher than that of negative control group [(17.49 + 3.13)%, P=0.001] and blank control group [(16.9 3 + 2.95)%, P=0.001].4, two target gene prediction sites showed that the anti apoptosis protein BCL2L2 gene is a potential target gene of miR-378. Dual luciferase results showed that, compared with the control group, miR-378 plasmid and BCL2L2 plasmid were co transfected with wild-type 3'UTR luciferase activity group significantly decreased (P0.05). The expression of miR-378 increased MDS in cells, no significant changes in BCL2L2mRNA level (P0.05), BCL2L2 protein expression was significantly reduced. In addition, we also found that miR-378 can reduce the expression of another miR-378 target gene CDC40 protein. Conclusion the expression of miR-378 is lower in MDS, MI up-regulated the expression of R-378 can inhibit the growth of MDS cells, promoting MDS cell apoptosis and caused cell cycle arrest. Its function and possible mechanism of targeting BCL2L2 and CDC40.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R551.3

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