小鼠小肠铁代谢相关miRNA筛选及miR let-7d靶向调控IEC-6细胞Slc11a2基因的研究

发布时间：2018-05-10 21:19

本文选题：地中海贫血 + 铁过载　；参考：《广西医科大学》2017年博士论文

【摘要】：地中海贫血(thalassemia)是一种遗传性慢性溶血性贫血,重型β-地中海贫血需要依靠长期输血和去铁治疗以维持生命。长期输血以及小肠上皮细胞对铁的吸收增加,可导致地中海贫血病人体内铁过载。铁过载引起肝脏、心脏、内分泌等多个器官功能衰竭,是地中海贫血患者死亡的主要原因,去铁治疗是重度β-地中海贫血治疗的重要组成部分。铁在人体内的可溶性极低(10-18 M),还原铁Fe++与过氧化氢反应生成的羟基自由基,在游离铁浓度过高时,高度反应活性的自由基能够破坏的DNA,脂质和蛋白质,可损害全身各组织器官,具有潜在的毒性,因此保持人体的铁稳态显得尤其重要。MicroRNAs(miRNAs)是一类非编码RNA,最早在真核生物中发现,miRNAs具有内源性的特点,可调控多种生物功能。Micro RNAs也是调节铁稳态的调控因子之一,miRNA对铁代谢具有靶向调控作用。本课题从小肠是铁元素吸收的主要器官这一角度出发,通过构建铁过载动物模型,筛选出小肠上皮细胞铁代谢相关蛋白的差异表达miRNA,对mi RNA和靶基因的调控关系进行验证,在ICE-6细胞中的过表达mi RNA,研究该mi RNA调控IEC-6细胞的铁代谢功能。预计本研究可为铁代谢的miRNA调控、以及地中海贫血患者的去铁治疗提供研究思路和方向。第一章铁过载小鼠模型的构建目的构建铁过载小鼠模型,为下一步的研究提供合格实验标本方法采用6-8周龄的KM雄性小鼠随机分为4组,腹腔注射不同剂量右旋糖酐铁,观察小鼠生长发育情况,每周称重并记录。造模结束后采集小肠、肝、脾、心脏、肾等组织及血清铁,测量铁元素,同时采集肝、脾、心脏、肾等组织制作HE和铁染色切片。结果铁过载小鼠出现外观改变,生长发育落后。观察各组织器官可见形态颜色及质地改变,呈现明显的铁沉积,检测结果显示血清铁蛋白及小肠、肝、脾、心脏、肾等组织铁含量增加,程度和注射铁的剂量成正比。HE切片发现组织器官有病理改变,铁染色切片看见有不同程度的铁沉积,与注射铁的剂量成正比。结论腹腔注射右旋糖酐铁可以构建符合后期实验要求的铁过载小鼠。第二章铁过载小鼠小肠上皮细胞差异表达miRNA的筛选与验证研究一铁过载小鼠小肠上皮细胞差异表达miRNA的筛选目的比较正常对照组和高铁组小鼠小肠细胞的高通量小RNA测序结果,筛选出铁代谢相关基因的差异表达miRNA。方法提取对照组和高铁组两组小鼠小肠上皮细胞的总RNA,构建RNA文库,选择合格文库进行高通量小RNA测序。评估测序质量、分析其特有序列,对测序数据进行基因组比对、miRNA的差异分析、差异表达miRNA的靶基因预测、GO富集分析、KEGG代谢通路分析,筛选出差异表达miRNA。对差异表达的miRNA采用Tagretscan等多个数据库进行交叉分析比较,筛选出可能靶向调控小肠铁代谢相关基因的miRNA。结果共有正常组、对照组各3个文库进行测序,共筛选出9个差异表达miRNA,通过对这9个miRNA进行比较分析,最终选择miR-143,miR let-7d进行下一步的验证。结论对两组标本进行小RNA高通量测序,对测序结果进行生物信息学分析及靶基因预测,筛选出小鼠小肠细胞铁代谢相关miRNA。研究二 miR let-7d靶向调控Slc11a2基因及miR 143靶向调控Slc40a1基因的研究目的探讨miR let-7d靶向调控Slc11a2基因及miR 143靶向调控Slc40a1基因的关系方法分别构建Slc11a2基因和Slc40a1基因3’-UTR的野生型和突变型载体,同时合成mi R let-7d mimics和mi R 143 mimics,将mi RNA与构建好的报告基因载体共转染至293T细胞,通过检测报告基因相对荧光值的改变,以此验证该mi RNA和该靶基因的调控关系。结果双荧光素酶的表达分析显示,mi R143对Slc40al基因野生型载体的荧光值没有下调作用。说明在该实验模型下,mi R143可能不能通过Slc40al基因上相应的3’UTR位点调控报告基因的表达。而mi R let-7d对Slc11a2野生型载体的荧光值有明显下调作用,下调差别有统计学意义(P0.01)。说明在该实验模型下,mi R let-7d可以通过Slc11a2基因上相应的3’UTR位点调控报告基因的表达。结论首次通过构建动物模型筛选出mi R let-7d,并证实其可以通过与Slc11a2基因上相应的3’UTR位点调控报告基因的表达,Slc11a2基因是miR let-7d的靶基因。miR143不能通过Slc40al基因上相应的3’UTR位点调控报告基因的表达,不能确定Slc40al是miR143的靶基因。第三章 miR let-7d靶向调控IEC-6细胞铁代谢功能的研究目的研究在ICE-6细胞中过表达mi R let-7d是否能靶向调控Slc11a2基因的m RNA和蛋白表达,及调控ICE-6细胞对铁的吸收功能。方法在IEC-6细胞中过表达miR let-7d,荧光显微镜观察IEC-6细胞的转染效率,采用Western Blot实验及q RT-PCR实验,检测转染后的Slc11a2蛋白表达量和扩增溶解曲线。使用含100mM的Fe SO4高铁DMEM-H培养基和普通DMEM-H培养基培养IEC-6细胞,并转染mi R let-7d mimics到细胞内,完成转染后收集细胞,对细胞进行计数后,原子吸收法测定细胞铁含量。另取培养板培养细胞进行铁染色,在倒置显微镜下观察细胞染色改变。结果转染miR let-7d mimics后荧光显微镜下观察ICE-6细胞的转染效率在75%以上,说明mi R let-7d转染IEC-6细胞成功。过表达后的Western Blot结果显示空白对照组和处理组的蛋白表达量有明显区别,转染组的Slc11a2蛋白条带量减少。q RT-PCR检测Slc11a2基因mRNA的表达量,提示转染组mRNA的表达量下降,和空白对照组比较差异有统计学意义。结论首次发现了miR let-7d可转染IEC-6细胞,miR let-7d通过靶向调控Slc11a2基因,抑制IEC-6细胞对铁的吸收。
[Abstract]:Thalassemia (thalassemia) is a hereditary chronic hemolytic anemia. Severe beta thalassemia needs to rely on long-term blood transfusions and iron removal to maintain life. Long-term blood transfusion and increased absorption of iron by small intestinal epithelial cells can lead to iron overload in patients with thalassemia. Iron overload causes liver, heart, endocrinology and so on. Organ failure is the main cause of death in patients with thalassemia. Iron removal is an important part of the treatment of severe beta thalassemia. The soluble iron is extremely low in the human body (10-18 M), the reduction of iron Fe++ and hydrogen peroxide in the hydroxyl radical, when the free iron concentration is too high, the highly reactive free radical can be found. The damaged DNA, lipid and protein can damage the organs and organs of the whole body and have potential toxicity. Therefore, it is particularly important to maintain the iron homeostasis of the human body..MicroRNAs (miRNAs) is a class of non coded RNA. The earliest found in eukaryotes that miRNAs has endogenous characteristics and can regulate a variety of biological functions.Micro RNAs as well as regulating iron homeostasis. One of the regulatory factors, miRNA has a targeted regulation of iron metabolism. From the point of view of the small intestine as the main organ of iron absorption, the differential expression of iron metabolism related proteins in small intestinal epithelial cells is screened by constructing an iron overload animal model, and the regulatory relationship between MI RNA and target genes is verified, and ICE-6 cells are used in ICE-6 cells. The overexpression of MI RNA, in which the MI RNA regulates the iron metabolism of IEC-6 cells, is expected to provide research ideas and directions for the regulation of miRNA in iron metabolism, as well as for the treatment of iron removal in thalassemia patients. KM male mice of 6-8 weeks of age were randomly divided into 4 groups. Different doses of iron dextran was injected into the abdominal cavity. The growth and development of the mice were observed and weighed and recorded every week. The small intestine, liver, spleen, heart, kidney and other tissues and serum iron were collected after the model was finished, and the iron elements were measured, and the liver, spleen, heart, kidney and other tissues were collected to make HE and iron staining. The results showed that the iron content of serum ferritin and small intestine, liver, spleen, heart, kidney and other tissues increased, the degree of range and the dose of injection iron were in direct proportion to.HE slices found in tissues and organs. There is a pathological change. Iron staining section shows a different degree of iron deposition and is proportional to the dose of the iron injection. Conclusion intraperitoneal injection of dextran iron can construct the iron overload mice that meet the requirements of the later experiment. Second the screening and verification of differential expression of miRNA in small intestinal epithelial cells of iron overload mice Screening of differential expression of miRNA in skin cells compared the high throughput small RNA sequencing results of small intestinal cells in normal control group and high iron group, screening the differential expression of iron metabolism related genes by differential expression miRNA. method to extract the total RNA of small intestinal epithelial cells of two groups of mice in the control group and the high iron group, construct the RNA library and select the qualified library for high flux. Small RNA sequencing, evaluation of the quality of sequencing, analysis of its unique sequence, genomic alignment of sequencing data, miRNA difference analysis, differential expression of miRNA target gene prediction, GO enrichment analysis, KEGG metabolic pathway analysis, screening and screening of differential expression miRNA. for miRNA acquisition of different expressions of Tagretscan and other databases for cross analysis and comparison. The miRNA. results that could be targeted to regulate the genes related to iron metabolism in the small intestine were selected. The control group was sequenced in 3 libraries, and 9 different expressions of miRNA were screened. Through the comparison and analysis of the 9 miRNA, the final selection of miR-143, miR let-7d was verified by the next step. Conclusion high throughput sequencing of the two groups of specimens was carried out. Bioinformatics analysis and target gene prediction of sequencing results were used to screen the iron metabolism related miRNA. study of mouse small intestinal cells two miR let-7d target regulation Slc11a2 gene and miR 143 targeted Slc40a1 gene, and the relationship between miR let-7d targeting Slc11a2 gene and miR 143 target to control Slc40a1 gene The wild type and mutant vector of Slc11a2 gene and Slc40a1 gene 3 '-UTR were constructed respectively, and MI R let-7d mimics and MI R 143 mimics were synthesized, and MI RNA and constructed reporter gene carrier were co transfected to the cell. The expression analysis of double luciferase showed that MI R143 had no down-regulation on the fluorescence value of the Slc40al gene, which indicated that MI R143 could not regulate the expression of the reporter gene by the corresponding 3 'UTR locus on the Slc40al gene. And the MI R let-7d decreased the fluorescence of the Slc11a2 wild type carrier. The difference was statistically significant (P0.01). Under this experimental model, MI R let-7d could regulate the expression of the reporter gene through the corresponding 3 'UTR locus on the Slc11a2 gene. Conclusion the first animal model was first screened for MI R let-7d, and it was confirmed that it could be regulated by the corresponding 3' UTR locus on the Slc11a2 gene. Because of the expression, the Slc11a2 gene is the target gene of miR let-7d,.MiR143 can not regulate the expression of the reporter gene by the corresponding 3 'UTR locus on the Slc40al gene, and the target gene of Slc40al is not determined. The aim of the third chapter miR let-7d to regulate the metabolic function of IEC-6 cells is to study the overexpression in the ICE-6 cells. To regulate the expression of M RNA and protein of Slc11a2 gene, and to regulate the absorption function of ICE-6 cells to iron. Methods the expression of miR let-7d in IEC-6 cells was overexpressed in IEC-6 cells. The transfection efficiency of IEC-6 cells was observed by fluorescence microscopy. Western Blot experiment and Q RT-PCR experiment were used to detect the expression of the protein and the expansion and dissolution curve of the transfected protein. IEC-6 cells were cultured with 100mM Fe SO4 high iron DMEM-H medium and ordinary DMEM-H medium, and IEC-6 cells were transfected with MI R let-7d mimics. After transfection, the cells were collected. After counting the cells, the iron content was measured by atomic absorption spectrometry. The culture plate culture cells were also stained with iron, and the cell staining changes were observed under the inverted microscope. Results after transfection of miR let-7d mimics, the transfection efficiency of ICE-6 cells was more than 75% under the fluorescence microscope, indicating that MI R let-7d transfected to IEC-6 cells successfully. The results of Western Blot after overexpression showed that the protein expression of the blank control group and the treated group was distincently different, and the Slc11a2 protein band in the transfected group was reduced by.Q. The expression of gene mRNA indicated that the expression of mRNA in the transfected group decreased, and there was a significant difference between the control group and the blank control group. The conclusion was that miR let-7d could be transfected to IEC-6 cells for the first time, and miR let-7d regulated the Slc11a2 gene by targeting and inhibited the absorption of iron by IEC-6 cells.

【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R556.61

【参考文献】