【作者】 罗维；

【导师】 苏金为； 胡宝成；

【作者基本信息】 福建农林大学 ， 细胞生物学， 2012， 硕士

【摘要】 细胞外基质磷酸糖蛋白(MEPE)是细胞外基质蛋白，它可以调节体内磷酸盐平衡，以及在骨骼和牙本质形成的形成过程中起到重要的作用。2000年，Rowe等首次在瘤源性软骨症中(TIO)的细胞中发现了细胞外基质磷酸糖蛋白。Petersen等人也在当年发现了细胞外基质磷酸糖蛋白基因，最初命名这个基因为成骨细胞/骨细胞因子45(Osteoblast/Osteocyte Factor45, OF45)，后来又发现发现成骨细胞/骨细胞因子45与人MEPE同源，因此细胞外基质磷酸糖蛋白基因也称为Mepe/Of45。微小分子RNA (microRNA, miRNA)是形成于内源性发卡结构转录本的长度为19到25核苷酸的单链RNA。通过和mRNAs的结合，从而抑制mRNA的翻译，达到降解mRNA的目的，还有抑制转录的目的。我们实验室的前期研究发现了MEPE/OF45蛋白参与了DNA损伤应答反应，并且能够提高大鼠及人细胞对DNA损伤诱导剂的耐受性。对MEPE/OF45功能的研究能够为以后寻找理想的放化疗药物提供理论基础。近年来，大量研究结果表明：miRNA在生物体很大范围的生物过程中发挥着重要作用，至少调控了30%的动物基因的表达。本研究旨在发现靶向Mepe的miRNA，并研究其在DNA损伤应答过程中所发挥的功能，为进一步研究MEPE/OF45及MEPE/OF45相关蛋白所涉及的细胞内信号通路机制奠定基础。首先，通过NCBI检索人源Mepe的3’UTR，我们利用miRNA预测工具TargetScan预测可能靶向Mepe所有可能的microRNA，可能存在36条靶向Mepe的miRNA，根据预测分值的高低，选择6条miRNA进行验证，通过双荧光素酶报告基因实验，检测筛选出来的6条miRNA与Mepe3’UTR的结合情况，从而初步筛选出可能靶向Mepe的miRNA。研究发现与转染空载体pGL3-cm的相对荧光素值相比，转染miR-376a的相对荧光素值降低较为明显，而当Mepe3’UTR与miR-376a结合位点突变后，miR-376a则不能抑制荧光素酶的活性。同时，Western印迹分析结果显示miR-376a能够明显抑制MEPE蛋白的表达，miRNA-376a可能是靶向Mepe基因的miRNA。随后，我们对于miR-376a的功能进行了探索，在细胞生长周期的实验中，我们发现对细胞辐射后12h，转染miR-376a的细胞与转染空载体pcDNA对照的细胞相比G2期阻滞更弱；而后我们又进行了MTT实验，实验表明在DNA损伤诱导剂处理后，转染miR-376a的HeLa细胞对DNA损伤诱导剂的耐受性减弱，变得更敏感。实验表明miR-376a可能抑制MEPE蛋白的表达，从而降低了细胞对DNA损伤诱导剂的耐受性。本研究通过生物信息学及实验生物学方法，获得了一个靶向Mepe的microRNA，为进一步研究MEPE在DNA损伤应答中的作用机制奠定了基础，也为研发新的miRNA放化疗药物奠定了理论基础。更多还原

【Abstract】 Matrix Extracellular Phosphoglycoprotein (MEPE) had been limited to regulation ofbone metabolism, phosphate homeostasis, and dentin. MEPE was first found intumor-induced osteomalacia (the tumor-induced osteomalacia, TIO) by Rowe in2000, inthe same year Petersen and his colleague found the gene which was originally named asOsteoblast/Osteocyte Factor45(OF45) when they screened significant differentiallyexpressed novel gene in a rat differentiated and undifferentiated osteoblasts. MEPE andOF45are homologous genes, Mepe/Of45. Subsequently MEPE/Mepe homologousprotein in mice was also found.microRNAs (miRNAs) represent a class of naturally occurring small noncodingRNA molecules, distinct from but related to siRNAs. Mature miRNAs are19-to25-nucleotide-long molecules cleaved from70-to100-nucleotide hairpin pre-miRNAprecursors. In animals, single-stranded miRNAs bind, through partial sequence homology,to the3’-UTR of target mRNAs and cause a block of translation or, less frequently,mRNA degradation.We previously reported an important function of MEPE/OF45in protecting cellsfrom DNA damage induced killing, which would provide MEPE/OF45as a new targetfor sensitizing tumor cells to radiotherapy or chemotherapy.miRNAs are estimated to target more than30%of animal genes, suggesting thatthey may exert a large combinatorial outcome in biological processes. In this study, wewill focus on selection of miRNAs targeting Mepe and its function in DNA damageresponse, it will be basis to study the signal pathway of MEPE/OF45and its relatedproteins in the cells.Here Human Mepe-3’UTR was searched by NCBI website, and Targetscan was usedto predict the candidate miRNAs targeting Mepe,36candidate miRNAs targeting Mepewere found, and only6predicated miRNAs targeting Mepe were selected by contextscore percentile and consequential pairing and were identified by using dual luciferaseassay. Luciferase analysis showed that the activity of wild type3’UTR reporter wassignificantly suppressed by miR-376a, suppression by miR-376a depends on the wildtype miR-376a complementary sites, and was not longer observed in reporter in whichmiR-376a complementary sites were mutated, so miR-376a directly targets Mepe and miR-376a can repress the translation of Mepe gene in HeLa cells by Western blotting.The results show that introduction of miR-376a expression in human cells resulted indecrease of G2phase arrest following ionizing radiation (IR) by the flow cytometry, andoverexpression of miR-376a sensitized the cells to DNA damage inducers. All theseresults proved that miR-376a sensitized the cells to DNA damage by repressing theexpression of MEPE.In this study, a miRNA targeting Mepe was obtained by bioinformatics andexperimental biology, it will be basis to study potential drugs for radiotherapy orchemotherapy.更多还原