M.Tech. / RNA interference (RNAi) is a gene silencing mechanism that uses short RNA duplexes to block gene expression. This mechanism has been widely explored to determine functions of genes. Furthermore, this phenomenon has been used to silence unwanted genes such as viral genes. RNAi has been successfully employed in non-mammalian organisms such as plants, where long dsRNAs (more than 30 bp) have been used without inducing non-specific effects. However, in mammalian cells, cytoplasmic dsRNAs of more than 30 bp trigger non-specific induction of many genes, which may result from the activation of dsRNA-dependent protein kinase (PKR) and 2’,5’-oligoadenylate synthetase (2’,5’-OAS), via the interferon response pathway. In this study, we describe a novel and simple strategy to overcome nonspecific effects induced by longer RNA duplexes. This strategy uses sodium bisulphite which is a mutagen that deaminates cytosine residue to uracil residues in order to introduce mutations in the sense strand of the duplex. Introduction of these mutations results in the formation of G:U pairings between the sense and antisense strands of the long hairpin RNA. RNA duplexes with mismatches have been shown to be able to prevent interferon induction in mammalian cells. According to the obtained results, long hairpins RNA with and without mismatches were unable to inhibit the expression of the target region, which was the U5 region of the HIV-1 subtype C LTR. The U5 region of the LTR is actively involved in the reverse transcription of HIV-1. Therefore silencing of this region would have led to the inhibition or reverse transcription blockage. Furthermore, data showed that the interferon response was induced when using these long hairpin RNA duplexes. Due to the sensitivity of mammalian cells, the action of sodium bisulphite could have stimulated certain genes of the interferon pathway. Even though hairpins constructed in this study were unable to prevent the induction of the interferon response pathway and also could not silence the target, this strategy of using sodium bisulphite has a great potential as shown by its ability to induce changes in cytosine residues and leaving other nucleotides unchanged.

No description available.

成人骨量的更新与维持通过骨重塑来实现。骨重塑包括骨吸收与骨形成两个偶联的过程，其中成骨细胞介导骨形成，破骨细胞介导骨吸收，当偶联的骨吸收超过骨吸收就会导致骨量丢失，进而导致发生骨质疏松症的发生。目前，临床治疗骨质疏松的药物如阿仑膦酸盐、雌激素受体调节剂、活性维生素D、雌激素替代治疗、降钙素、骨化三醇等都是基于针对破骨细胞的调控来抑制骨吸收，但是对于已经丢失的骨量无法恢复。唯一被美国FDA批准用来通过刺激新骨形成来恢复丢失的骨量的治疗药物就是甲状旁腺激素。然而，这种药物在刺激新骨形成的同时也刺激了骨吸收，即：在使用18个月后有明显促进骨吸收的副作用。 / 酪蛋白激酶相互作用蛋白-1（CKIP-1）基因是一个新发现的骨形成的负调控基因，CKIP-1基因敲除小鼠在骨发育和正常骨代谢过程中均未发现激活骨吸收。CKIP-1敲除导致小鼠胫骨近端骨量与胫骨皮质骨形成速率显著高于野生型，且这一差异随着小鼠的增龄而显著，而骨外器官没有发现异常表型，提示CKIP-1是潜在相对安全的治疗骨质疏松的靶向基因。特别是我们最近研发的一种天门冬氨酸－丝氨酸－丝氨酸重复三肽修饰的脂质体递送（(Asp-Ser-Ser)₆-liposome）系统能够实现靶向骨形成表面的小干扰核酸的递送，并明显减少了小干扰核酸在非骨组织的分布。因此，提出本课题的研究假设：特异性静默骨内CKIP-1可以促进骨形成而不刺激骨吸收，从而为骨质疏松的临床治疗提供安全有效的治疗手段。 / 为了确定CKIP-1基因表达在老年绝经后妇女骨骼中与骨形成内在联系，首先，我们通过对发生骨折的老年绝经后妇女的骨痂标本中CKIP-1 mRNA和蛋白表达的测定，发现CKIP-1基因mRNA和蛋白表达水平与骨形成能力负相关。并且，这种相关性在骨质疏松动物模型中进一步得到证实。其次，针对我们研究假设，从一组针对大鼠、小鼠、猴和人类的成骨样细胞的CKIP-1 mRNA的跨种属siRNA序列中筛选出体外静默效率最高CKIP-1小干扰核酸序列si-3。接着，体内外实验证实si-3序列在健康动物体内的静默效率和促进成骨的功能。同时，确定尾静脉注射（Asp-Ser-Ser）₆-liposome 包裹的CKIP-1小干扰核酸在 大鼠和小鼠为的最佳剂量分别为3.75mg/kg和7.5mg/kg以及注射周期为每两周一次。最后，为了检验CKIP-1 小干扰核酸是否可通过促进骨形成从而逆转绝经后骨质疏松症中的骨丢失，我们分别以绝经后骨质疏松大鼠和小鼠为实验动物模型，通过测定骨形态计量学参数、骨量和骨结构等来评价骨靶向递送系统（(Asp-Ser-Ser)₆-liposome）递送的CKIP-1 siRNA对老年绝经后骨质疏松症的治疗效果。动态活体CT分析结果表明，与0周未治疗的基础值相比，经6周治疗骨密度（BMD）, 相对骨体积分数（BV/TV）和骨小梁厚度（Tb.Th）在小干扰核酸治疗组显著增加。此外，在治疗6周后小干扰核酸治疗组骨密度，相对骨体积和骨小梁厚度显示较高于模型对照组。0周与其它检测时间点之间的对比分析较显示，小干扰核酸治疗组的新生骨显著高于模型组或假手术组。组织形态学分析结果表明在治疗6周后，无论是股骨远端或中段的矿化沉积率（MAR）、骨形成速率（BFR） 和组的骨形成表面（Ob.S/ BS）在OVX组和siRNA组均显著高于模型对照组，而模型对照组和小干扰核酸治疗组的骨吸收表面（Oc.S/ BS）之间无显著性差异。 / 结论：CKIP-1基因小核酸干扰治疗在老年绝经后骨质疏松中能够显著促进骨形成并不会加剧骨吸收，该药物具有显著逆转骨丢失的作用。 / Osteoporosis is characterized by an imbalance between bone formation and bone resorption. Therefore, promoting bone formation and inhibiting bone resorption are the two major therapeutic strategies in the treatment of osteoporosis. Currently, the only Food and Drug Administration (FDA)-approved anabolic agent capable of stimulating bone formation is parathyroid hormone (PTH). However, dominant bone resorption after 18-month treatment with PTH is a great concern (Rubin and Bilezikian 2003). Thus, development of alternative bone anabolic agents is highly desirable. / Casein kinase-2 interacting protein-1 (CKIP-1), which is encoded by Plekho1, and thus also known as Plekho1, is a newly discovered negative regulator of bone formation during bone development and subsequent bone maintenance that does not activate bone resorption (Lu, Yin et al. 2008). Specifically, CKIP-1 protein functions as the auxiliary factor of ubiquitin ligase Smad ubiquitylation regulatory factor 1 (Smurf1) to interrupt the bone anabolic BMP-signalling pathway, which has been demonstrated to be a specific suppressor of bone formation (Yamashita, Ying et al. 2005). In a previous study, we found that CKIP-1 expression in female rat bone increases with aging, whereas bone formation decreases with aging (Guo, Zhang et al. 2010). Systemic examination of the tissue distribution of CKIP-1 expression has revealed that is abundantly expressed in the musculoskeletal system but sparingly expressed in the liver, lungs, kidneys, pancreas, and other organs (Zhang, Tang et al. 2007). In addition, an abnormal tissue phenotype in heart, liver, spleen, lung, and kidney tissue has not been observed in CKIP-1 gene knockout mice (KO), even at an advanced age (Lu, Yin et al. 2008). Thus, CKIP-1 gene silencing might be a potential strategy for promoting bone anabolic action in reversing bone loss. / RNA interference (RNAi), a natural cellular process that regulates gene expression by a highly precise mechanism of sequence-directed gene silencing at the stage of translation by degrading specific messenger RNA and then blocking translation of the specific gene, has been employed for gene silencing in vivo (Frank-Kamenetsky, Grefhorst et al. 2008). Accordingly, RNAi should be an appropriate target for CKIP-1 gene silencing in vivo. / We raised the hypothesis that therapeutic RNAi targeting of CKIP-1 might promote bone formation for reversing postmenopausal bone loss. To test the hypothesis, we performed several studies to achieve the following specific aims: (1) To explore the relationship between CKIP-1 expression and bone formation in aged postmenopausal osteoporosis; (2) To Identify a cross-species CKIP-1 siRNA sequence with high knockdown efficiency; (3) To validate of the identified CKIP-1 siRNA in healthy rodents in vivo; (4) To examine the anabolic effect of the identified CKIP-1 siRNA on bone in osteoporotic animal models. / The relationship between CKIP-1 gene expression and bone formation in bone specimens from aged postmenopausal women: To explore the association between CKIP-1 gene expression and bone formation in bone specimens from aged postmenopausal women, the gene expression of CKIP-1 and ALP in the bone specimens from aged female patients were examined. We found the protein expression of CKIP-1 increased during aging and negatively correlate to bone formation as indicated by the mRNA expression of ALP (Guo., Zhang. et al. 2011). Further, we also found the decreased bone formation during aging was partly rescued in Ckip-1 KO mice during aging. / A cross-species CKIP-1 siRNA sequence: Recently, we identified a specific CKIP-1 siRNA sequence (CKIP-1 siRNA si-3) with high knockdown efficiency across rat, mouse, rhesus, and human osteoblast-like cells that does not induce immunostimulatory activity and promotes osteoblast differentiation across the species in vitro and bone formation in rats in vivo (Guo, Zheng et al. 2012). / Validation of the CKIP-1 siRNA si-3 capsulated by bone-targeted siRNA delivery system in healthy rodents in vivo: We developed a bone-targeting siRNA delivery system (tripeptide aspartate-serine-serine linked with liposome, i.e. (Asp-Ser-Ser)₆-liposome) that can remarkably reduce the exposure of non-bone tissue to CKIP-1 siRNA (Zhang, Guo et al. 2012). To validate the identified CKIP-1 siRNA in healthy rodents in vivo, the established continuous CKIP-1 gene silencing protocol is optimized in adult rats and mice in vivo by hydrodynamic tail vein injection of 3.75mg/kg for rats and 7.5 mg/kg for mice every 2 weeks (Guo, Zhang et al. 2010). The osteogenic effects of CKIP-1 siRNA in both rats and mice were further validated in vivo. / Anabolic effect of CKIP-1 siRNA si-3 on bone in aged postmenopausal osteoporosis: For evaluation of the anabolic effect of CKIP-1 siRNA si-3 on reversing bone loss due to osteoporosis in an animal model, we intravenously injected ovariectomized (OVX) rats and mice with CKIP-1 siRNA delivered by the (Asp-Ser-Ser)₆-liposome, a liposome linked with six repeated aspartate-serine-serine moiety, every 2 weeks for 6 weeks. In vivo and ex vivo microCT analysis demonstrated a change over time in the variables examined and different change patterns over time among the groups examined after administration. We found that the siRNA group had experienced a significant increase in bone mineral density (BMD), relative bone volume (BV/TV), and trabecular thickness (Tb.Th) between weeks 0 and 6; had a higher BMD, BV/TV, and Tb.Th compared to the OVX group at week 6; and had a similar Tb.Th to that of the SHAM group at week 6. Registration analysis between week 0 and other time points revealed that the siRNA had a greater number of newly formed bone than the OVX and SHAM groups. Histomorphometric analysis showed that the siRNA group had a significantly higher mineralization rate (MAR), a significantly higher bone-formation rate (BFR), a significantly larger osteoblast surface (Ob.S/BS) at both the distal and mid-shaft femur compared to the OVX group after 6 weeks of treatment but not a significantly different Oc.S/BS. / Significance: Confirmation of our hypothesis by our results helps establish CKIP-1’s role as a pivotal negative regulator of bone formation in the aging skeleton and provides evidence that inhibiting CKIP-1 is a novel anabolic treatment for osteoporosis, indicating great potential for the use of therapeutic RNAi in orthopaedics and traumatology. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Guo, Baosheng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves [132-150]). / Abstract also in Chinese. / Declaration --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / 论文摘要 --- p.vii / Table of Content --- p.ix / Abbreviations --- p.xvii / List of Figures --- p.xix / List of Tables --- p.xxii / Chapter CHAPTER 1 --- Review of recent anabolic therapy for osteoporosis --- p.1 / Chapter 1.1. --- Epidemiology of postmenopausal osteoporosis --- p.1 / Chapter 1.1.1. --- Definition of osteoporosis --- p.1 / Chapter 1.1.2. --- Epidemiology and health challenge of postmenopausal osteoporosis --- p.2 / Chapter 1.2. --- General pathophysiological understanding of osteoporosis and current challenge for osteoporosis treatment --- p.3 / Chapter 1.2.1. --- Bone modeling and remodeling --- p.3 / Chapter 1.2.2. --- Pathophysiological process of osteoporosis --- p.4 / Chapter 1.2.3. --- Systemic risk factors in the pathophysiology of osteoporosis --- p.5 / Chapter 1.2.4. --- Local risk factors in the osteoporosis pathophysiology --- p.6 / Chapter 1.2.5. --- Two therapeutic strategies for osteoporosis treatment --- p.7 / Chapter 1.3. --- Current and potential anabolic agents for osteoporosis treatment --- p.8 / Chapter 1.3.1. --- PTH analogues --- p.8 / Chapter 1.3.2. --- Potential concerns regarding PTH administration --- p.9 / Chapter 1.3.3. --- Potential PTH alternatives --- p.10 / Chapter 1.3.4. --- Modulation of Wnt/β-cateinin pathway --- p.10 / Chapter 1.3.5. --- Aptamer-based technology in osteoporosis treatment --- p.14 / Chapter 1.4. --- CKIP-1: A novel negative regulator of bone formation --- p.15 / Chapter 1.4.1. --- TGF-β/BMP signaling pathways involved in regulating bone formation --- p.15 / Chapter 1.4.2. --- CKIP-1 interrupts BMP signaling pathway --- p.16 / Chapter 1.4.3. --- CKIP-1 negatively regulates bone formation without activating bone resorption --- p.17 / Chapter 1.5. --- RNA interference strategy in anabolic therapy of osteoporosis --- p.18 / Chapter 1.5.1. --- siRNA-mediated gene silencing in osteoporosis treatment --- p.18 / Chapter 1.5.2. --- MicroRNAs as potential therapeutic targets in the anabolic treatment of osteoporosis --- p.20 / Chapter 1.5.3. --- Bone targeted RNAi-based anabolic-agents delivery --- p.23 / Chapter 1.6. --- Summary --- p.24 / Chapter CHAPTER 2 --- The relationship between CKIP-1 expression and bone formation in aged postmenopausal osteoporosis --- p.26 / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and methods --- p.28 / Chapter 2.2.1 --- Bone specimen collection from aged postmenopausal women --- p.28 / Chapter 2.2.2 --- Total RNA extraction, reverse transcription and quantitative real-time PCR --- p.28 / Chapter 2.2.3 --- Total protein extraction and western blot analysis --- p.30 / Chapter 2.2.4 --- CKIP-1 expression in bone and other tissues --- p.31 / Chapter 2.2.5 --- Relationship between CKIP-1 expression and bone formation in aged ovariectomized rats --- p.31 / Chapter 2.2.6 --- Role of CKIP-1 in regulating bone formation in aged ovariectomized mice --- p.32 / Chapter 2.2.7 --- Statistics --- p.32 / Chapter 2.3 --- Results --- p.33 / Chapter 2.3.1 --- Correlation analysis between CKIP-1 expression and bone formation-related gene expression in bone specimens from agedd postmenopausal women across age --- p.33 / Chapter 2.3.2 --- CKIP-1 gene expression pattern in bone and other tissues --- p.37 / Chapter 2.3.3 --- Correlation between CKIP-1 expression and bone formation in rat bone --- p.38 / Chapter 2.3.4 --- CKIP-1 negatively regulates bone formation in aged ovariectomized mice by using CKIP-1 knockout mice --- p.39 / Chapter 2.4 --- Summary --- p.41 / Chapter CHAPTER 3 --- Identification of a cross-species CKIP-1 siRNA sequence --- p.43 / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.2 --- Materials and methods --- p.44 / Chapter 3.2.1 --- Design rationale and modification for cross-species CKIP-1 siRNA --- p.44 / Chapter 3.2.2 --- In vitro screening for cross-species CKIP-1 siRNA sequences --- p.45 / Chapter 3.2.3 --- Investigation of the effects of the identified CKIP-1 siRNA on the expression of osteoblast phenotype genes --- p.47 / Chapter 3.2.4 --- Total RNA extraction, reverse transcription and quantitative real-time PCR --- p.47 / Chapter 3.2.5 --- Western blot analysis --- p.51 / Chapter 3.2.6 --- Evaluation of calcium deposition --- p.51 / Chapter 3.2.7 --- BMP-2 reporter activity assay in MC3T3-E1 cells --- p.52 / Chapter 3.2.8 --- Isolation of the primary human blood monocytes and IFN-α and TNF-α measurement --- p.53 / Chapter 3.2.9 --- Statistics --- p.54 / Chapter 3.3 --- Results --- p.54 / Chapter 3.3.1 --- Bio-informatic analysis of the designed CKIP-1 siRNA sequences --- p.54 / Chapter 3.3.2 --- Identified the cross-species CKIP-1 siRNA sequences by In vitro screening --- p.56 / Chapter 3.3.3 --- Effects of the identified CKIP-1 siRNA on the expression of osteoblast phenotype genes --- p.60 / Chapter 3.3.4 --- Effects of the identified CKIP-1 siRNA on matrix mineralization --- p.65 / Chapter 3.3.5 --- Effect of the identified CKIP-1 siRNA on BMP signaling --- p.67 / Chapter 3.3.6 --- Effects of the identified CKIP-1 siRNA on the ratio of RANKL/OPG --- p.67 / Chapter 3.3.7 --- Effects of the identified CKIP-1 siRNA on immunostimulatory activity --- p.68 / Chapter 3.4 --- Summary --- p.71 / Chapter 3.4.1 --- CKIP-1 siRNA si-3 as the identified sequence --- p.71 / Chapter 3.4.2 --- CKIP-1 siRNA si-3 promoted osteoblast differentiation in vitro --- p.72 / Chapter CHAPTER 4 --- Validation of the identified CKIP-1 siRNA in healthy rodents in vivo --- p.74 / Chapter 4.1 --- Introduction --- p.74 / Chapter 4.2 --- Materials and methods --- p.74 / Chapter 4.2.1 --- Localization of intraosseous siRNA delivered by (Asp-Ser-Ser)₆-liposome --- p.75 / Chapter 4.2.2 --- Cell-selective delivery in vivo of CKIP-1 siRNA --- p.76 / Chapter 4.2.3 --- Dose-response study of CKIP-1 siRNA --- p.77 / Chapter 4.2.4 --- Time-course study of CKIP-1 siRNA --- p.77 / Chapter 4.2.5 --- Examination of the effect of the identified siRNA on the expression of osteoblast phenotype genes --- p.78 / Chapter 4.2.6 --- Measurement for serum PINP and urinary DPD --- p.80 / Chapter 4.2.7 --- 5’-RACE Analysis --- p.81 / Chapter 4.2.8 --- Laser captured micro-dissection (LCM) --- p.82 / Chapter 4.2.9 --- Evaluation the anabolic effect of the identified siRNA on healthy rat bone --- p.82 / Chapter 4.2.10 --- Evaluation the anabolic effect of the identified siRNA on healthy mouse bone --- p.84 / Chapter 4.2.11 --- Micro CT analysis --- p.84 / Chapter 4.2.12 --- Dynamic bone histomorphometric analysis --- p.85 / Chapter 4.2.13 --- Statistics --- p.86 / Chapter 4.3 --- Results --- p.87 / Chapter 4.3.1 --- Rationale of bone targeted delivery of CKIP-1 siRNA by (Asp-Ser-Ser)₆-liposome --- p.87 / Chapter 4.3.2 --- Intraosseous distribution of siRNA delivered by (Asp-Ser-Ser)₆-liposome --- p.89 / Chapter 4.3.3 --- Optimal dosage and duration for CKIP-1 siRNA administration in vivo --- p.92 / Chapter 4.3.4 --- Knockdown efficiency of CKIP-1 siRNA in osteoblasts by LCM in combination with Q-PCR --- p.94 / Chapter 4.3.5 --- Examination of the effect of the identified siRNA on the expression of osteoblast phenotype genes --- p.96 / Chapter 4.3.6 --- RNAi mechanism of CKIP-1 siRNA action in vivo --- p.99 / Chapter 4.3.7 --- Anabolic effect of the identified siRNA on healthy rat bone --- p.101 / Chapter 4.3.8 --- Anabolic effect of the identified siRNA on healthy mouse bone . --- p.104 / Chapter 4.4 --- Summary --- p.107 / Chapter 4.4.1 --- Intraosseous localization of CKIP-1 siRNA after systemic administration --- p.107 / Chapter 4.4.2 --- Evidence of RNAi in bone tissue from systemic administration of CKIP-I siRNA --- p.107 / Chapter 4.4.3 --- CKIP-1 siRNA si-3 promots bone formation in rats and mice in vivo --- p.108 / Chapter CHAPTER 5 --- Anabolic effect of the identified CKIP-1 siRNA on bone in postmenopausal osteoporostic animal models --- p.110 / Chapter 5.1. --- Introduction --- p.110 / Chapter 5.2. --- Materials and Methods --- p.110 / Chapter 5.2.1. --- Evaluation of anabolic effect of CKIP-1 siRNA on osteoporotic mouse bone --- p.111 / Chapter 5.2.2. --- Evaluation of anabolic effect of CKIP-1 siRNA on osteoporotic rat bone --- p.112 / Chapter 5.2.3. --- In vivo micro-CT analysis and registration of proximal tibia from osteoporotic rats --- p.112 / Chapter 5.2.4. --- Ex vivo micro-CT analysis of the distal femur and 5th lumbar vertebrae body of osteoporotic rats --- p.115 / Chapter 5.2.5. --- Ex vivo micro-CT analysis of distal femur from osteoporotic mice --- p.115 / Chapter 5.2.6. --- Bone histomorphometric analysis --- p.116 / Chapter 5.2.7. --- Mechanical testing --- p.117 / Chapter 5.2.8. --- Statistics --- p.118 / Chapter 5.3. --- Results --- p.116 / Chapter 5.3.1. --- Anabolic effect of CKIP-1 siRNA si-3 on osteoporotic mouse bone --- p.118 / Chapter 5.3.2. --- In vivo microCT data of proximal tibia from aged osteoporotic rats --- p.121 / Chapter 5.3.3. --- Ex vivo microCT data of distal femur from aged osteoporotic rats --- p.124 / Chapter 5.3.4. --- Ex vivo microCT data of 5th LV body from aged osteoporotic rats --- p.126 / Chapter 5.3.5. --- Bone histomorphometric analysis of aged osteoporotic rats --- p.129 / Chapter 5.3.6. --- Mechanical testing of the mid-shaft femur of aged osteoporotic rats --- p.132 / Chapter 5.4. --- Summary --- p.134 / Chapter CHAPTER 6 --- Discussions --- p.134 / Chapter 6.1 --- CKIP-1 siRNA design rationale and further modification --- p.135 / Chapter 6.1.1 --- Specificity design rationale of the CKIP-1 siRNA --- p.135 / Chapter 6.1.2 --- Stability enhancing modification of CKIP-1 siRNA --- p.136 / Chapter 6.1.3 --- Safety concerns with CKIP-1 siRNA therapy --- p.136 / Chapter 6.2 --- Development of bone-targeted siRNA delivery --- p.136 / Chapter 6.3 --- Prospects for and limitation of application of study findings to clinical therapeutics --- p.137 / References --- p.139 / Publications --- p.159

By performing microRNA microarrays to globally detect any novel miRNAs in the limbus, eleven microRNAs (hsa-miR-136, hsa-miR-373*, hsa-miR-150, hsa-miR-143, hsa-miR-455, hsa-miR-145, hsa-miR-381, hsa-miR-224, hsa-miR-338, hsa-miR-154, hsa-miR-377) were found to be upregulated while two microRNAs (hsa-miR-122a and hsa-miR-425-3p) were identified as downregulated by more than 2 folds. Among these, hsa-miR-143 and hsa-miR-145 were distingushed to be the most significantly up-regulated limbal miRNAs. Individual assessement of the microarray results of a recently reported stem cell specific microRNA, hsa-miR-21, were also upregulated by more than two thousand fold when comparing limbus and cornea. miR-21, miR-143 and miR-145 were therefore selected as the most likely microRNA candidates in the present study. The expression level of these miRNA candidates were validated and confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). To localize these candidates, we performed in situ hybridization on frozen corneal rim sections using locked nucleic acid (LNA)-modified oligonucleotide probes. Results showed that miR-2I, 143 and 145 were confined in the limbal region with gradation of expression level along the basal-suprabasal line. / Functional roles of these microRNAs were then deciphered by overexpressing human corneal epithelial cell line (HCE) with precursor microRNAs (pre-miRs) through lipophilic transfection. Results showed that high endogenous level of miR-145 could inhibit cell proliferation by 3.5 fold as shown from MTT proliferation assay at day 5, and could generate discrete spherical colonies that resembles the morphology of holoclones at day 8, but not the other two candidate miRNAs. / In conclusion, 1 have identified three novel microRNAs (hsa-miR-21, 143, 145) which were precisely upregulated in the limbus region, while miR-145 was being the most limbal specific. In addition, the functions of miR-145 were found to be inhibitory on cell proliferation, possibly through the indirect regulation of IFNB1. These unprecedented results may suggest a therapeutic potential of miR-145 on limbal stem cell deficiency and limbal tumors because miR-145 can affect cell survival and proliferation. / MicroRNAs is a family of small non-coding RNAs that, in human, binds imperfectly to the 3' untranslated region (UTR) of target mRNAs for translational repression or negative regulation. Recent studies have shown that such negative regulatory pathways may play pivotal roles in the maintenance of asymmetric cell division in embryonic and tissue specific stem cells. Human corneal epithelial progenitor cells (CEPC), a tissue specific stem cell lineage residing between cornea and conjunctiva in the Palisade of Vogt of the limbus region, is known to maintain corneal homeostasis throughout human life. They respond to injury and normal wearing by rapid proliferation and differentiation into transit amplifying cells (TACs) and eventually corneal epithelial cells, though the biological factors controlling this homeostatic switch are still largely unknown. Here I hypothesized that microRNAs can participate in CEPC regulation. Experiments elucidating the anatomical distribution and functional roles of microRNAs on the human cornea rims were performed to testify this proposition. / Protocols aim at enriching the CEPC population were then devised. For the first time a four parameter cell sorting system utilizing ABCG2, Connexin 43, Notch 1 and pyronin Y as markers was established for the prospective in vitro study. Nevertheless, manual microdissection isolating the limbus region and the cornea region was employed for the present study of microRNAs. / This study begins with the phenotypic validation of human cornea rims recruited from the Chinese Hong Kong population using immunohistochemistry. Conventional CEPC markers (p63, EGFR, cytochrome oxidase and cytokeratin 15), embryonic stem cell marker (stat1) and cancer stem cell markers (p73, MDM2 and pStat1) were expressed in the limbus region, suggesting that these specimens contained a source of CEPC for attesting our hypothesis. / To determine the mRNA targets of candidate microRNAs in HCE cells, Whole Human Genome Oligo Microarray Kits (Agilent Technologies) which contained 41K human genes and transcripts were employed. When compared to the scrambled control, HCE cells over-expressed with hsa-miR-21, 143 or 145 revealed differential expression of genes that participate in cell activation, motility and proliferation. Of note, interferon beta 1 fibroblast (IFNB1), a gene that is often deleted or rearranged in cancers, was significantly upregulated by a medium of 1093 fold in pre-miR-145 treated cells as confirmed by real time PCR assays. / Lee, Sharon Ka-wai. / "December 2009." / Advisers: Calvin Chi-Pui Pang; Gary Hin Fai Yam. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 216-252). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Based on matching analysis between different EBV strains, we found two nucleotide variations in miR-BART21 and four nucleotide changes in miR-BART22. Interestingly, two nucleotide variations upstream of mature miR-BART22 likely favor its biogenesis by Drosha/DGCR8 processing and we experimentally confirmed this augmentation by in-vitro Drosha digestion, and thus may underline the high and consistent expression of miR-BART22 in NPC tumors. / Infection with the Epstein-Barr virus (EBV) is a strong predisposing factor in the development of nasopharyngeal carcinoma (NPC). Many viral gene products including EBNA1, LMP1 and LMP2 have been implicated in NPC tumorigenesis, although the de novo control of these viral oncoproteins remain largely unclear. / MicroRNAs (miRNAs) are a class of small, non-coding RNAs with a size around 18--24 nucleotides with significant roles in regulating gene expression by either transcriptional silencing or translational suppression. As gene regulators, recent miRNA studies have emphasized the contribution of aberrant miRNA expression in cancer development. The recent discovery of EBV encoded viral miRNAs (ebv-miRNAs) in lymphoid malignancies has prompted us to examine the NPC-associated EBV miRNAs. In this study, we have systematically examined the NPC associated EBV genome for viral-encoded miRNA expression. By constructing small cDNA libraries from a native EBV positive NPC cell line (C666-1) and a xenograft (X2117), we screened about 3000 clones and detected several small EBV fragments, within which two novel ebv-miRNAs in the BARTs region were identified. These two newly identified miRNAs, now named miR-BART21 and miR-BART22, were proven to be abundantly expressed in most NPC samples by both Northern blot and QRT-PCR analysis. / Taken together, this thesis shows that two newly identified EBV-encoded miRNAs are highly expressed in latent EBV infection in NPC. Frequent expression of miR-BART22 can be explained partially by a specific EBV strain that is associated with NPC in our locality. Our findings emphasize the role of miR-BART22 in modulating LMP-2A expression. Because LMP-2A is a potent immunogenic viral antigen that is recognized by the cytotoxic T cells (CTLs), down-modulation of LMP-2A expression by mir-BART22 may permit escape of EBV-infected cells from host immune surveillance. / We attempted to predict the potential viral and cellular targets of miR-BART21 and miR-BART22 by public available computer programs, miRanda and RNAhybrid. A number of potential cellular mRNA targets were suggested, although many failed to be validated by luciferase reporter assay. However, we found a putative miR-BART22 binding site in the LMP2A-3'UTR. Although the LMP-2A transcript is consistently detected in NPC, only 6 out of 26 (23%) primary NPC tumors show weak LMP-2A expression by immunohistochemistry (IHC). The expression levels of miR-BART22 and LMP-2A mRNA have also been determined in eleven of these tumors. Interestingly, the LMP-2A mRNA expression level did not directly correlate with protein expression, and relatively low expression levels of miR-BART22 miRNA were observed in all 3 LMP-2A positive-primary tumors. The suppressive effect of miR-BART22 on LMP-2A was also experimentally validated by a series of dual luciferase reporter assays using reporter constructs containing the putative or mutated recognition site at the LMP-2A 3'UTR. By co-transfection of different amounts of miR-BART22 with the LMP-2A-3'UTR expression vector in reporter assay, we confirmed that miR-BART22 suppressed the LMP-2A protein level in a dose-dependent manner. Furthermore, transfection of miR-BART22 into HEK293 cells that had been stably transfected with pcDNA3.1-LMP-2A, which contains a complete LMP-2A ORF and 3'UTR, readily suppressed levels of the LMP-2A protein. / Lung, Wai Ming Raymond. / Adviser: To Ka Fai. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 197-226). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Yip, Wing Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 100-119). / Abstract also in Chinese. / Abstract (English version) --- p.i / Abstract (Chinese version) --- p.iii / Acknowledgments --- p.v / Table of Contents --- p.vii / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Hepatocellular Carcinoma --- p.1 / Chapter 1.1.1 --- Epidermiology --- p.1 / Chapter 1.1.2 --- Etiology --- p.1 / Chapter 1.2 --- Hepatitis B Virus --- p.3 / Chapter 1.2.1 --- The Epidermiology of Hepatitis B Virus Infection --- p.3 / Chapter 1.2.2 --- The Morphology and Genome of Hepatitis B Virus --- p.4 / Chapter 1.2.3 --- HBV Genotypes and Their Significance --- p.8 / Chapter 1.3 --- Hepatitis B Virus X Protein --- p.9 / Chapter 1.3.1 --- HBx Alters Various Signal Transduction Pathways --- p.10 / Chapter 1.3.2 --- HBx Interacts with Various Transcription Factors and Co-activators --- p.12 / Chapter 1.3.3 --- HBx Induces Epigenetic Alterations --- p.14 / Chapter 1.3.4 --- Identification of COOH-terminal Truncated HBx in Liver Tumors --- p.15 / Chapter 1.4 --- MicroRNAs --- p.17 / Chapter 1.4.1 --- Transcriptional Regulation and Biogenesis of MicroRNAs --- p.18 / Chapter 1.4.2 --- MicroRNAs and Cancer --- p.21 / Chapter 1.4.3 --- MicroRNAs and HCC --- p.25 / Chapter 1.5 --- Hypothesis and Aims of the Study --- p.29 / Chapter CHAPTER 2 --- MATERIALS and METHODS --- p.30 / Chapter 2.1 --- Patients --- p.30 / Chapter 2.2 --- Cell Lines --- p.30 / Chapter 2.3 --- Cloning of Various HBx Constructs --- p.32 / Chapter 2.3.1 --- PCR Amplification of HBx Fragments --- p.32 / Chapter 2.3.2 --- Cloning of HBx Fragments into TA-vectos --- p.33 / Chapter 2.3.3 --- Heat Shock Transformation --- p.33 / Chapter 2.3.4 --- Sub-cloning of HBx Fragments into Lentiviral Vectors --- p.34 / Chapter 2.4 --- Generation of Lentivirus --- p.37 / Chapter 2.4.1 --- Lentivirus Infection --- p.37 / Chapter 2.5 --- RNA Extraction --- p.38 / Chapter 2.6 --- Western Blot Analysis --- p.39 / Chapter 2.7 --- MiRNA Microarray --- p.40 / Chapter 2.7.1 --- Cyanine3-pCp Labeling of RNA Samples --- p.40 / Chapter 2.7.2 --- Sample Hybridization --- p.41 / Chapter 2.7.3 --- Microarray Wash --- p.41 / Chapter 2.7.4 --- Array Slide Scanning and Processing --- p.41 / Chapter 2.8 --- Detection of HBx Gene Deletion by PCR --- p.43 / Chapter 2.9 --- Immunohistochemistry --- p.44 / Chapter 2.10 --- Quantitative Real-time PCR --- p.45 / Chapter 2.11 --- Proliferation Assay --- p.47 / Chapter 2.12 --- Cell Cycle Analysis --- p.48 / Chapter 2.13 --- Annexin V Apoptosis Assay --- p.49 / Chapter 2.14 --- Colony Formation Assay --- p.50 / Chapter 2.15 --- Statistical Analysis --- p.51 / Chapter CHAPTER 3 --- RESULTS --- p.52 / Chapter 3.1 --- Detection of Full-length and COOH-terminal Truncated HBx in HCC Tissues --- p.52 / Chapter 3.2 --- Confirmation of HBx Expression in HCC Tissues --- p.55 / Chapter 3.3 --- Comparison of HBx from Different HBV Genotypes for Study --- p.61 / Chapter 3.4 --- Functional Characterization of COOH-tterminal Truncated HBx --- p.64 / Chapter 3.4.1 --- Selection of COOH-terminal Truncated HBx --- p.64 / Chapter 3.4.2 --- Generation of Various HBx-expressing Hepatocyte Cell Lines --- p.66 / Chapter 3.4.3 --- Effect of Full-length and COOH-terminal Truncated HBx on Cell Proliferation --- p.69 / Chapter 3.4.4 --- Effect of Full-length and COOH-terminal Truncated HBx Cell Cycle --- p.34 / Chapter 3.4.5 --- Effect of Full-length and COOH-terminal Truncated HBx on Apoptosis --- p.45 / Chapter 3.5 --- MicroRNA Profiling of Various HBx-expressing Hepatocyte Cell Lines --- p.76 / Chapter 3.5.1 --- Identification of Deregulated MicroRNAs by Microarray --- p.76 / Chapter 3.5.2 --- Validation of Deregulated MicroRNAs by Real-time PCR Analysis --- p.80 / Chapter 3.5.3 --- Confirmation of Deregulated MiRNAs in HCC and Adjacent Non-tumor Tissues --- p.84 / Chapter 3.5.4 --- Potential Downstream Targets of the HBx-deregulated MiRNAs --- p.87 / Chapter CHAPTER 4 --- DISCUSSION --- p.91 / Chapter 4.1 --- The Impact of COOH-terminal Truncated HBx in HCC --- p.91 / Chapter 4.2 --- The Biological Significance of COOH-terminal Truncated HBx Induced MiRNAs --- p.94 / Chapter 4.3 --- Limitations of the Present Study --- p.97 / Chapter 4.4 --- Future Studies --- p.98 / Chapter CHAPTER 5 --- CONCLUSION --- p.99 / REFERENCES --- p.100

Over 500 million people worldwide suffer from disease associated with intestinal inflammation, including gastric cancer, inflammatory bowel disease, h. pylori infections, and numerous viral and bacterial infections. Although potentially effective therapeutics exist for many of these pathologies, delivery challenges thwart their clinical viability. The objective of this work was to develop drug delivery platforms that could target toxic immunomodulatory therapeutics to diseased intestinal tissues. To meet this objective, we developed an oral delivery vehicle for siRNA and an NF-κB inhibiting nanoparticle that reduces drug-resistance. Small interfering RNA (siRNA) represents a promising treatment strategy for numerous gastrointestinal (GI) diseases; however, the oral delivery of siRNA to inflamed intestinal tissues remains a major challenge. In this presentation, we describe a delivery vehicle for siRNA, termed thioketal nanoparticles (TKNs), that can orally deliver siRNA to sites of intestinal inflammation, and thus inhibit gene expression in diseased intestinal tissue. Using a murine model of ulcerative colitis, we demonstrate that orally administered TKNs loaded with TNFα-siRNA (TNFα-TKNs) diminish TNFα messenger RNA (mRNA) levels in the colon and protect mice from intestinal inflammation. Activation of nuclear factor-κB (NF-κB) results in the expression of numerous prosurvival genes that block apoptosis, thus mitigating the efficacy of chemotherapeutics. Paradoxically, all conventional therapeutics for cancer activate NF-κB, and in doing so initiate drug resistance. Although adjuvant strategies that block NF-κB activation could potentiate the activity of chemotherapeutics in drug resistant tumors, clinical evidence suggests that current adjuvant strategies also increase apoptosis in non-malignant cells. In this presentation, we present a nanoparticle, formulated from a polymeric NF-κB-inhibiting prodrug, that target the chemotherapeutic irinotecan (CPT-11) to solid tumors, and thus abrogates CPT-11-mediated drug resistance and inhibits tumor growth. In order to maximize the amount of NF-κB inhibitor delivered to tumors, we synthesized a novel polymeric prodrug, termed PCAPE, that releases the NF-κB inhibitor caffeic acid phenethyl ester (CAPE) as its major degradation product. Using a murine model of colitis-associated cancer, we demonstrate that when administered systemically, CPT-11-loaded PCAPE-nanoparticles (CCNPs) are three time more effective than a cocktail of the free drugs at reducing both tumor multiplicity and tumor size.

Small silencing RNAs function in almost every aspect of cellular biology. Argonaute proteins bind small RNA and execute gene silencing. The number of Argonaute paralogs range from 5 in Drosophila melanogaster , 8 in Homo sapiens to an astounding 27 in Caenorhabditis elegans. This begs several questions: Do Argonaute proteins have different small RNA repertoires? Do Argonaute proteins behave differently? And if so, how are they functionally and mechanistically distinct? To address these questions, we examined the thermodynamic, kinetic and functional properties of fly Argonaute1 (dAgo1), fly Argonaute2 (dAgo2) and mouse Argonaute2 (mAGO2). Our studies reveal that in fly, small RNA duplexes sort into Argonaute proteins based on their intrinsic structures: extensively paired siRNA duplex is preferentially sorted into dAgo2 while imperfectly paired miRNA duplex is channeled into dAgo1. The sorting of small RNA is uncoupled from its biogenesis. This is exemplified by mir-277, which is born a miRNA but its extensive duplex structure licenses its entry into dAgo2. In the Argonaute protein, the small RNA guide partitions into functional domains: anchor, seed, central, 3' supplementary and tail. Of these domains, the seed initiates binding to target. Both dAgo2 and mAGO2 (more closely related to and a surrogate for dAgo1 in our studies) bind targets at astonishing diffusion-limited rates (~107–108 M−1s−1). The dissociation kinetics between dAgo2 and mAGO2 from their targets, however, are different. For a fully paired target, dAgo2 dissociates slowly (t½ ~2 hr) but for a seed-matched target, dAgo2 dissociates rapidly (t½ ~20 s). In comparison, mAGO2 does not discriminate between either targets and demonstrates an equivalent dissociation rate (t½ ~20 min). Regardless, both dAgo2 and mAGO2 demonstrate high binding affinity to perfect targets with equilibrium dissociation constants, KD ~4–20 pM. Functionally, we also showed that dAgo1 but not dAgo2 silence a centrally bulged target. By contrast, dAgo2 cleaved and destroyed perfectly paired targets 43-fold faster than dAgo1. In target cleavage, dAgo2 can tolerate mismatches, bulged and internal loop in the target but at the expense of reduced target binding affinities and cleavage rates. Taken together, our studies indicate that small RNAs are actively sorted into different Argonaute proteins with distinct thermodynamic, kinetic and functional behaviors. Our quantitative biochemical analysis also allows us to model how Argonaute proteins find, bind and regulate their targets.

piRNAs guide PIWI proteins to silence transposons in animal germ cells. In Drosophila, the heterochromatic piRNA clusters transcribe piRNA precursors to be transported into nuage, a perinuclear structure for piRNA production and transposon silencing. At nuage, reciprocal cycles of piRNA-directed RNA cleavage—catalyzed by the PIWI proteins Aubergine (Aub) and Argonaute3 (Ago3) in Drosophila—destroy the sense transposon mRNA and expand the population of antisense piRNAs in response to transposon expression, a process called the Ping-Pong cycle. Heterotypic Ping-Pong between Aub and Ago3 ensures that antisense piRNAs predominate. My thesis research mainly focuses on two fundamental questions about the piRNA production: How does the germ cell differentiate piRNA precursor from mRNAs for piRNA biogenesis? And what is the mechanism to impose Aub Ping-Pong with Ago3? For the first question, we show that the HP1 homolog protein Rhino marks the piRNA cluster regions in the genome for piRNA biogenesis. Rhino seems to anchor a nuclear complex that suppresses cluster transcript splicing, which may differentiate piRNA precursors from mature mRNAs. Moreover, LacI::Rhino fusion protein binding suppresses splicing of a reporter transgene and is sufficient to trigger de novo piRNA production from a trans combination of sense and antisense transgenes. For the second question, we show that Qin, a new piRNA pathway factor contains both E3 ligase and Tudor domains, colocalizes with Aub and Ago3 in nuage, enforces heterotypic Ping- Pong between Aub and Ago3. Loss of qinleads to less Ago3 binding to Aub, futile Aub:Aub homotypic Ping-Pong prevails, antisense piRNAs decrease, many families of mobile genetic elements are reactivated, DNA damage accumulates in the germ cells and flies are sterile.

Selective gas-phase dissociation strategies have been developed for the characterization of cross-linked peptides and proteins in quadrupole ion trap mass spectrometers. An infrared chromogenic cross-linker (IRCX) containing a phosphotriester afforded rapid differentiation of cross-linked peptides from unmodified ones in proteolytic digests of cross-linked proteins by selective infrared multiphoton dissociation (IRMPD). Only the cross-linked peptides containing the chromogenic phosphate underwent IRMPD and unmodified peptides were not affected by IR irradiation. IRMPD of IRCX-cross-linked peptides yielded uncross-linked y-ion sequence tags of the constituent peptides due to secondary dissociation of all primary product ions which contained the chromophore, thus allowing successful de novo sequencing of the cross-linked peptides. Peptides cross-linked via a two-step conjugation strategy through the formation of a bis-arylhydrazone (BAH) bond were selectively dissociated by ultraviolet radiation at 355 nm. The BAH-cross-linked peptides could be distinguished from not only unmodified peptides but also dead-end modified peptides based on the selectivity of ultraviolet photodissociation. In a complementary approach, electron transfer dissociation of BAH-cross-linked peptides resulted in preferential cleavage of the hydrazone bond which produced two modified peptides. These modified peptides were subsequently interrogated by CID which allowed for the original site of cross-linking to be pinpointed. IRMPD was implemented in a dual pressure linear ion trap to demonstrate successful photodissociation of peptides having modest absorptivities. Peptides were observed to efficiently dissociation by IR irradiation exclusively in the low pressure cell whereas no dissociation was observed in the high pressure cell due to extensive collisional cooling. IRMPD provided greater sequence coverage of the peptides than CID and yielded product ion mass spectra which were predominantly composed of singly charged product ions which simplified spectral interpretation. IRMPD was further applied for the sequencing of small-interfering RNA. Complete sequence coverage was obtained and the results were compared to CID. / text