Introduction

RNA interference (RNAi) was first discovered in 1998 by Fire et al. in Caenorhabditis elegans, and has been found in evolutionally diverse organisms, such as plants, flies and protozoan. It is a naturally occurring mechanism that controls gene expression at the post-transcriptional level caused by double-stranded RNAs (dsRNAs). Soon after its discovery, RNAi was shown to be able to achieve in mammalian cells using small-interfering RNAs (siRNAs), an intermediate product in the RNAi pathway that could bypass the interferon response, which is known to be induced by long dsRNAs. In RNAi, dsRNAs were cleaved into of 21-23 nucleotides (nts) siRNAs by Dicer, a member of the RNAse III family. These siRNAs are then incorporated into RNA-Induced Silencing Complexes (RISCs), which then bind to the target mRNA having sequences complementary to the incorporated siRNA, leading to the degradation of the target mRNA, and subsequently, resulted in the selective silencing of specific proteins. This characteristic of RNAi makes it a valuable tool both in laboratory research and therapeutic field.

In term of laboratory research, the use of RNAi has already led to the characterization of novel oncogenes, including protein kinase C・ (PRKCE) in breast cancer, abnormal spindle homologue, microcephaly associated (ASPM) in glioblastoma, and mutant KRAS in non-small-cell lung cancer.

Meanwhile, in the therapeutic field, besides its high target specificity and knock down effect, the relatively short turnaround time for efficacy testing of potential therapeutic RNAi molecules, and the fact that even newly discovered pathogens are theoretically amenable to rapid targeting have caused great excitement about the potential of RNAi for treating a wide range of diseases. In fact, the first clinical applications of RNAi have been directed at the treatment of wet, age-related macular degeneration (AMD) and respiratory syncytial virus (RSV) infection. Furthermore, therapies based on RNAi are also in preclinical development for other viral disease, neurodegenerative disorders and cancers.

In the first part of this thesis, I described the application of RNAi technology in studying genes involved in the transcriptional activity induction of p73, a structural homologue of p53 gene, and I found a novel cooperation of Yin Yang 1 (YY1) and E2F1 in inducing it transcriptional activity. In the second part, I described an example for the application of RNAi-based technology in therapeutic field, i.e., using RNAi for silencing Prolyl Hydroxylase Domain 2 (PHD2) gene, and subsequently, enhanced angiogenesis.

Yin Yang 1 induces transcriptional activity of p73 through cooperation with E2F1

Yin Yang 1 (YY1) is a multifunctional transcription factor, which can act as transcriptional activator, repressor, or initiator element binding protein. Nowadays, YY1 has already been known to have a fundamental role in normal biological processes such as embryogenesis, differentiation, DNA replication, and cellular proliferation. Additionally, YY1 has also been shown to be involved in neuronal differentiation. Recently, YY1 has been reported to possess a potential function in cancer biology. It is important to explain the biological function of YY1 via studying the putative interactions between YY1 and cell cycle regulators, death genes, and furthermore, transcription factors and cofactors in the suppression or progression of various physiological phenomena.

Transcription factor p73 is a structural homologue of tumor suppressor gene p53 and plays an important role in tumorigenesis, cellular differentiation and development. Although recently transcription factor E2F1 has been identified as a vital regulatory factor for p73 transcription, the upstream and downstream regulation of p73 pathway remains to be understood.

To understand the upstream regulation of p73 pathway, I established a p73-promoter based luciferase reporter system for performing screening using siRNA expression vectors. During the initiation process of searching genes related with E2F1, I identified the transcription factor YY1 as a novel regulator of p73 transcription. In contrast to its function in p53 pathway, in which knocking down of YY1 resulted in the enhance of p21, a p53 downstream gene, I found that co-transfecting YY1-siRNA vectors and p73 luciferase reporter vectors led to the decrease of both p73 luciferase reporter activity and p73 message level for around 60%, indicating that YY1 might act as a positive regulator of p73 promoter. Consistently, overexpression of YY1, as well as E2F1, increased the activity of the p73 luciferase reporter vectors in a plasmid-dose dependent manner. Together, these results had shown the unsuspected role of YY1 in regulating p73 transcription level, i.e., the possibility that YY1 upregulate the p73 promoter activity.

On the other hand, it has been reported that E2F1 could recruit the p73 promoter and activate its transactivation. To further reveal the regulatory mechanism of p73 by YY1, we overexpressed YY1 and E2F1, and found out that compared to the overexpression of either YY1 or E2F1 alone, the transcriptional activity of p73 was further enhanced when both of them were overexpressed together, suggesting that YY1 could regulate p73 transcriptional activity in a synergistic fashion with E2F1. This synergistic effect of YY1 and E2F1 on p73 promoter is also implicated in physiological event, as in the cells in which YY1 or E2F1 was knocked down, the induction of the activation of p73 luciferase reporter using a doxorubicin, a DNA damaging reagent which has been known to activate p73 promoter E2F1-dependently could be suppressed to 50%. Moreover, through immunofluorescence staining and immunoprecipitation experiments, we found that YY1 and E2F1 sublocalized together mainly in the nuclei, and associated physically.

These data demonstrated two novel roles of YY1. First, YY1 could function as a cooperator of E2F1, a mediator of cellular proliferation. The cooperation between E2F1 and YY1 may be at least partially responsible for determining the physiological function (i.e. oncogenic or tumor suppressive function) of E2F1 by modulating specificity and sensitivity of E2F1 on different promoter. Second, YY1 could be involved in the regulation of p73, which has been reported to be able to promote cellular growth in a synergistic manner with the proto-oncogene c-Jun. Data in this study that tumor activator gene YY1 up-regulates p73 transcription is also consistent with this result. Altogether, I demonstrated an example for using RNAi technology in analyzing biological pathway, and uncovered a novel function of YY1 on the E2F1-mediated p73 regulation, indicating its roles in tumorigenesis, development and differentiation.

Enhancement of Angiogenesis through Stabilization of Hypoxia Inducible Factor-1 by Silencing Prolyl Hydroxylase Domain 2 Gene

Therapeutic angiogenesis aims to improve neovascularization in ischemic tissues by delivery of angiogenic factors or DNA vectors encoding those proteins. Vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) are pro-angiogenic molecules and well investigated for therapeutic angiogenesis in previous studies. Therapeutic angiogenesis using these growth factors has been extensively investigated. However, although phase I clinical trials already had shown that the delivery of angiogenic proteins was safe, phase II trials have not demonstrated efficacy as expected, suggesting that the administration of a single angiogenic growth factor might be insufficient to induce functional vessels. Based on these disappointing results, therapeutic angiogenesis studies were altered into the combinatorial application of angiogenic growth factors. Combination of VEGF plus FGF2, VEGF plus angiopoietin-1 (Ang1), VEGF plus platelet-derived growth factor-BB (PDGF-BB), and PDGF-BB plus FGF2 have been reported to have a synergistic effect on neovascularization in experimental animal models. Collectively, these results showed that angiogenesis is a complex process, which includes the temporally and spatially orchestrated expression of multiple angiogenic factors induced by multiple cell types.

Hypoxia inducible factor-1 (HIF-1) is a heterodimeric α/β transcription factor that functions as a central regulator of oxygen homeostasis, which is degraded O2-dependently. In oxygenated cells, HIF-1 has a very short half-life (less than 5 min at 21% O2), while the reduction of O2 concentration enhances HIF-1 stability by suppressing its ubiquitin-proteasome degradation. More than 60 HIF-1 target genes have been identified so far, including VEGF and other angiogenic factors. Transgenic expression of HIF-1 in mouse skin induced the increased vascularization without excessive permeability. Furthermore, constitutively active form of HIF-1 is sufficient to induce angiogenesis in nonischemic animal tissue.

Meanwhile, in mammalian cells, three prolyl hydroxylase domain (PHD) protein isoforms, PHD1, PHD2 and PHD3 have been determined to hydroxylate two prolyl residues (P402 and/or P564) in the oxygen-dependent degradation domain (ODDD) of HIF-1. Under the normoxic condition, PHD2 is the key limiting enzyme targeting HIF-1α for hydroxylation, which subsequently led to the ubiquitination and proteosomal degradation of HIF-1α

Here I demonstrated the possibility of a potent molecular therapy for therapeutic angiogenesis using RNAi technology, i.e., by silencing PHD2 in order to enhance the expression of angiogenic growth factors and to induce neovascularization through HIF-1 activation. siRNA and siRNA expression vectors were used for silencing murine PHD2 expression, which resulted in the stabilization of HIF-1, but not HIF-2, and subsequently, in the upregulation of multiple angigogenic growth factors, especially VEGF and FGF2, in mouse fibroblast cells. Moreover, by using PHD2-siRNA vector, the increase of VEGF secretion was observed for as long as 18 days post-transfection. Furthermore, in vitro treatment of human umbilical vein endothelial cells (HUVECs) with conditioned medium from PHD2-siRNA transfected NIH3T3 cells increased cells proliferation.

Finally, in vivo experiment showed that implantation of Matrigel plugs mixed with NIH3T3 cells transfected with PHD2-siRNA vector is sufficient to induce angiogenesis in mice.

These results indicated that PHD2 silencing induces expressions of multiple angiogenic growth factors by stabilizing HIF-α, and that implantation of PHD2-knocked down cells is sufficient to induce angiogenesis in vivo. Thus, RNAi-mediated PHD2 silencing might offer a potential tool for angiogenic therapy.

YY1 Interacts with Hypoxia-Inducible Factor 1α and Inhibits Transcriptional Activity of HIF-1α

Hypoxia inducible factor 1 (HIF-1) is a heterodimeric α/β transcription factor that functions amaster regulator of oxygen homeostasis in metazoan species. HIF-1 is expressed in most tissues, but HIF-1 activity is determined by the expression and activity of itsα subunit. The half-life of the HIF-1α is determine by oxygen-dependent prolyl hydroxylation, which is required for hydroxylation of Prolyl hydroxylase domain proteins (PHD) and binding of the von Hippel-Lindau protein (VHL), the recognition component of an E3 ubiquitin ligase that targets HIF1-a for ubiquitination and degradation. Here, we demonstrate that Yin Yang 1, the protein product of a widely expressed gene, is involved in the functional activation of HIF1α. YY1 gain-of-function promotes the inhibition of HIF-1-mediated transcription. YY1 loss-of-function caused by RNA interference increases HIF-1α protein levels, HIF-1-mediated transcription under nonhypoxic conditions. These data indicate that YY1 is an essential component of a multiprotein complex that regulates HIF-1α levels in an O2-dependent manner, and YY1 has an important role in the hypoxia signaling.

Conclusions and Future Perspectives

In this thesis, firstly, I developed a method for analyzing the genes involved in the E2F1 signal introduction by using the vector-based shRNA library. During the initiation process of searching genes related with E2F1, I found the previously unsuspected role of YY1 on the E2F1-mediated p73 regulation. The fact that YY1 affects p53 family members opened up an attractive possibility that needs further investigation, that is, YY1 might function as a key integrator or modulator of various pathways in the network that includes p53 family members and regulators concerned with various biological processes. Then, YY1 might be a good tarter for cancer therapy. Here, I also showed that the genome-wide RNAi approach is a powerful tool in gene function analysis and target identification. As for the disease therapy, this technique enable high-throughput screening, thus, the cost, time, validation and others aspects of drugs discovery could be significantly reduced. In addition, in chapter four of this thesis, by using gene-loss and gene-gain assay our data firstly indicate that YY1 is an essential component of a multiprotein complex that regulates HIF-1α levels in an O2-dependent manner, and YY1 has an important role in the hypoxia signalling. The further study is required for analyzing the role of YY1 in hypoxia signalling.

In the RNAi-based angiogenesis study, we have demonstrated that implantation of cells, of which PHD2 were silenced, induced neovascularization in the in vivo Matrigel plug assay, providing a basis for further therapeutic application. For practical therapeutic applications, the delivery systems should be developed to deliver the siRNA expression vector and/or siRNA to the target sites and to have them function in an efficient and safe manner. In this regard, our laboratory have explored polymeric micelle-based nanocarriers for plasmid DNA and siRNA, and demonstrated their in vitro and in vivo efficacies, including efficient gene transfer to primary cells, in vivo transfection to a rabbit carotid artery, and transfection-mediated bone regeneration. Thus, I will further investigate the therapeutic effects of PHD2-silencing on neoangiogenesis by using polymeric micelle-based nanocarriers.

Collectively, RNAi not only has a profound effect on studies of gene regulation, it has also led to the development of a new class of therapeutic agents based on small dsRNA. I believe that the results shown in this thesis contribute to the further application of RNAi in both gene regulation and RNAi-based therapeutic fields in human disease.

RNA干渉(RNA interference, RNAi)は、二本鎖RNAによる転写後の遺伝子発現抑制現象であり、標的遺伝子を特異的かつ効率にノックダウンできることから、遺伝子機能の解析手段として使われるだけでなく、疾患の治療法としても大きな注目を集めている。本論文では、上記のRNAiに関して、細胞内シグナル伝達経路の解析への応用と血管新生療法への展開という二つの新規課題に取り組んだ。さらに、転写因子であるYin Yang 1(YY1)と血管新生のキーファクターであるHypoxia Inducible Factor-1α(HIF-1α)の関連性に着目し、YY1が血管新生に関わる可能性及びそのメカニズムについて検討した。本論文は全5章より構成されている。

第一章では、序論としてRNAiの歴史、メカニズム、RNAiを利用した医薬品開発の現状を概説し、本研究の背景及び目的を述べている。

第二章では、siRNA 発現ベクターを用いて転写因子であるYin Yang 1(YY1)とp73の関連性を解析している。p73は、高度に保存されているp53ファミリーに属する転写因子で、p53と同様、癌抑制活性を持っていると予測されており、幾つかのp53に対する応答性を持つ遺伝子を活性化することが知られている。最近、p73は細胞周期の停止やアポトーシスを誘導することも報告されている。このような類似点が幾つかあるものの、p73は、p53と特にDNA損傷のシグナルで異なっていることが報告された。p73の機能及び上流遺伝子の制御には未知な部分が多いため、本論文では、siRNAによる遺伝子ノックダウンを利用して、p73の上流因子として、新たにYin Yang 1 (YY1)を同定し、また、p73を調節する因子として知られているE2F1との関係について解析した結果について報告している。本研究によって、転写因子YY1とE2F1が相互作用し、相乗的に、DNA損傷で誘導されるp73パスウエイに関与していることが初めて示された。本結果は、YY1がp53非依存性的なパスウエイの役割を担っている可能性を示唆すると同時に、p73の上流制御の解明と神経分化などのパスウエイの解析にもつながるとものと考えられる。

第三章では、RNAiの血管新生療法への応用に関して、その基本概念を実験的に明らかにしている。血管新生療法は、虚血性疾患に対する治療法として重要である。VEGFやFGF2など血管新生因子を単独で利用した場合、十分な治療効果が得られないことが臨床的に明らかにされつつあり、現在は複数の血管新生因子の組み合わせた治療法が検討されるようになってきている。そこで本論文では、血管新生因子の遺伝子群の転写因子であるHIF-1αに注目し、その分解を促進するPHD2を、RNAiを用いてノックダウンすることで、HIF-1αが安定化させ、血管新生因子群が発現し、in vivoで血管新生が誘導されることを初めて明らかにした。本システムはRNAiを利用した新しい血管新生療法として臨床応用が期待される。

第四章では、転写因子YY1と血管新生のキーファクターであるHIF-1αの関連性について述べている。YY1は、活性因子としても抑制因子としても作用し、どちらとして作用するかは細胞種類やその細胞内環境に大きく依存する。本論文では、癌抑制因子p53が正常の細胞とp53欠損細胞で、YY1がHIF-1αの転写を正と負のパスウエイで調節する可能性について示した。また、その制御は、p53を介したHIF-1αの分解を阻害し、HIF-1αを正に制御する系と、HIF-1αの機能を直接に低下させる負の制御系を介する可能性についても示唆している。すなわち、YY1は、HIF-1αを介して血管新生などの生命現象に関与している可能性が考えられる。

第五章では、第二章から第四章までの研究を総括し、本論文の成果を含めて今後の展望について論じている。

以上のように、本論文では、RNAiの新しい応用として遺伝子機能解析と血管新生療法への展開について記述されていた。本論文は、RNAiを利用した生命現象の解明と医薬品開発につながるものであり、極めて秀逸である。

よって本論文は博士(工学)の学位請求論文として合格と認められる。