環形RNA 由於不具自由的末端，因此無法被核醣核酸外切酶剪切，相對於線性RNA有更長的半衰期。環形RNA本身不具有5’端帽及3’polyA尾端，當含有internal ribosome entry site (IRES)時可以以cap-independent方式進行轉譯，因此可透過形成環形RNA來建立外源基因長效表現系統。為了在細胞內形成環形RNA，我們分別採用ribozyme剪切及intron反向剪接方式進行環化。其中ribozyme 進行剪切後，在5’端形成OH基及3’端形成2’,3’環磷酸根可由細胞內的RtcB蛋白辨識將兩端連接。在intron反向剪接的方式，透過同源臂將兩端拉近，在基因下游splice donor site可與上游splice acceptor site 進行反向剪接，形成環形RNA。運用綠色螢光觀察環形RNA的表現，實驗結果顯示兩種方式皆無法延長表現。透過qPCR絕對定量測定環化效率，環化效率皆在≈ 0.1-1.0 %，以此兩種方式表現少量環形RNA皆無法有效延長目標基因表現。
除此之外，當microRNA binding sites 存在於環形RNA時，可作為microRNA 海綿體來進行基因調控。先前研究指出骨質疏鬆患者有異常表現的microRNA，其中，發現microRNA-214 在骨質疏鬆的大鼠脂肪間葉幹細胞(OVX-ASC)異常表現，會具有抑制往骨分化及增強蝕骨細胞成熟能力。從實驗結果發現以ribozyme系統能有效形成小片段環形RNA，因此以ribozyme 剪切方式形成環形microRNA-214 perfect match 海綿體，透過比較不同binding sites數量形成環形RNA 比例，實驗結果顯示在擁有4個binding sites 環形比例為15 %。在以桿狀病毒載體轉導送入到OVX-ASC後，線性海綿體與環形海綿體抑制microRNA-214效果接近，從此結果並未觀察到形成環形microRNA海綿體時，可提升對目標microRNA 抑制效果。

Circular RNA does not have free ends, so it cannot be cleaved by exonuclease and has longer life-time compared to linear RNA. Also, circular RNA itself does not have 5’cap and 3’poly A tail, and it can only be translated in cap-independent manner when it contains an internal ribosome entry site (IRES). In this research, we try to establish the long-term expression system through the formation of circular RNA. In order to form circular RNA in cells, ribozyme splicing and intron back-splicing were used to induce circularization. The green fluorescence protein can be expressed by circular RNA in cap-independent manner. Through oberserving the ratio of cells expressing green fluorescence from day to day, the result showed that the expression can not be prolonged by these two methods. By absolute qPCR quantification measurement, the forming efficiency of RNA circularization is only 0.1-1.0 %. Circular RNA can’t be formed efficiently by these two ways, and the long term expression can’t be enhanced.
When microRNA binding sites exist in the circular RNA, it can act as microRNA sponge for gene regulation. Recently, studies have shown that abnormal microRNA expression is in osteoporosis patients. When high microRNA-214 expression is in osteoporotic rat adipose mesenchymal stem cells (OVX-ASC), cells can’t easily differentiate into osteoblasts. In the research, circular microRNA-214 perfect match sponges were formed by the ribozyme splicing method. By comparing the number of binding sites, the percentage of circular sponges for 4 binding sites is 15 %. After transducing OVX-ASC by linear or circular sponges, microRNA-214 inhibition is similar. The inhibition effect of microRNA can’t be enhanced by circular microRNA sponge.

[1] W. R. Jeck and N. E. J. N. b. Sharpless, "Detecting and characterizing circular RNAs," vol. 32, no. 5, p. 453, 2014.
[2] W. R. Jeck et al., "Circular RNAs are abundant, conserved, and associated with ALU repeats," vol. 19, no. 2, pp. 141-157, 2013.
[3] C. Cocquerelle, B. Mascrez, D. Hétuin, and B. J. T. F. J. Bailleul, "Mis‐splicing yields circular RNA molecules," vol. 7, no. 1, pp. 155-160, 1993.
[4] E. Lasda and R. J. R. Parker, "Circular RNAs: diversity of form and function," vol. 20, no. 12, pp. 1829-1842, 2014.
[5] J. J. T. i. g. Salzman, "Circular RNA expression: its potential regulation and function," vol. 32, no. 5, pp. 309-316, 2016.
[6] Y. Enuka, M. Lauriola, M. E. Feldman, A. Sas-Chen, I. Ulitsky, and Y. J. N. a. r. Yarden, "Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor," vol. 44, no. 3, pp. 1370-1383, 2016.
[7] T. B. Hansen et al., "Natural RNA circles function as efficient microRNA sponges," vol. 495, no. 7441, pp. 384-388, 2013.
[8] J. U. Guo, V. Agarwal, H. Guo, and D. P. J. G. b. Bartel, "Expanded identification and characterization of mammalian circular RNAs," vol. 15, no. 7, p. 409, 2014.
[9] R. A. Wesselhoeft, P. S. Kowalski, and D. G. J. N. c. Anderson, "Engineering circular RNA for potent and stable translation in eukaryotic cells," vol. 9, no. 1, pp. 1-10, 2018.
[10] A. Bonizzato, E. Gaffo, G. Te Kronnie, and S. J. B. c. j. Bortoluzzi, "CircRNAs in hematopoiesis and hematological malignancies," vol. 6, no. 10, pp. e483-e483, 2016.
[11] S. Petkovic and S. J. N. a. r. Müller, "RNA circularization strategies in vivo and in vitro," vol. 43, no. 4, pp. 2454-2465, 2015.
[12] P. G. J. M. Zaphiropoulos and c. biology, "Exon skipping and circular RNA formation in transcripts of the human cytochrome P-450 2C18 gene in epidermis and of the rat androgen binding protein gene in testis," vol. 17, no. 6, pp. 2985-2993, 1997.
[13] Y. Wang and Z. J. R. Wang, "Efficient backsplicing produces translatable circular mRNAs," vol. 21, no. 2, pp. 172-179, 2015.
[14] G. DI SEGNI, L. BORGHESE, S. SEBASTIANI, and G. P. J. R. TOCCHINI-VALENTINI, "A pre-tRNA carrying intron features typical of Archaea is spliced in yeast," vol. 11, no. 1, pp. 70-76, 2005.
[15] V. M. Reyes and J. J. C. Abelson, "Substrate recognition and splice site determination in yeast tRNA splicing," vol. 55, no. 4, pp. 719-730, 1988.
[16] P. P. Chan and T. M. J. N. a. r. Lowe, "GtRNAdb: a database of transfer RNA genes detected in genomic sequence," vol. 37, no. suppl_1, pp. D93-D97, 2009.
[17] O. Fedorova, M. Gottikh, T. Oretskaya, Z. J. N. Shabarova, Nucleotides, and N. Acids, "Cyanogen bromide-induced chemical ligation: Mechanism and optimization of the reaction conditions," vol. 15, no. 6, pp. 1137-1147, 1996.
[18] M. J. Moore, "Joining RNA molecules with T4 DNA ligase," in RNA-Protein Interaction Protocols: Springer, 1999, pp. 11-19.
[19] M. Puttaraju and M. J. N. a. r. Been, "Group I permuted intron-exon (PIE) sequences self-splice to produce circular exons," vol. 20, no. 20, pp. 5357-5364, 1992.
[20] J. O. Westholm et al., "Genome-wide analysis of drosophila circular RNAs reveals their structural and sequence properties and age-dependent neural accumulation," vol. 9, no. 5, pp. 1966-1980, 2014.
[21] J. E. J. R. b. Wilusz, "Circular RNAs: unexpected outputs of many protein-coding genes," vol. 14, no. 8, pp. 1007-1017, 2017.
[22] J. E. J. M. g. e. Wilusz, "Repetitive elements regulate circular RNA biogenesis," vol. 5, no. 3, pp. 39-45, 2015.
[23] A. E. Brinegar and T. A. J. B. r. Cooper, "Roles for RNA-binding proteins in development and disease," vol. 1647, pp. 1-8, 2016.
[24] S. J. Conn et al., "The RNA binding protein quaking regulates formation of circRNAs," vol. 160, no. 6, pp. 1125-1134, 2015.
[25] R. Ashwal-Fluss et al., "circRNA biogenesis competes with pre-mRNA splicing," vol. 56, no. 1, pp. 55-66, 2014.
[26] C. Daniel, M. Behm, M. J. C. Öhman, and M. L. Sciences, "The role of Alu elements in the cis-regulation of RNA processing," vol. 72, no. 21, pp. 4063-4076, 2015.
[27] K.-Y. Lee and B.-J. J. M. Lee, "Structural and biochemical properties of novel self-cleaving ribozymes," vol. 22, no. 4, p. 678, 2017.
[28] R. M. Jimenez, J. A. Polanco, and A. J. T. i. b. s. Lupták, "Chemistry and biology of self-cleaving ribozymes," vol. 40, no. 11, pp. 648-661, 2015.
[29] S. Moteki and D. J. M. c. Price, "Functional coupling of capping and transcription of mRNA," vol. 10, no. 3, pp. 599-609, 2002.
[30] N. Sonenberg and A. G. J. C. Hinnebusch, "Regulation of translation initiation in eukaryotes: mechanisms and biological targets," vol. 136, no. 4, pp. 731-745, 2009.
[31] Y. Yang and Z. J. J. o. M. C. B. Wang, "IRES-mediated cap-independent translation, a path leading to hidden proteome," vol. 11, no. 10, pp. 911-919, 2019.
[32] G. J. Belsham and J. K. J. J. o. v. Brangwyn, "A region of the 5'noncoding region of foot-and-mouth disease virus RNA directs efficient internal initiation of protein synthesis within cells: involvement with the role of L protease in translational control," vol. 64, no. 11, pp. 5389-5395, 1990.
[33] K. Tsukiyama-Kohara, N. Iizuka, M. Kohara, and A. J. J. o. v. Nomoto, "Internal ribosome entry site within hepatitis C virus RNA," vol. 66, no. 3, pp. 1476-1483, 1992.
[34] C. B. Buck, X. Shen, M. A. Egan, T. C. Pierson, C. M. Walker, and R. F. J. J. o. v. Siliciano, "The human immunodeficiency virus type 1gag gene encodes an internal ribosome entry site," vol. 75, no. 1, pp. 181-191, 2001.
[35] A. Kozomara and S. J. N. a. r. Griffiths-Jones, "miRBase: integrating microRNA annotation and deep-sequencing data," vol. 39, no. suppl_1, pp. D152-D157, 2010.
[36] M. C. Siomi, K. Sato, D. Pezic, and A. A. J. N. r. M. c. b. Aravin, "PIWI-interacting small RNAs: the vanguard of genome defence," vol. 12, no. 4, pp. 246-258, 2011.
[37] A. Esquela-Kerscher and F. J. J. N. r. c. Slack, "Oncomirs—microRNAs with a role in cancer," vol. 6, no. 4, pp. 259-269, 2006.
[38] G. Meister, M. Landthaler, Y. Dorsett, and T. J. R. Tuschl, "Sequence-specific inhibition of microRNA-and siRNA-induced RNA silencing," vol. 10, no. 3, pp. 544-550, 2004.
[39] J. Krützfeldt et al., "Silencing of microRNAs in vivo with ‘antagomirs’," vol. 438, no. 7068, pp. 685-689, 2005.
[40] U. A. Ørom, S. Kauppinen, and A. H. J. G. Lund, "LNA-modified oligonucleotides mediate specific inhibition of microRNA function," vol. 372, pp. 137-141, 2006.
[41] M. S. Ebert, J. R. Neilson, and P. A. J. N. m. Sharp, "MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells," vol. 4, no. 9, pp. 721-726, 2007.
[42] M. S. Ebert and P. A. J. R. Sharp, "MicroRNA sponges: progress and possibilities," vol. 16, no. 11, pp. 2043-2050, 2010.
[43] H. Mizuno, M. Tobita, H. Orbay, A. C. Uysal, and F. Lu, "Adipose-derived stem cells as a novel tool for future regenerative medicine," in Stem Cells and Cancer Stem Cells, Volume 12: Springer, 2014, pp. 165-174.
[44] C.-Y. Lin et al., "The use of ASCs engineered to express BMP2 or TGF-β3 within scaffold constructs to promote calvarial bone repair," vol. 34, no. 37, pp. 9401-9412, 2013.
[45] Y. Wang et al., "Modification of human BMSC with nanoparticles of polymeric biomaterials and plasmid DNA for BMP-2 secretion," vol. 183, no. 1, pp. 8-17, 2013.
[46] W. Zhang et al., "VEGF and BMP-2 promote bone regeneration by facilitating bone marrow stem cell homing and differentiation," vol. 27, no. 12, pp. 1-11, 2014.
[47] K. Y.-Z. Zheng et al., "Salidroside stimulates the accumulation of HIF-1α protein resulted in the induction of EPO expression: A signaling via blocking the degradation pathway in kidney and liver cells," vol. 679, no. 1-3, pp. 34-39, 2012.
[48] D. Zou et al., "Repair of critical‐sized rat calvarial defects using genetically engineered bone marrow‐derived mesenchymal stem cells overexpressing hypoxia‐inducible factor‐1α," vol. 29, no. 9, pp. 1380-1390, 2011.
[49] Y. Nakamura, J. B. Inloes, T. Katagiri, T. J. M. Kobayashi, and c. biology, "Chondrocyte-specific microRNA-140 regulates endochondral bone development and targets Dnpep to modulate bone morphogenetic protein signaling," vol. 31, no. 14, pp. 3019-3028, 2011.
[50] F. Dai et al., "Downregulation of MiR-199b-5p inducing differentiation of bone-marrow mesenchymal stem cells (BMSCs) toward cardiomyocyte-like cells via HSF1/HSP70 pathway," vol. 24, p. 2700, 2018.
[51] U. L. Rahbek et al., "Bioresponsive hyperbranched polymers for siRNA and miRNA delivery," vol. 18, no. 10, pp. 812-820, 2010.
[52] J. Jin, M. Cid, C. B. Poole, and L. A. J. B. McReynolds, "Protein-mediated miRNA detection and siRNA enrichment using p19," vol. 48, no. 6, pp. xvii-xxiii, 2010.
[53] Y. Ohnishi et al., "Small RNA class transition from siRNA/piRNA to miRNA during pre-implantation mouse development," vol. 38, no. 15, pp. 5141-5151, 2010.
[54] R. Jankovic, S. Radulovic, and M. J. J. o. B. O. o. j. o. t. B. U. o. O. Brankovic-Magic, "siRNA and miRNA for the treatment of cancer," vol. 14, p. S43, 2009.
[55] L. T. Riedmann and R. Schwentner, "miRNA, siRNA, piRNA and argonautes: news in small matters," ed: Taylor & Francis, 2010.
[56] K. J. Airenne et al., "Baculovirus: an insect-derived vector for diverse gene transfer applications," vol. 21, no. 4, pp. 739-749, 2013.
[57] L.-Y. Sung et al., "Enhanced and prolonged baculovirus-mediated expression by incorporating recombinase system and in cis elements: a comparative study," vol. 41, no. 14, pp. e139-e139, 2013.
[58] J. Zeng, J. Du, Y. Zhao, N. Palanisamy, and S. J. S. C. Wang, "Baculoviral vector‐mediated transient and stable transgene expression in human embryonic stem cells," vol. 25, no. 4, pp. 1055-1061, 2007.
[59] K. J Airenne, K.-E. Makkonen, A. J Mahonen, and S. J. C. g. t. Yla-Herttuala, "In vivo application and tracking of baculovirus," vol. 10, no. 3, pp. 187-194, 2010.
[60] S. Wang and G. J. C. g. t. Balasundaram, "Potential cancer gene therapy by baculoviral transduction," vol. 10, no. 3, pp. 214-225, 2010.
[61] Y. C. J. A. i. v. r. Hu, "Baculovirus vectors for gene therapy," vol. 68, pp. 287-320, 2006.
[62] Y.-C. J. C. g. t. Hu, "Baculoviral vectors for gene delivery: a review," vol. 8, no. 1, pp. 54-65, 2008.
[63] L.-Y. Sung et al., "Efficient gene delivery into cell lines and stem cells using baculovirus," vol. 9, no. 8, p. 1882, 2014.
[64] M.-W. Lin et al., "Synthetic switch-based baculovirus for transgene expression control and selective killing of hepatocellular carcinoma cells," vol. 46, no. 15, pp. e93-e93, 2018.
[65] W. Luo et al., "Development of the hybrid Sleeping Beauty-baculovirus vector for sustained gene expression and cancer therapy," vol. 19, no. 8, p. 844, 2012.
[66] J. L. Litke and S. R. J. N. b. Jaffrey, "Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts," vol. 37, no. 6, pp. 667-675, 2019.
[67] Z. Lu et al., "Metazoan tRNA introns generate stable circular RNAs in vivo," vol. 21, no. 9, pp. 1554-1565, 2015.
[68] S. Müller and B. J. R. b. Appel, "In vitro circularization of RNA," vol. 14, no. 8, pp. 1018-1027, 2017.
[69] D. Sui, K. Wang, H. Park, and J. J. B. e. o. Chae, "Bright field microscopic cells counting method for BEVS using nonlinear convergence index sliding band filter," vol. 13, no. 1, p. 147, 2014.
[70] D. Lozano et al., "Osteostatin-loaded onto mesoporous ceramics improves the early phase of bone regeneration in a rabbit osteopenia model," vol. 8, no. 6, pp. 2317-2323, 2012.
[71] J. J. Noto, C. A. Schmidt, and A. G. J. R. b. Matera, "Engineering and expressing circular RNAs via tRNA splicing," vol. 14, no. 8, pp. 978-984, 2017.
[72] E. K. Borchardt et al., "Inducing circular RNA formation using the CRISPR endoribonuclease Csy4," vol. 23, no. 5, pp. 619-627, 2017.
[73] K.-C. Li, Y.-H. Chang, M.-N. Hsu, S.-C. Lo, W.-H. Li, and Y.-C. J. S. r. Hu, "Baculovirus-mediated miR-214 knockdown shifts osteoporotic ASCs differentiation and improves osteoporotic bone defects repair," vol. 7, no. 1, pp. 1-13, 2017.
[74] A. Lavenniah et al., "Engineered circular RNA sponges act as miRNA inhibitors to attenuate pressure overload induced cardiac hypertrophy," 2020.
[75] H. A. Vincent, B. Ziehr, E. M. Lenarcic, and N. J. J. V. Moorman, "Human cytomegalovirus pTRS1 stimulates cap-independent translation," vol. 537, pp. 246-253, 2019.