Field of researchNon-codingRNA, Cancer Biology Topic of researchRoleof circular RNA (circRNA) in the development and progression of nasopharyngealcarcinoma. Statement of problem:Epstein-BarrVirus (EBV), also known as human gamma herpes virus that consists of 172 kb oflinear double stranded DNA genome. Genetic content is enclosed within anicosahedral capsid, surrounded by an envelope.

EBV is one of the most commonhuman viruses as it infects approximately 95% of the world’s population. Human usuallyget infected through parental exposure or exposure to infected body fluids suchas blood, saliva, semen and breast milk. The infected person will carry thevirus DNA for his entire life (Young et al., 2016).  PrimaryEBV infection is usually asymptomatic or causes infectious mononucleosis.Immune system of healthy individual controls the pathological effects of EBVinfection, leading to asymptomatic infection.

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In immunocompromised individuals,EBV infection can cause fatal lymphoproliferative diseases and tumor growth.EBV is the first discovered human tumor virus that accounts for variousmalignancies, including Burkitt’s lymphoma (BL) and nasopharyngeal carcinoma(NPC). Epithelial cells and B lymphocytes are the targets of EBV for infection.EBV remains latent in infected B lymphocytes possibly throat epithelium, withperiodic reactivation of lytic replication (Matsuura et al., 2010). Duringacute infection, expression of EBV gene is under control.

During latency, EBVgenome is circularized and form episomes. EBV has a unique replicationmechanism which is used for establishment and maintenance during latencyperiod. There are 6 EBV-encoded nuclear antigens (EBNA), 3 latent membraneproteins (LMP) and several RNA species. These factors are essential foractivation of quiescent B lymphocytes from G0 phase into cell cycle,initiation of proliferation and maintenance of episomal viral genome. EBNA-2and LMP-1 protein is essential for transformation process (Young et al.

, 2016).   Besidescoding RNA, non-coding RNA is also suggested to play a vital role indevelopment of EBV-related diseases. Non-coding RNA is a functional RNAmolecule that is transcribed from DNA but do not encode for a protein. TwoEBV-encoded RNA, EBER-1 and EBER-2 with 167 and 172 nucleotides respectively,are well-known non-coding RNA that are abundantly expressed in all form oflatency, as well as in lytic growth. Studies found that EBERs increasetumorigenicity, enhance cell survival and induce interleukin-10 production in Burkitt’slymphoma, whereas promotes proliferation of NPC cells (Young and Rickinson, 2004, Daker et al., 2013). Other than EBERs, microRNA (miRNA),a 17–23 nucleotide-long non-coding RNA can also be found within EBV genomewhich acts as a modulator for protein production through transcriptionalrepression of mRNAs.

EBV miRNAs are postulated to have important role in theinitial stages of B cells transformation (Klinke et al., 2014).  Moreover,there is a recently discovered class of non-coding RNA, circular RNA (circRNA)which its expression is widespread and more than 20% of expressed genes inexamined cells and tissues are able to produce these transcripts. Nevertheless,there is no study has reported the expression of circRNA in EBV. The 3′ ends ofan exon turns back and ligate with 5′ ends of other upstream exons to form aclosed loop, forming circRNA  A dozens ofcircular RNA are reported to be highly expressed in tissue or developmentalstage specific manners. circRNAs can be derived from either exons or introns orboth, with different lengths. circRNAs exhibit highly conserved region andabundantly expressed in cytoplasmic region.

Several biological functions ofcircRNAs have been proposed, including acts as miRNA sponges, promoting parentgenes transcription and mRNA traps (Huang et al., 2017). Research ObjectiveTodetermine the genes and/or pathway regulated by circLMP2 via ectopic expressionof circLMP2 in NPC cells. Research MethodologyCloning Cloningof pcDNA-Tet-Puro-circLMP2 will be carried out. pcDNA3 containing circLMP2 geneis used as the vector for cloning to produce a Tet-on inducible system.Tet-pLKO-puro plasmid is used as the template.

Two tet operon genes will beinserted to the upstream of circLMP2 gene. Neomycin resistant gene in pcDNA3plasmid will be replaced with tet repressor gene and puromycin resistant gene.  Ectopic expression of circLMP2 Ectopicexpression of circLMP2 on pre-malignant EBV-ve NP460hTert and NPC EBV+ve C666-1cells will be carried out to determine the gene and/or pathway regulated bycircLMP2 in this study. pcDNA-Tet-Puro-circLMP2 will be used. An empty vector willbe used as negative control. The expression of circLMP2 will be measured usingRT-qPCR to ensure satisfactory circLMP2 expression.

mRNA and small RNA-sequencingTotalcellular RNA will be extracted from 2 stable cell lines over-expressingcircLMP2 and negative control before mRNA and small RNA-sequencing. RIN score>7 will be send for library preparation and RNA-sequencing.  RNA-seq data analysisThetop affected genes and/or small RNAs will be subjected to ingenuity pathwayanalysis (IPA) to identify significantly impacted pathways and gene ontology(GO terms) associated with circLMP2.  Significant of the researchCircularRNA which was regarded as “junk” for almost two decades, are now becoming oneof the popular molecules in research focus. Identification of EBV circRNA mayprovide a better understanding of its role in the development and progressionof nasopharyngeal carcinoma as circRNA has been reported to have variousfunctions in regulation of gene and disease development.  

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