Field infected B lymphocytes possibly throat epithelium, with

Field of research

RNA, Cancer Biology

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Topic of research

of circular RNA (circRNA) in the development and progression of nasopharyngeal


Statement of problem:

Virus (EBV), also known as human gamma herpes virus that consists of 172 kb of
linear double stranded DNA genome. Genetic content is enclosed within an
icosahedral capsid, surrounded by an envelope. EBV is one of the most common
human viruses as it infects approximately 95% of the world’s population. Human usually
get infected through parental exposure or exposure to infected body fluids such
as blood, saliva, semen and breast milk. The infected person will carry the
virus DNA for his entire life (Young et al., 2016).


EBV infection is usually asymptomatic or causes infectious mononucleosis.
Immune system of healthy individual controls the pathological effects of EBV
infection, leading to asymptomatic infection. In immunocompromised individuals,
EBV infection can cause fatal lymphoproliferative diseases and tumor growth.
EBV is the first discovered human tumor virus that accounts for various
malignancies, 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, with
periodic reactivation of lytic replication (Matsuura et al., 2010).


acute infection, expression of EBV gene is under control. During latency, EBV
genome is circularized and form episomes. EBV has a unique replication
mechanism which is used for establishment and maintenance during latency
period. There are 6 EBV-encoded nuclear antigens (EBNA), 3 latent membrane
proteins (LMP) and several RNA species. These factors are essential for
activation of quiescent B lymphocytes from G0 phase into cell cycle,
initiation of proliferation and maintenance of episomal viral genome. EBNA-2
and LMP-1 protein is essential for transformation process (Young et al., 2016). 


coding RNA, non-coding RNA is also suggested to play a vital role in
development of EBV-related diseases. Non-coding RNA is a functional RNA
molecule that is transcribed from DNA but do not encode for a protein. Two
EBV-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 of
latency, as well as in lytic growth. Studies found that EBERs increase
tumorigenicity, enhance cell survival and induce interleukin-10 production in Burkitt’s
lymphoma, 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 genome
which acts as a modulator for protein production through transcriptional
repression of mRNAs. EBV miRNAs are postulated to have important role in the
initial stages of B cells transformation (Klinke et al., 2014).


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 in
examined 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 of
an exon turns back and ligate with 5′ ends of other upstream exons to form a
closed loop, forming circRNA  A dozens of
circular RNA are reported to be highly expressed in tissue or developmental
stage specific manners. circRNAs can be derived from either exons or introns or
both, with different lengths. circRNAs exhibit highly conserved region and
abundantly expressed in cytoplasmic region. Several biological functions of
circRNAs have been proposed, including acts as miRNA sponges, promoting parent
genes transcription and mRNA traps (Huang et al., 2017).


Research Objective

determine the genes and/or pathway regulated by circLMP2 via ectopic expression
of circLMP2 in NPC cells.


Research Methodology


of pcDNA-Tet-Puro-circLMP2 will be carried out. pcDNA3 containing circLMP2 gene
is 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 be
inserted to the upstream of circLMP2 gene. Neomycin resistant gene in pcDNA3
plasmid will be replaced with tet repressor gene and puromycin resistant gene.


Ectopic expression of circLMP2

expression of circLMP2 on pre-malignant EBV-ve NP460hTert and NPC EBV+ve C666-1
cells will be carried out to determine the gene and/or pathway regulated by
circLMP2 in this study. pcDNA-Tet-Puro-circLMP2 will be used. An empty vector will
be used as negative control. The expression of circLMP2 will be measured using
RT-qPCR to ensure satisfactory circLMP2 expression.

mRNA and small RNA-sequencing

cellular RNA will be extracted from 2 stable cell lines over-expressing
circLMP2 and negative control before mRNA and small RNA-sequencing. RIN score
>7 will be send for library preparation and RNA-sequencing.


RNA-seq data analysis

top affected genes and/or small RNAs will be subjected to ingenuity pathway
analysis (IPA) to identify significantly impacted pathways and gene ontology
(GO terms) associated with circLMP2.


Significant of the research

RNA which was regarded as “junk” for almost two decades, are now becoming one
of the popular molecules in research focus. Identification of EBV circRNA may
provide a better understanding of its role in the development and progression
of nasopharyngeal carcinoma as circRNA has been reported to have various
functions in regulation of gene and disease development.



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