First Record of Identification of RPMS1 Gene Variations in Vietnamese Nasopharyngeal Carcinoma Patients
First Record of Identification of RPMS1 Gene Variations in Vietnamese Nasopharyngeal Carcinoma Patients
Asian Journal of Pharmaceutical Research and Health Care, Vol 10(4), 97-103, 2018 DOI: 10.18311/ajprhc/2018/23576
Abstract
Epstein-Barr Virus (EBV) infection and EBV genes’ variation are considered as the etiological factors contributed to Naso-
Pharyngeal Carcinoma (NPC). In the latent EBV stage, not only the latent membrane proteins but also RPMS1 expression
has been confirmed in all EBV-associated tumors. In Vietnam, an Asian country with the high incidence, mortality rate of
NPC, had limit research on the RMPS1 gene variation. Therefore, the objects of current study were to identify the pattern of
RMPS1 variations in Vietnamese NPC patients for its value further applied in NPC patients. In this study, thirty NPC biopsy
samples and thirty non-cancerous swab specimens were collected from local patients, analyzed by PCR, sequencing and
compared to previous B95-8 sequence. As the results, the strongly association between the detection of RPSM1 gene and
NPC incidence in Vietnamese NPC patients was determined. Additionally, the RMPS1 gene variants, including wild-type,
RMPS1-B, RMPS1-C, RMPS1-C*, were identified. Among them, RMPS1-C/C* was preferential in Vietnamese nasopharyngeal
cancer. In conclusion, those data is the first dataset on the polymorphism in the RPMS1 gene in Vietnamese NPC patients,
and could be utilized as a promising biomarker for prognosis, diagnosis and therapy for NPC based on the EBV gene
variations.
the member of the BamHI-A Rightward Transcripts HDDD, was obtained from the Medical Ethics Committee
(BARTs) family, RPMS1 expression has been confirmed of the Cho Ray Hospital, Ho Chi Minh City, Vietnam. All
in all EBV-associated tumors9-13. A number of studies the biopsy samples used in current study were collected
have reported attempts to identify the NPC associated from participants, who agreed and signed on the consent
EBV genes’ variations, including EBV Nuclear Antigens forms.
(EBNAs), Latent Membrane Proteins (LMPs) and EBV-
encoded small nuclear RNAs (EBERs), have been shown 2.2 Samples Collection, and DNA Isolation
to be associated with NPC pathogenesis14-17. However,
Thirty nasopharyngeal carcinoma biopsy samples were
few studies have investigated the single nucleotide
collected from local patients, in Cho Ray Hospital, Ho
polymorphisms (SNPs) of RPMS1 gene, as the reported,
Chi Minh City, Vietnam. The entire sample was submitted
only one SNP site (locus: 155391 G > A, named
to the histopathological diagnosis center to confirm the
G155391A) is identified and significantly associated with
NPC. Notably, all of those biopsies were collected from
NPC incidence16,18. They found that the RPMS1 variation
patients, who were obeyed to ethical approval for study
(G155391A) functionally relevant to regulating the
human samples, and patients agreed with purpose of the
PRMS1 protein stability and over expressed in vitro16.
study. In addition, thirty nasopharyngeal swab samples,
Vietnam, located in Southern Asia, is well known as
which were collected from healthy donors, used as
the high incidence and mortality rate of nasopharyngeal
negative-nasopharyngeal carcinoma control.
carcinoma in the world. According to EBV infection, the
For DNA isolation, biopsies were lysed in lysis buffer,
rate of EBV positive in NPC patients is high17. However,
containing 10 mM Tris-HCl pH=8, 10 mM EDTA,
limited studies were carried on the genotyping of EBV as
150 mM NaCl, 2% SDS, and 0.1mg/ml Proteinase K,
well as the variations of EBV genes. In previous studies,
incubated in 56oC, overnight. Total of genomic DNA
we established the protocol for genotyping of EBNA-1
of clinical samples were extracted by using Phenol/
subtype from of nasopharyngeal biopsy samples collected
Chloroform solution, pH = 8. The purification of DNA
from Vietnamese nasopharyngeal carcinoma patients, and
was done by Ethanol solution 99%. The quality and purity
the results suggested the V-val subtype is the preferentially
of DNA isolates were measured by the evaluation of A260/
subtype associated with nasopharyngeal carcinoma. To
A280 proportion. The DNA solution was stored in Tris-
further identify the EBV variations linked to NPC risks,
EDTA 0.5M, at -20oC for further assays.
we conducted the analysis on the RPMS1 gene, a member
of BARTs family, in the comparison to the prototype
2.3 PCR-sequencing of RPMS1 Gene
B95-B strain, starting from NPC biopsy samples and
healthy controls in the Vietnamese population. Therefore, Multiplex PCR assay was applied for the amplification
our objective was to develop the simple method to of RPMS1 gene and beta-actin gene, which served as the
identify the RPMS1 gene’s polymorphisms at Vietnamese internal control. The sequence and position of primers
population. were shown in (Table 1).
The PCR assay was performed in a total of 15 µL,
containing 100 ng genomic DNA templates, 0.25 µM
2. Materials and Methods each primer, and 7.5 µL MyTaqTM Mix (Bioline, Cat No.
BIO-25041). PCR amplification was performed with an
2.1 Ethics Statement
initial denaturation at 94oC for 5 minutes; followed by 45
Institutional Ethics Board approval, the decision number cycles of denaturation at 94oC for 30 seconds, annealing at
of the permission from Ethical committee: 516/BVCR- 60oC for 30 seconds, extension at 72oC for 30 seconds; and
98 Vol 10 (4) | 2018 | www.informaticsjournals.org/index.php/ajprhc Asian Journal of Pharmaceutical Research and Health Care
Duc Thuan Lao, Danh Hoang Nguyen, Minh Trong Quang, Hue Hong Thieu and Thuy Ai Huyen Le
elongation step at 72oC for 10 minutes. The PCR products extracted DNA and PCR assay. Overall, the frequency of
were visualized and analyzed by electrophoresis through RPMS1 in NPC samples was 66.67% (20 of 30 samples).
stained with Ethidium bromide, and 2.0% agarose gel. In non-NPC swab samples, no RPMS1-positive case was
For RPMS1 gene sequencing, 30 µL products were sent detected. Based on the statistical analysis, the presence
to Nam Khoa Biotek Co. Ltd. (Vietnam) for directly of RPMS1 was found to be significantly associated with
sequencing in both directions with primer of RPMS1. NPC (p < 0.05). Additionally, the 733-bp product, which
indicated the presence of RMPS1, was determined by
2.4 Determination and Analysis of RPMS1 Sanger sequencing. The signals of peaks in PCR product
sequencing were good for nucleotide reading (Figure 2).
Variations According to BLAST results, candidate gene’s sequence
The results of RPMS1 sequencing was read by Chromas was similar to Human gammaherpes virus 4 (Accession
2.6.4 (Technelysium) and checked for sequencing number: MH144220) within the Total score = 1216, ident
homology by using BLAST (NCBI). For determination of = 99.85%, E-value = 0.0.
RPMS1 variations, the sequencing of RPMS1 of current
study were compared to the B95-8 strain (Genbank 3.2 Identification of RPMS1 Gene
accession number: NC_007605; Gene RPMS1’s Location
Variations: RPMS1-C Variation was
138352..160531). The alignment between data sequence
and B95-8 strain sequence were analyzed by using BioEdit Preferential in Vietnamese Nasopharyngeal
sequence alignment editor. Cancer
RPMS1 gene was successfully amplified, observed in
a distinct band with 733 bps, shown in Figure 1 and
3. Results
sequenced in 20 of 30 NPC samples. The nucleotide
3.1 Association between RMPS1 and NPC variations were determined by mapping and comparing
Incidence to the RPMS1 gene of wild-type EBV genome (GenBank
Accession No. NC_007605: location 138352..160531).
The frequency of RPMS1 gene in NPC biopsy samples and As the results, nice NPC samples (counting for 45.0%)
healthy samples were detected by PCR method. The PCR were similar to the wild-type EBV’s RPMS1 gene. The
product sizes, 319-bps and 733-bps bands, were different polymorphisms point G155391A, classified as RPMS1-C,
which is easy to be distinguished on electrophoresis results, was discovered in 10 NPC samples (counting for 50.0%).
shown in (Figure 1). The human beta-actin PCR product Of these, 1 NPC sample differentially distributed
of 319 bps gave the indication on the good quality of the substitution G155391A and one silent polymorphism
Figure 1. Electrophoresis of Nested-PCR products of representative clinical samples: T4, T5, T6, T7, T11: clinical biopsy
samples; H1, H2, H3, H4, H5: Healthy (non-NPC) swab samples; N: negative control; MW: molecular weight 100 bps.
Vol 10 (4) | 2018 | www.informaticsjournals.org/index.php/ajprhc Asian Journal of Pharmaceutical Research and Health Care 99
First Record of Identification of RPMS1 Gene Variations in Vietnamese Nasopharyngeal Carcinoma Patients
(A)
(B)
(C)
Figure 2. Validation of the RPMS1 single nucleotide polymorphism (A) G155391A; (B) T155384C; (C) C155389T in
representative NPC samples by Sanger sequencing. (a) wild type RPMS1 gene (NC_007605); (b) representative SNPs identified
samples.
100 Vol 10 (4) | 2018 | www.informaticsjournals.org/index.php/ajprhc Asian Journal of Pharmaceutical Research and Health Care
Duc Thuan Lao, Danh Hoang Nguyen, Minh Trong Quang, Hue Hong Thieu and Thuy Ai Huyen Le
site as T155384C, classified as RPMS1-C* (Figure 2A, B). the current study is the initial study, which recorded the
Moreover, only one sample, counting for 5.00%, within determination of RPMS1 polymorphisms in total of 20
the substitution C155389T, classified as RPMS1-B, was RPMS1-positive samples collected from Vietnamese NPC
determined (Figure 2C) (Table 2). Therefore, RPMS1-C patients. As the results, T155384C variant was identified
variation was preferential in Vietnamese nasopharyngeal as the novel variant of RPMS1 variations from Vietnamese
cancer. nasopharyngeal carcinoma patients. However, this
All the nucleotide variations of sequencing, encoded variant did not effected the primary structure of
the amino acid residue 48, 50, 51 of RPMS1 gene, of 20 translated protein, due to the silence substitution
cases were determined by comparing with the B95- (CCT: Pro>CCC:Pro). Therefore, in summary, three
8. As the results, three patterns of RPMS1, including patterns, RPMS1-B, RPMS1-C/C*, and Wt, were
RPMS1-B, RPMS1-C, RPMS1-C*, and wild type (Wt), identified. In previous studies, four RPMS1 patterns
were determined (Table 2). In detail, Wt (amino acid were determined and clustered as RPMS1-A, RPMS1-B,
at 51 (GAC:Asp), representatively reported by T6), RPMS1-C and RPMS1-D (compared to wild type EBV
RPMS1-B (the amino acid change was at residue 50 genome). A RPMS1-C pattern variation was represented
(CCA:Pro>CTA:Leu), representatively T22), RPMS1-C by the RPMS1 SNP G155391A (amino acid residue
and RPMS1-C* (the amino acid change was at residue GAC:Asp>AAC:Asn). This variation has been reported
51 (GAC:Asp>AAC:Asn), representatively reported by to be associated with a high risk of NPC16. According to
T5), were similar to previous studies. The RPMS1-C Feng et al., they suspected that the variation of G155391A
and RPMS1-C* shared the same pattern, in spite of the from Guanine to Adenine, leading to the amino acid
T155384C occurred in RPMS1. change from Aspartic acid (Asp) to Asparagine (Asn),
might related to the expression of RPMS116. In detail,
the variations of G155391A are functionally relevant to
4. Discussion the stability of RPMS1, which is in a longer half life of
In current study, we successfully amplified RPMS1 gene RPMS1 protein and may exhibit stronger carcinogenesis
from NPC biopsy samples by multiplex-PCR with the potential. Additionally, the oncogenic RPMS1 has shown
internal control – beta actin. Notably, to our knowledge, to promote the cell differentiation and proliferation, thus,
Table 2. Nucleotide and amino acid substitution in RPMS1 gene compared to wild type B95-8 sequence
Patterns Nucleotide Position Frequency
155382
155383
155384
155388
155389
155390
155391
155392
155393
Wild type (NC_007605) C C T C C A G A C
Pro Pro Asp
Codon 48 50 51
RPMS1-B T22 . . . . T . . . . 5.0%
* Leu *
RPMS1-C T5 . . . . . . A . . 50.0%
* * Asn
RPMS1-C* T9 . . C . . . A . .
+ * Asn
Wt T6 . . . . . . . . . 45.0%
* * *
Note: The top four rows were corresponding to the nucleotide, amino acid position, nucleotide and amino acid of B95-8 prototype
sequence. In each row, the upper character denoted the nucleotide, which differed to the B95-8 prototype sequence. Conversely,
the dot (.) denoted the same nucleotide compared to the referent sequence. The below characters indicated the amino acid written
in three letter code that differed to the referent sequence. On the contrary, the “*” character indicated the unchanged amino acid,
and the “+” character indicated the silent amino acid changes.
Vol 10 (4) | 2018 | www.informaticsjournals.org/index.php/ajprhc Asian Journal of Pharmaceutical Research and Health Care 101
First Record of Identification of RPMS1 Gene Variations in Vietnamese Nasopharyngeal Carcinoma Patients
102 Vol 10 (4) | 2018 | www.informaticsjournals.org/index.php/ajprhc Asian Journal of Pharmaceutical Research and Health Care
Duc Thuan Lao, Danh Hoang Nguyen, Minh Trong Quang, Hue Hong Thieu and Thuy Ai Huyen Le
11. Marquitz AR, Raab-Traub N. The role of miRNAs and EBV Q, Cao SM, Jia WH, Bei JX, Zeng YX. A single nucleo-
BARTs in NPC. Semin Cancer Biol. 2012; 22(2):166−72. tide polymorphism in the Epstein-Barr virus genome is
https://doi.org/10.1016/j.semcancer.2011.12.001. PMid: strongly associated with a high risk of nasopharyngeal car-
22178394, PMCid: PMC3340885. cinoma. Chin. J. Cancer. 2015; 34(12):563−72. https://doi.
12. Gilligan KJ, Rajadurai P, Lin JC, Busson P, Abdel-Hamid org/10.1186/s40880-015-0073-z.
M, Prasad U, et al. Expression of the Epstein-Barr virus 17. Lao TD, Nguyen DH, Nguyen TM, Le TAH. Molecular
BamHI A fragment in nasopharyngeal carcinoma: evi- Screening for Epstein-Barr virus (EBV): Detection of
dence for a viral protein expressed in vivo. J Virol. 1991; Genomic EBNA-1, EBNA-2, LMP-1, LMP-2 Among
65(11):6252−59. Vietnamese Patients with Nasopharyngeal Brush Samples.
13. Smith PR, de Jesus O, Turner D, Hollyoake M, Karstegl Asian Pac. J. Cancer Prev. 2017; 18(6):1675−1679.
CE, Griffin BE, Karran L, Wang Y, Hayward SD, Farrell 18. Cui Q, Feng FT, Xu M, Liu WS, Yao YY, Xie SH, Li XZ,
PJ. Structure and coding content of CST (BART) family Ye ZL, Feng QS, Chen LZ, Bei JX, Feng L, Huang QH,
RNAs of Epstein-Barr virus. J. Virol. 2000; 74(7):3082−92. Jia WH, Cao SM, Chang ET, Ye W, Adami HO, Zeng
https://doi.org/10.1128/JVI.74.7.3082-3092.2000. PMid: YX. Nasopharyngeal carcinoma risk prediction via sali-
10708423, PMCid: PMC111807. vary detection of host and Epstein-Barr virus genetic
14. Hui KF, Chan TF, Yang W, Shen JJ, Lam KP, Kwok H, Sham variants. Oncotarget. 2016; 8(56):95066−74. https://doi.
PC, Tsao SW, Kwong DL, Lung ML, Chiang AKS. High org/10.18632/oncotarget.11144.
risk Epstein-Barr virus variants characterized by distinct 19. Yap YY, Hassan S, Chan M, Choo PK, Ravichandran
polymorphisms in the EBER locus are strongly associated M. Epstein-Barr virus DNA detection in the diagnosis
with nasopharyngeal carcinoma. Int. J. Cancer. 2018; [Epub of nasopharyngeal carcinoma. Otolaryngol Head Neck
ahead of print]. https://doi.org/10.1002/ijc.32049. Surg. 2007; 136(6):986−91. https://doi.org/10.1016/j.
15. Chao M, Wang HN, Lu YJ, Chang YS, Yu JS. The V-val otohns.2006.11.027. PMid: 17547993.
subtype Epstein-Barr virus nuclear antigen 1 promotes 20. Zhang J, Chen H, Weinmaster G, Hayward SD. Epstein-
cell survival after serum withdrawal. Oncol Rep. 2015; Barr virus BamHi-a rightward transcript-encoded RPMS
33(2):958−66. https://doi.org/10.3892/or.2014.3625. PMid: protein interacts with the CBF1-associated corepres-
25434292. sor CIR to negatively regulate the activity of EBNA2
16. Feng FT, Cui Q, Liu WS, Guo YM, Feng QS, Chen LZ, Xu and NotchIC. J. Virol. 2001; 75(6):2946−56. https://doi.
M, Luo B, Li DJ, Hu LF, Middeldorp JM, Ramayanti O, Tao org/10.1128/JVI.75.6.2946-2956.2001. PMid: 11222720,
PMCid: PMC115921.
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