SIMULTANEOUSLY BOTH EXPRESSION OF LMP-1 AND METHYLATION OF E-CADHERIN: MOLECULAR BIOMARKER IN STAGE IV OF NASOPHARYNGEAL CARCINOMA PATIENTS
Lao TD1, Truong PK1, Thieu HH1, Nguyen DH2, Nguyen MT3, Le TAH1,*
*Corresponding Author: Associate Professor Thuy A.H. Le, Department of Pharmaceutical and Medical Biotechnology, Ho Chi Minh City Open University, 35-37 Ho Hao Hon Street, Ho Chi Minh City, Vietnam. Tel.: +84-905-784-471. E-mail: thuy.lha@ou.edu.vn
page: 57

MATERIALS AND METHODS

Ethics Statement, Sample Collection. Institutional Ethics Board approval was obtained from the Medical Ethics Committee of the Cho Ray Hospital, Ho Chi Minh City, Vietnam [the decision number of the permission: 516/BVCR-HDDD]. All the samples used in this study were approved by Cho Ray Hospital and obtained from all participants from January 2017 to December 2018. The patients enrolled in this study were required to sign consent forms to approve the usage of the samples for laboratory studies and analyses. A total of 93 archived NPC biopsy tissues that were confirmed by immunohistochemistry, were obtained from the Cho Ray Hospital, Ho Chi Minh City, Vietnam. Notable clinicopathological characteristics, including patients’ gender, age, and histological type were recorded. One hundred non cancerous swab samples were collected from volunteers. In brief, a 15 cm-long cotton stick was inserted into the nasal cavity and moved toward the nasopharyngeal wall; it was then swept over the surface of the posterior and lateral nasopharyngeal wall. The cotton stick was withdrawn and immediately immersed in phosphate-buffered saline stored at –20 °C for further experiments. Nucleic Acid Isolation and Bisulfite Modification. Total RNA was isolated using TRIzol™ Reagent (Cat: 15596026; Thermo Fisher Scientific Inc., Waltham, MA, USA). cDNA was reverse-transcribed from using ~1 μg isolated RNA using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific Inc.). The reverse-transcription assay was performed according to according to the manufacturer’s guideline. Total of genomic DNA was isolated from biopsy and swab samples by the phenol/chloroform method. Then, total genomic DNA was isolated and purified using standard phenol-chloroform and ethanol precipitation. The bisulfite conversion of 500 ng purified DNA was performed using EpiJET Bisulfite Conversion Kit (Thermo Fisher Scientific Inc.). The final precipitation was eluted in a volume of 20 μL and stored at –20 °C for further studies. Quantitative Polymerase Chain Reaction and Nested-Methylation-Specific Polymerase Chain Reaction. For evaluation of LMP-1 expression, quantitative polymerase chain reactions (qPCRs) were done by means of a qSYBR-green and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous control. The internal control candidate was used to normalize the cycle threshold (Ct) values of each LMP-1. The primers, which were used in the current study, were obtained by the previous studies and are shown in Table 1 [16,18,19]. The methylation status of the E-cadherin gene promoter in the clinical samples was examined by two-stage nestedmethylation- specific PCR (nested-MSP). The primers of stage 1 and stage 2 PCR are shown in Table 1. The primers of stage 1 PCR were used for preceding amplification that recognize the bisulfite-modified template, and notably, do not discriminate between methylated and un-methylated sequences. In stage 2 PCR, two pairs of primer were used to amplify the regions of interest. One pair recognized a sequence in which CpG sites were methylated (unmodified by bisulfite treatment). Another pair recognized a sequence in which CpG sites were unmethylated (modified to UpG treatment). Each stage of PCR was performed in a total of 15 μL containing 3 μL bisulfite-modified template DNA (in case of stage 1 PCR) or 3 μL stage 1 PCR product (in case of stage 2 PCR), 0.75 unit iTaq DNA polymerase (Bio-Rad Laboratories, Hercules, CA, USA), 0.5 μM each primer, 7.5 μL MyTaqTM Mix (Bioline Reagents Ltd., London, UK). Thermal cycling was initiated at 95 °C for 5 min., followed by 40 cycles of denaturation at 95 °C for 30 seconds, annealing at X °C for 30 seconds, extension at 72 °C for 30 seconds, and a final extension at 72 °C for 10 min. (X °C was the specific annealing temperature for each primer, shown in Table 1). Finally, the methylated and unmethylated PCR products were separated on 2.0% agarose gel and visualized by ethidium bromide staining. Representative nested-MSP products were sequencing to confirm the specificity of primers, examine the efficiency of bisulfite modification and the hypermethylation status of target gene. Statistical Analyses. Statistical analyses were performed by Medcalc® Version 12.7.0.0 (MedCalc Software bv, Ostend, Belgium). The average frequency of methylation and LMP-1 expression was calculated. The χ2 test was used to determine the association between the expression of LMP-1, E-cadherin methylation and NPC status. Moreover, the association between hypermethylation of E-cadherin, expression of LMP-1 and risk of cervical cancer was estimated by computing the odds ratio (OR) and 95% confidence interval (CI).



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