NPC is happening by insertion of EBV-DNA viruses. EBV infects B lymphocyte by expressed the LMP1 (oncogene) to increase B-cell proliferation by making a similar signal to activated B-cell by upper cell molecule B-cell, CD40. In another hand, LMP-1 is prevented apoptosis by activated BCL2. Then, the proliferation will be uncontrolled . To achieve long-term in vivo, the EBV express the latent gene which causes latent infection .
The drugs of choice in NPC therapy was cisplatin, an inorganic metal drugs as an alkylating agent. As an alkylating agent, it works by inhibiting cross-linking in the N7 alkyl group of guanines and makes uncoil strand . The issue of drugs resistance in cancer therapy have been investigated after cisplatin therapy . Molecular mechanism of cisplatin resistance has been investigated caused by a kinase, such as Akt and YAP1. Alteration of the kinase expressions has been influenced by the expression of a tumor suppressor gene. MicroRNA, the single-strand non-coding RNA has been worked in post-transcriptional mechanism and could be suppressed the expression of tumor suppressor gene .
miR-141 have been investigated in NPC proliferation cells by downregulated PTEN, a tumor suppressor gene as an antagonist in the AKT/PI3K pathway . miR-141 have been investigated a role in resistance to cisplatin in esophageal squamous cell carcinoma by targeting YAP1 . In ovarian cancer have been investigated the role of miR-141 in cisplatin resistance by targeting KEAP1 . Induction of NPC in Akt/FoxO pathway is known to lead to their resistance to cisplatin .
A research study in NPC cells has investigated the role of miR-141 and mRNA PTEN in NPC pathogenesis. miR-141 has been investigated as oncomiR and mRNA PTEN was targetted . In order to support the noninvasive biomarker agent in drugs resistance, research on miR-141 and mRNA PTEN expression was conducted in blood plasma of NPC patients. At the same time, inhibition of EBV-miR to mRNA PTEN also done to investigate the miRNA-miRNA interaction by in silico study.
2. Materials and Methods
Sample collections used was some blood patients with NPC (pre and post therapy) and plasma of normal people from Dharmais Cancer Hospital and diagnoses of NPC has been done by Cita Herawati. All sample experiments were approved by the ethical committee of Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada.
Micropipet tube, microRNA mini spin column BF (Exiqon), vortex, centrifuge, spin down, thermal cyder Biorad c1000, qRT-PCR Biorad.
Alcohol 70 %, isopropanol p.a, rDNAse, miRCURY RNA isolation kit- Biofluid Exiqon (Lysis solution BF, Protein precipitation solution BF, Wash solution 1 dan 2 BF, RNAse free water), kit universal cDNA synthesis kit II, 8-64 rxns (cat no. 2-33-1, exiqon), 5 times of reaction buffer, nuclease free water, spike in (Sp6), enzyme mix, ice pack. miRCURY LNA Universal RT micro RNA PCR, LNA PCR Primer Set, hsa-miR-141-3p UAACACUGUCUGGUAAAGAUG
For mRNA: KAPA SYBRⓇ FAST One-Step qRT-PCR Kit Universal (KAPA SYBR FAST qPCR master mix, ROX Reference Dye High, ROX Reference Dye Low, dUTP, KAPA RT mix), primer set mRNA PTEN (forward: GGGTCTGAGTCGCCTGTCA, reverse: CCGTGTTGGAGGCAGTAGAAG), primer beta-actin (forward: GGGAATTCAAAACTGGAACGGTGAAGG, reserve: GGAAGCTTATCAAAGTCCTCGGCCACA). The primer of mRNA PTEN was available from Vinciguerra, et al..
Blood sample in vacutainer EDTA has been centrifuged at 1 500 rad · s in 10 min and collect the plasma. Total RNA from blood plasma was extracted using the miRCURY RNA Isolation Kit-Biofluid (Cat No. 300112, Exiqon). Complementary DNA was prepared from DNAse-treated RNA samples with Universal cDNA Synthesis kit II, 8-64 rxns (Cat. No. 203301, Exiqon). Quantitative PCR was performed on a StepOnePlus real-time PCR system (Applied Biosystems) using Exilent SYBR Green master mix (Cat No. 203402, Exiqon). mRNA PTEN was detected using KAPA SYBR ⓇFAST One-Step qRT-PCR KIT Universal, All instructions according to the manufacturer's instructions.
Quantitative real-time PCR
Total RNA from blood plasma was extracted using the miRCURY RNA Isolation Kit-Biofluid (Cat No. 300112, Exiqon). Complementary DNA was prepared from DNAse-treated RNA samples with Universal cDNA Synthesis kit II, 8-64 rxns (Cat. No. 203301, Exiqon). Quantitative PCR was performed on a StepOnePlus real-time PCR system (Applied Biosystems) using Excellent SYBR Green master mix (Cat No. 203402, Exiqon). mRNA PTEN was detected using KAPA SYBR ⓇFAST One-Step qRT-PCR KIT Universal
Table 1 describes the expression of miR-141 and mRNA PTEN in the sample. The expression of miR-141 has been upregulated after cisplatin therapy, but the expression of mRNA PTEN have been downregulated after therapy. Its indicated that the therapy was not responded.
NPC happen because of EBV genome insertion. Then we conducted interaction between EBV-miR with mRNA PTEN by in silico study with Vienna Package Software (Figure 1). The study has been done for investigated the reason of down-regulated of mRNA PTEN after therapy. All of EBV-miR have been targeted mRNA PTEN, which means that its interaction may cause the down-regulated of mRNA PTEN.
miR-141 have been investigated as oncomir by down-regulated mRNA PTEN in blood plasma NPC. PTEN plays a role in conducting dephosphorylation of PIP3 into PIP2 on Akt phosphorylation of PI3K pathway . However, miR-141 as oncomir can inhibit the expression of PTEN in post-transcriptional processes. The expression of oncomir also decreased after drugs therapy, which it can increase the expression of mRNA targetted. However, the expression of mRNA PTEN was upregulated after therapy although the expression of miR-141 was down-regulated. It has meant that the therapy was not responded and there might be cisplatin resistance.
In cellular respond, cisplatin can be interacted with secondary thiols, like glutathione and metallothionein. Glutathione-cisplatin conjugated exit from the cell by ATP- dependent pump. After cisplatin interacted with DNA, it will inhibit proliferation by activated DNA damage. Kinase such as Akt, PKC, and ERK is also involved in the regulation of cell death that induced by cisplatin. miR-214 was promoted cisplatin resistance by downregulating PTEN and Akt . miR-141 might be promoted cisplatin resistance by downregulating PTEN and Akt activated similar as miR-214.
The inhibition in mRNA PTEN by another oncomir, like EBV-miR, makes mRNA PTEN down-regulated and not responded to cisplatin therapy. miRNA-miRNA interaction may be potential on complex network regulation of biological processed . Another miRNA can influence any miRNA by regulated their targeted and modulated their activity .
miR-141 is one of the important factors in cisplatin drugs resistance through inhibition of mRNA PTEN and Akt activated signaling. mRNA PTEN in NPC might be bound to EBV-miR that makes mRNA PTEN down-regulated and not responded to cisplatin therapy