Ju-Hong Yang1, Chun-Sheng Lu1 and Song Zhang2*
1Department of blood transfusion, Guang Ming New District People’s Hospital of Shenzhen, Shenzhen, Guang Dong, TX 518106, PR China
2Department of Otolaryngology, Guang Ming New District People’s Hospital of Shenzhen, Shenzhen, Guang Dong, TX 518106, PR China
Received: 22 May, 2015; Accepted: 06 July, 2015; Published: 09 July, 2015
Song Zhang, Department of Otolaryngology, Guang Ming New District People’s Hospital of Shenzhen, Shenzhen, Song Bai Road 339#, Guang Dong, TX 518106, PR China; Email:
Yang JH, Lu CS, Zhang S (2015) Alteration of the Methylation Status of Urokinase Plasminogen Activator (uPA) is involved in Proliferation and Invasion of Nasopharyngeal Cancer Cells. Arch Otolaryngol Rhinol 1(1): 023-027. DOI: 10.17352/2455-1759.000004
© 2015 Yang JH, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Nasopharyngeal cancer; Urokinase plasminogen activator (uPA); Methyl binding domain protein 2 (MDB2); siRNA; CNE2 cell line; Methylation
The extracellular matrix degradation is the most important step in the process of tumor cell metastasis. Urokinase-type plasminogen activator (uPA) can catalyzes the conversion of the inactive zymogen plasminogen to the active broad-spectrum plasmin, which degrades a number of matrix proteins and also activates other proteases, including some matrix metalloproteinases. In this study, we evaluated that effect of methylation status of urokinase plasminogen activator (uPA) on invasion and metastasis of nasopharyngeal cancer cells. Methylation status of urokinase plasminogen activator (uPA) was detected by Methylation-specific PCR in the nasopharyngeal carcinoma cells. Reverse transcription-PCR was used to detect the expression of uPA mRNA in the nasopharyngeal carcinoma cell. Invasive and proliferation capacity of CNE2 cell was detected by using Boyden chamber Matrigel invasion assay and Cell proliferation assay after MBD2 knockdown. Our data demonstrated that uPA gene promoter hypomethylation is related to strong uPA mRNA expression in nasopharyngeal cancer cell, uPA mRNA expression was significantly reduced after MBD2 knockdown in the CNE2 cells, the proliferation and invasion capacity of CNE2 cells was significantly inhibited after MBD2 knockdown. Our data suggest that reversal of uPA gene hypomethylation will become a novel therapeutic approach for blocking nasopharyngeal cancer progression and metastatic.
A major feature of cancer cells is their ability to migrate and to invade and develop in surrounding or distant tissues. Tumor cells metastasis to distant organs involves four major steps [11. Mollabashy A, Scarborough M (2000) The mechanism of metastasis. Orthop Clin North Am 31: 529–535.]: adhesion of tumor cells to the extracellular matrix (ECM), ability of tumor cells to degrade the ECM and intravasate into surrounding blood vessels, survival against the natural host defenses and settling at the preferred organ site, and extravasation into the organ and formation of new tumors. The extracellular matrix degradation is the most important step in the process of tumor cell metastasis. Urokinase-type plasminogen activator (uPA) is a member of the serine protease family. It catalyzes the conversion of the inactive zymogen plasminogen to the active broad-spectrum plasmin, which degrades a number of matrix proteins and also activates other proteases, including some matrix metalloproteinases [22. Morgan H, Hill PA (2005) Human breast cancer cell mediated bone collagen degradation requires plasminogen activation and matrix metalloproteinase activity. Cancer Cell Int 5:1.]. It has been implicated that uPA plays an important role in the process of invasion, metastasis and angiogenesis of several malignancies [33. Blasi F, Carmeliet P (2002) uPAR: a versatile signaling orchestrator. Nat Rev Mol Cell Biol 3: 932–943.-55. Rabbani SA, Mazar AP (2001) The role of the plasminogen activation system in angiogenesis and metastasis. Surg. Oncol Clin N Am 10: 393–415.].
Nasopharyngeal cancer (NPC) is a common tumor in head and neck. It has an unusually high incidence in southern China. Cervical lymph node metastasis is a predominant clinical characteristic in the earlier period of nasopharyngeal cancer. It is not very clear that the mechanism of nasopharyngeal cancer metastasis. We have previous study showed that methylation status of Death-associated protein kinase (DAPK), a tumor suppress gene, plays an important role in nasopharyngeal carcinoma progression and metastasis [66. Kong WJ, Zhang S, Guo CK, Wang YJ, Chen X, Zhang SL, et al. Effect of methylation-associated silencing of the deathassociated protein kinase gene on nasopharyngeal carcinoma. Anti-Cancer Drugs17: 251–259, 2006.]. Recent studies have found that the hypomethylation of oncogene plays a causal role in tumor metastasis. Pakneshan found that hypomethylation is responsible for uPA activation and plays a role in metastasis, inhibition of demethylation result in methylation and inactivation of uPA and reversal of metastasis in breast cancer [77. Pakneshan P, Szyf M (2004) Farias-Eisner R, Rabbani S A. Reversal of the Hypomethylation Status of Urokinase (uPA) Promoter Blocks Breast Cancer Growth and Metastasis. J Bio chem 279: 31735-31744.].
In the present study, we demonstrate that methylation state and expression of uPA gene, and its hypomethylated state could be reversed pharmacologically by agents that modulate methylation like an antisense inhibitor of Methyl-binding protein 2 (MDB2) in nasopharyngeal cancer cell line CNE2. This study confirms that uPA is activated by hypomethylation in nasopharyngeal cancer cells, and uPA plays a important role in proliferation and invasion of nasopharyngeal cancer cell. It suggests that pharmacological reversal of uPA gene hypomethylation can be used as a novel therapeutic approach for blocking nasopharyngeal cancer progression.
Materials and Methods
Cell culture and treatment
Human nasopharyngeal cancer cell line CNE2 was obtained from the China Center for Type Culture Collection (CCTCC). CNE2 cell lines were maintained in RPMI 1640 with 10% Fetal bovine serum (FBS), 2 mM L-glutamine adjusted to contain 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate. Cells were incubated at 37°C in 5% CO2.
The targeting sequences corresponding to the siRNA for MBD2 were selected from GeneBank Accession No. NM_003927.4 (GAAGTTCAGAAGCAAGCCT). The pEGFP-C1–U6 plasmid, which had been constructed from pEGFP-C1 and U6 promoter by cloning technique, was digested with BamHI and HindIII. Then, the annealed oligos were ligated into the plasmid. The sequence is BamHI + sense + loop + antisense +termination signal + SalI + HindIII 5'-GATCC GAAGTTCAGAAGCAAGCCTTTCAAGACGAGGCTTGCTTCTGAACTTCTTTTTTGTCGACA-3'. The sequence encoding siRNA with 19 nt of homology to MBD2 was inserted to the down-stream of U6 promoter between BamHI and HindIII. The constructed plasmid was confirmed by DNA sequencing and named pEGFP-C1-U6-MBD2. The target sequence of unrelated siRNA controls (UC) was GACTTCATAAGGCGCATGC. The plasmid constructed using the unrelated siRNA controls sequence was named pEGFP-C1-U6-UC.
CNE2 cells were seeded at an initial density of 104 per well in six well plates. When the cells reached 30% to 50% confluency, cells were transfected with appropriate plasmids (pEGFP-C1-U6-MBD2 or pEGFP-C1-U6-UC) by using Lipofectamine 2000 (Invitrogen, USA) in accordance with the manufacturer's protocol. At 48 hours post-transfection, 0.8mg/ml G418 (Sigma, USA) was added to select positive cell clones. Stable cell clones were isolated after 8 weeks. 104 cells without transfection, cells transfected with control vector or cells transfected with siRNA were seeded into a 6-well cell culture plate, respectively. Cells of each group were harvested after culture for 72 h. It was used for followed experiment.
Methylation-specific PCR (MSP)
Nasopharyngeal cancer cell genomic DNA was extracted using the DNeasy tissue kit and was modificated using the Zymo golden methylation kit (Zymo, Orange, CA) following the manufacturer's instructions. Methylation-specific PCR was done to amplify the indicated region of uPA gene. The methylated primer: forward 5'-AGC GTT GCG GAA GTA CGCGG-3'and reverse 5'-AAA CCC GCC CCG ACG CCG CC-3'). The unmethylated primer: forward 5'-AGT GTT GTG GAA GTA TGT GG-3' and reverse 5'-AAA CCC ACC CCA ACACCA CC-3'). The above uPA promoter using Platinum Taq DNA polymerase (Invitrogen) according to the manufacturer's recommendations under the following same cycle conditions: 95℃ for 3 min; 10 cycles of 95℃ for 30 s, 52℃ for 30 s and 72℃ for 45 s; 20 cycles of 95℃ for 30 s, 50℃ for 30 s and 72℃ for 45 s; a final extension of 72℃ for 5 min. The amplified products were fractionated on 2% agarose gels.
Reverse transcription-PCR (PT-PCR)
Total RNA was isolated from nasopharyngeal cancer cells using Trizol regents. It was used for reverse transcription and amplification. The following primers were used for RT-PCR. The uPA primer: 5-ACA TTC ACT GGT GCA ACT GC-3 (forward) and 5-TTT CCC CAG GCC CAG CTG AA-3 (reverse). The MBD2 primer: 5-AGC GAT GTC TAC TAC TTC AG-3 (forward) and 5-AGA TGT CTG CCA TCA GTG CT-3 (reverse). The β-actin primer: 5'-CGT CTG GAC CTG GCT GGC CGG GACC-3' (forward) and 5'-CTA GAA GCA TTT GCG GTG GAC GAT G-3' (reverse). The DNA was amplified under the following conditions: 95°C for 3 min, 30 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 5 min. The PCR products were then analyzed on a 1% agarose gel.
Boyden chamber Matrigel invasion assay
We examined the invasive capacity of control and experimental CNE2 cell using two compartments: Boyden chambers (Transwell Costar, Cambridge, MA) and basement membrane Matrigel (BD Biosciences, Mississauga, Ontario, Canada). All cells were analyzed for their viability and an equal number of viable cells (104) was added to the upper chamber and allowed to invade through the Matrigel onto the filters for 18 hours. At the end of the incubation period, the filters were washed, fixed, and stained. The invading cells were then examined and counted in 10 randomly selected fields under a light microscope at×400 magnification. Ten random fields for each set of experiments were analyzed and the average number of cells invaded was calculated.
Cell proliferation assay
Three kinds of cells (cells without transfection, cells transfected with control vector and cells transfected with siRNA), were seeded into five 6-well culture plates, with each plate having all three kinds of cells and each group consisted of two parallel wells. All cells, plated at exactly the same density of the viable cells per plate in triplicates, were trypsinized and the number of viable cells was counted daily throughout the 5-day treatment. The growth curve of each group was plotted on the basis of each day cells number.
Results are expressed as the mean±SE. Statistical comparisons were performed using Student's t-test analysis of variance. A probability value of p< 0.05 was considered to be significant.
Effect of MBD2 knockdown on status of uPA gene methylation
Methylation status of the uPA promoter was analyzed by using MSP in nasopharyngeal cancer cell line (CNE2). Hypomethylation of uPA gene promoter was found in CNE2 cells transfected with plasmids pEGFP-C1-U6-UC and without any transfection. Hypermethylation of uPA gene promoter was found in the CNE2 cells transfected with plasmids pEGFP-C1-U6-MBD2.These results showed that promoter hypomethylated uPA gene was re-methylated by transfected pEGFP-C1-U6-MBD2 in CNE2 cells (Figure 1).