adenosine is released into the extracellular space when the oxygen supply is decreased or energy consumption is increased [ 1 , 2 ] . in particular , significant levels of adenosine
are found in the extracellular fluid of solid tumors , suggesting a role for adenosine in tumor growth .
adenosine is an endogenous nucleoside that modulates many physiological processes through its interaction with at least four membrane receptors : a1 , a2a , a2b , and a3 [ 4 , 5 ] .
the a2 receptors are divided into two subtypes : high - affinity a2 receptors in rat striatum and low - affinity a2 receptors throughout the brain .
these high- and low - affinity receptor subtypes were later designated as a2a and a2b , respectively .
it was thought that the a2b receptor plays a small role in vivo because of its relatively low affinity for adenosine .
recently , spychala discovered that activation of adenosine receptors may be involved in tumor progression .   in a previous study , we collected 64 samples of hepatocellular carcinoma from clinical patients and found that a2b receptors were highly expressed in tumor tissue and expressed in peritumoral tissues to a lesser extent by real - time pcr , western blot , and immunohistochemical staining .
these results indicate that the a2b receptor may contribute to hepatic tumor progression and may represent a good therapeutic target . in recent years
, rna interference ( rnai ) has been widely used to study gene expression regulation in mammalian cells and therapeutic intervention for various diseases including cancer . in this study
our vector is based on the psilencer 3.1-h1 neo ( hind iii / bamh i ) vector ( ambion ) which contains the human h1 promoter and neomycin resistance gene to enable antibiotic selection in mammalian cells .
three pairs of complementary oligonucleotides ( shrna1 , shrna2 , and shrna3 ) were synthesized , targeting adenosine a2b receptor cdna ( genbank : nm_000676cds 3331331 ) at nucleotides 591610 , 856875 , and 909928 , respectively .
the oligonucleotides encoding the human adenosine a2b receptor shrna targeting the adenosine a2b receptor were as follows ( http://www.ambion.com/techlib/resources/rnai/index.html ) .  

shrna 1 
 5-gatcccgtgctggtgatctacattaattcaagagattaatgtagatcaccagcattttttggaaa-35-agcttttccaaaaaatgctggtgatctacattaatctcttgaattaatgtagatcaccagcacgg-3 

 
 5-gatcccgtgctggtgatctacattaattcaagagattaatgtagatcaccagcattttttggaaa-35-agcttttccaaaaaatgctggtgatctacattaatctcttgaattaatgtagatcaccagcacgg-3 
  5-gatcccgtgctggtgatctacattaattcaagagattaatgtagatcaccagcattttttggaaa-3 5-agcttttccaaaaaatgctggtgatctacattaatctcttgaattaatgtagatcaccagcacgg-3 
 shrna 2 
 5-gatcccgtcccattgtctatgcttacttcaagagagtaagcatagacaatggga ttttttggaaa-35-agcttttccaaaaaatcccattgtctatgcttactctcttgaagtaagcatagacaatgggacgg-3 

 
 5-gatcccgtcccattgtctatgcttacttcaagagagtaagcatagacaatggga ttttttggaaa-35-agcttttccaaaaaatcccattgtctatgcttactctcttgaagtaagcatagacaatgggacgg-3 
  5-gatcccgtcccattgtctatgcttacttcaagagagtaagcatagacaatggga ttttttggaaa-3 5-agcttttccaaaaaatcccattgtctatgcttactctcttgaagtaagcatagacaatgggacgg-3 
 shrna 3 
 5-gatcccgttatctccaggtatcttctttcaagagaagaagatacctggagataattttttggaaa-35-agcttttccaaaaaattatctccaggtatcttcttctcttgaaagaagatacctggagataacgg-3 

 
 5-gatcccgttatctccaggtatcttctttcaagagaagaagatacctggagataattttttggaaa-35-agcttttccaaaaaattatctccaggtatcttcttctcttgaaagaagatacctggagataacgg-3 
  5-gatcccgttatctccaggtatcttctttcaagagaagaagatacctggagataattttttggaaa-3 5-agcttttccaaaaaattatctccaggtatcttcttctcttgaaagaagatacctggagataacgg-3 all oligonucleotides were synthesized by sbs genetech , ltd .
the synthesized shrna cassette was annealed and cloned into the psilencer3.1-h1 neo vector according to the manufacturer 's instructions .
a blast search with the target sequences was performed to ensure that only the adenosine a2b receptor gene was targeted .
a scrambled control plasmid ( psilencer-3.1-y ) encoding a shrna contained a sequence not present in the mouse , human , or rat genome databases .
shrna 
 5-gatccagttcaacgaccagtagtcttcaagagagactactggtcgttgaactttttttggaaa-3 and   5-agcttttccaaaaaaagttcaacgaccagtagtctctcttgaagactactggtcgttgaactg-3. 

 
 5-gatccagttcaacgaccagtagtcttcaagagagactactggtcgttgaactttttttggaaa-3 and   5-agcttttccaaaaaaagttcaacgaccagtagtctctcttgaagactactggtcgttgaactg-3. 
  5-gatccagttcaacgaccagtagtcttcaagagagactactggtcgttgaactttttttggaaa-3 and    5-agcttttccaaaaaaagttcaacgaccagtagtctctcttgaagactactggtcgttgaactg-3. hepg2 cells were cultured in dulbecco 's modified eagle 's medium ( dmem , gibco ) , supplemented with 10% heat - inactivated fbs in a humidified incubator with 5% co2 at 37c .
cells were transfected with lipofectamine 2000 ( invitrogen ) according to the manufacturer 's instructions with 8  g psilencer 3.1-h1 neo - mix ( shrna1 , shrna2 , and shrna3 ) and psilencer-3.1-y .
one day after transfection , cells were screened in medium containing g418 ( 0.4  mg / ml ) and resistant clones were maintained in medium containing 0.4  mg / ml g418 .
stably transfected cells were plated in 60-mm dishes ( 1.5  10 per dish ) .
the treated cells were trypsinized and then centrifuged for 2 minutes at 12,000  rpm at 4c .
cell pellets were washed with pbs , collected , and lysed with trizol reagent ( gibco ) .
total rna was isolated by phenol / chloroform extraction , isopropanol precipitation , and 75% ethyl alcohol wash and dissolved in depc water .
the reverse transcription reaction was set up according to promega 's reverse transcription system protocol using primers for the a2b receptor ( forward primer : 5-tccatcttcagccttctggc-3 ; reverse primer : 5-aaaggcaaggacccagagga-3 ) and -actin ( forward primer : 5-gccctgaggcactcttcca-3 ; reverse primer : 5-gaaggtagtttcgtggatgcca-3 ) .
each pcr reaction was performed using an optimized number of cycles ( 25 cycles ) of 94c for 1 minute , 56c for 30 seconds , and 72c for 40 seconds , with a final extension of 72c for 7 minutes .
the pcr reactions were visualized on a 1.2% agarose gel containing 5  g / ml of ethidium bromide .
intensity of the dna bands was analyzed by glyko bandscan analysis software , using -actin as an internal standard .
cell monolayers in six - well culture plates were washed twice with ice - cold pbs and lysed with 150  l of ripa lysis buffer ( 0.05  m tris - hcl ( ph 7.4 ) , 0.15  m nacl , 0.25% deoxycholic acid , 1% np-40 , and 1  mm edta ) containing protease inhibitors ( 1  mm pmsf , 1  g / ml aprotinin , and 1  g / ml leupeptin ) .
protein concentration was measured ( bca protein assay , pierce ) , and samples were resolved by reducing sds - page , transferred to nitrocellulose , and incubated in blocking buffer ( 25  mm tris - hcl , ph 8.0 , 125  mm nacl , 0.05% tween 20 , and 5% bsa ) .
membranes were incubated with an antibody to the adenosine a2b receptor ( chemicon international , diluted 1  :  200 ) at 4c overnight .
the membranes were washed in blocking solution and incubated with an hrp - conjugated secondary antibody for 1 hour at room temperature .
blots were exposed to x - ray film for the appropriate time period . a total of 2  10 cells / well were suspended in complete medium containing 0.3% agarose ( gibco ) and seeded in triplicate in six - well plates onto a bottom layer of complete medium containing 0.6% agarose .
a total of 50,000 cells were used for fluorescence - activated cell sorter analysis ( facs ) .
briefly , the cells were harvested , washed twice with pbs buffer , and then incubated at room temperature for 45 minutes .
after two pbs washes , the cells were resuspended in pbs and filtered through spectra mesh filters ( spectrum ) .
data were analyzed by cellquest software ( becton - dickinson ) and the modfit / lt software . at total of 20,000 events
hepg2 cells were passed into 96-well plates ( 1,000 cells / well ) 24 hours after transfection .
viability and proliferation of cells were measured using mtt assays from the second until the seventh day after passage . for each group ,
experiments were carried out a total of three times , and the difference in average od ( 490  nm ) between treated groups was compared using the unpaired , two - tailed t - test .
results were expressed as mean  standard error ( se ) . values with p < 0.05 were considered significant .
four pairs of primers were cloned into psilencer3.1-h1 neo ( hind iii / bamh i ) vector to construct four interfering vectors : psilencer3.1-h1 neo-1 , psilencer3.1-h1 neo-2 , psilencer3.1-h1 neo-3 , and psilencer3.1-h1 neo - y .
plasmids were verified by sequencing ( figure 1 ) . to verify the silencing effect of the three sirna vectors , we transfected the three vector psilencer3.1-h1 neo-1 , 2 , 3 ( mixed ) and psilencer3.1-h1 neo - y ( as control ) into the hepg2 cell line .
rt- pcr showed that transfection of mixed vector could decrease the a2b receptor mrna about 66%  5% , while psilencer3.1-h1 neo - y had no effect ( figure 2 ) .  
western blot analysis of a2b receptor was comparable to the rt - pcr results ( figure 3 ) .
cells transiently transfected with psilencer3.1-h1 neo-1 , 2 , 3 decreased levels of adenosine a2b receptor ( about 70%  8% ) .
cells transfected with psilencer3.1-h1 neo - y had no effect on a2b receptor protein levels .
after seven days in culture , we compared the proliferation rate of the three groups of cells .
the cells transfected with psilencer3.1-neo - mix showed a significant decrease in the proliferation rate after five days in culture .
however , cells transfected with psilencer3.1-neo - y and untransfected cells had similar proliferation rates ( figure 4(a ) ) .   to determine anchorage - independent growth rates ,
control - transfected cells showed an average colony forming efficacy of 108  10.7% , compared to untransfected hepg2 cells (= 100% ) .
in contrast , stably transfected cells showed a decreased colony forming ability of 60.33  9.6% compared with hepg2 cells ( p < 0.05 ) ( figure 4 ( b ) ) . to explore the mechanism of growth suppression in a2br - silencing cells ,
the number of cells in go / g1 phase was 89.56 , 62.01 , and 56.19% for the psilencer3.1-neo - mix , psilencer3.1-neo - y , and untransfected cells , respectively ( figure 5 and table 1 ) .
liver cancer is the fifth most important cancer worldwide in terms of morbidity but the third in terms of mortality .
in addition , its mortality has increased in recent years , with 548,600 cases in the year 2000 .
little is known about the mechanisms of hepatocarcinogenesis which seem to differ according to the risk factors involved .
many genes are associated with liver cancer.the p53 protein is involved in cell cycle control , senescence , dna repair , genomic stability , and apoptosis , and different mutations of p53 are involved in hepatocellular carcinoma formation . the prb protein is phosphorylated during the g1 phase of the cell cycle by members of the cyclin - dependent kinase ( cdk )
the mutation of other genes , including -catenin , smad2 , smad4 , p16 ink4a , and cyclin d1 , may also be implicated in liver cancer .  
significant advances have been made in the understanding of the molecular pharmacology and physiological relevance of adenosine receptors , but little is known about a2b receptors .
a2b receptors have been implicated in mast cell activation   and asthma , vasodilation , regulation of cell growth , intestinal function , and modulation of neurosecretion . in a previous study , we found that a2b receptors were highly expressed in hepatocellular carcinoma tissue , indicating that expression of the a2b receptor may contribute to tumor progression and may be a good therapeutic target for liver cancer .
rna interference ( rnai ) is a natural silencing process first described in caenorhabditis elegans and drosophila melanogaster   by which double - stranded rna initiates and directs the degradation of homologous mrna .
specific inhibition of cellular mrna by rnai can be triggered in mammalian cells by the introduction of synthetic 21- to 23-nucleotide double - stranded small interfering rna ( sirna ) [ 26 , 27 ] or alternatively by the transcription of sirna from a dna construct driven by the rna polymerase cassette .
rnai technology has been widely used as an extremely powerful strategy for reverse functional genomics [ 29 , 30 ] and as an effective method for gene silencing - based therapeutics [ 31 , 32 ] .
rnai - mediated inhibition of virus infection or replication has been reported for numerous viruses , including several important human pathogens such as hiv-1 , hepatitis c virus , and dengue virus [ 3335 ] .
, we constructed three rnai vectors which target the a2b receptor gene to investigate if silencing can convert the tumor phenotype .
as shown by rt - pcr and western blot , transfected cells showed specific silencing of the a2b receptor gene without interrupting other molecular interactions . a stably transfected hepg2 cell line which silences the expression of a2b receptors
mtt and soft agar assays show that the proliferation rate of the stably transfected cells was significantly decreased compared with control - transfected or untransfected hepg2 cells .
fcm experiments revealed that 89.56  3.15% of stably transfected cells were in the g1 phase .
taken together , these results show that rnai silencing of the a2b receptor gene alters the phenotype of hepg2 cells , and this method might lead to development of novel therapies for the human hepatocellular carcinoma .
 to investigate the biological effect of adenosine a2b receptor ( a2br ) on the human hepatocellular carcinoma cell line hepg2 , three a2br sirna constructs were transiently transfected into hepg2 cells . 
 the results showed that a2br sirna reduced the levels of a2br mrna and protein . in order to further detect the function of a2br , we established a stable hepatocellular carcinoma cell line ( hepg2 ) expressing sirna targeting the adenosine a2b receptor . 
 targeted rnai significantly inhibited tumor cell growth in vitro , and flow cytometry ( fcm ) showed that significantly more cells expressing a2br sirna were in the g0/g1 phase compared to the untransfected group ( ( 89.56%  3.15% ) versus ( 56.19%  1.58% ) , p < 0.01 ) . 
 these results indicated that silencing the expression of adenosine a2b receptor in hepg2 cells can suppress cell growth effectively by blocking the cell cycle . 
 downregulation of adenosine a2b receptor gene expression with rna interference could be a new approach to hepatocellular carcinoma therapy . 