cancer is one of the leading causes of death worldwide and accounted for 7.6 million cases ( 13% of all deaths ) in 2008 ; in 2013 in the united states there are 12,340 estimated new cases and 4,030 deaths by cervical cancer [ american cancer society : cancer facts and figures 2013 ] .
meanwhile in romania , based on statistical data from 2012 , cervical cancer is ranked third place taking in account the women cancer incidence , after the breast and colorectal cancer .
cervical cancer develops in a multi - stage process , successively passing from a cervix with a normal epithelium to cervical intraepithelial neoplasia and finally leading to invasive cervical cancer . a relevant epidemiologic risk circumstance for the progress of the cervical cancer is represented by the presence of high risk human papillomavirus infections .
consequently , considerable importance has been given to the molecular premise which leads to the progression of cervical cancer .
rnai ( rna interference ) is a natural mechanism able to specific inhibit a target gene .
the prospective exploitation as a therapeutic has lately been strengthened by the small interfering rna ( sirna ) delivery to human tumours .
rnai has been demonstrated as a relevant investigation device for particular target genes by using exogenous sirnas .
sirnas are single - stranded non - coding rnas with 2023 nucleotides length , being able to negatively modulate gene expression profile at post - transcriptional level .
sirnas represent an encouraging novel type of medicaments for cancer therapy , as a result of targeting the mutated tumour suppressor genes or activated oncogenes .
many recent investigations have shown that sirna can efficiently inhibit oncogene expression in cancer cells .
various investigations demonstrated clearly that the deactivation of p53 constitutes a crucial step in carcinogenesis .
the present study was designed to investigate the potential use of p53 rnai to block p53 expression , as well as the subsequent effect on cell invasion and gene expression on the human cervical cancer cell .
hela is a human cervical carcinoma cell line ; this was incubated in a 5% co2 incubator at 37c . for maintenance
, cells were cultured in dmem medium , 10% fcs ( foetal bovine serum ) , 100 u / ml penicillin and 100 mg / ml streptomycin , 2 mm l - glutamine , 1% nonessential amino acids , all purchased from sigma - aldrich , bucharest , romania . for this investigations 510 hela cells
/ well were used for reverse - transfection , based on a preliminary optimised protocol for p53sirna , using siport neofx as transfection agent ( ambion , usa ) .
a validated p53sirna pool ( sc-29435 , santa cruz ) was used at a concentration of 50 nm in serum starvation condition using optimem medium ( gibco , invitrogen , romania ) , in accordance with the producer recommendations .
siport neofx 0.25% concentration was able to achieve the maximum inhibition of target gene with no effects on cell viability .
we performed the invasion assays on hela cells to evaluate the impact of p53sirna treatment using the cim - plate 16 with the xcelligence rtca dp instrument ( roche applied science ) according to the described protocol by .
the total rna for the cells treated with 50 nm p53sirna andthe control group respectively at 24 hours post treatment was extracted using trireagent ( sigma - aldrich , bucharest romania ) based on the producer recommended protocol for each triplicate specimen .
all of the samples had a rin ( rna integrity number ) higher than 7.5 .
the 8 genes selected were assessed using specific primer and upl based on in silico design . the total rna ( 500 ng ) from all the samples was reverse transcribed using the first strand cdna synthesis kit for rt - pcr ( roche , bucharest romania ) . for the gene amplification we used taqman universal pcr master mix , in a 20 l volume in a 96-well plate using the roche lightcycler 480 system .
the qrt - pcr reaction amplification program was as follows : 10 minutes at 95c for enzyme activation followed by 45 cycles of 15 seconds at 95c and 1 minute at 60c for the amplification step .
the data analysis was carried out to compare the gene expression values for the treated and untreated groups using ct method . as housekeeping gene
the evaluation of the vegf protein expression at 48 hours post treatment was done using human vegf quantikine elisa kit ( r&d , catalog no .
hela is a human cervical carcinoma cell line ; this was incubated in a 5% co2 incubator at 37c . for maintenance
, cells were cultured in dmem medium , 10% fcs ( foetal bovine serum ) , 100 u / ml penicillin and 100 mg / ml streptomycin , 2 mm l - glutamine , 1% nonessential amino acids , all purchased from sigma - aldrich , bucharest , romania . for this investigations 510 hela cells
/ well were used for reverse - transfection , based on a preliminary optimised protocol for p53sirna , using siport neofx as transfection agent ( ambion , usa ) .
a validated p53sirna pool ( sc-29435 , santa cruz ) was used at a concentration of 50 nm in serum starvation condition using optimem medium ( gibco , invitrogen , romania ) , in accordance with the producer recommendations .
siport neofx 0.25% concentration was able to achieve the maximum inhibition of target gene with no effects on cell viability .
we performed the invasion assays on hela cells to evaluate the impact of p53sirna treatment using the cim - plate 16 with the xcelligence rtca dp instrument ( roche applied science ) according to the described protocol by .
the total rna for the cells treated with 50 nm p53sirna andthe control group respectively at 24 hours post treatment was extracted using trireagent ( sigma - aldrich , bucharest romania ) based on the producer recommended protocol for each triplicate specimen . the rna concentrations and quality were assessed by the nanodrop-1100 agilent 2100 bioanalyzer spectrophotometer .
all of the samples had a rin ( rna integrity number ) higher than 7.5 .
the 8 genes selected were assessed using specific primer and upl based on in silico design .
the total rna ( 500 ng ) from all the samples was reverse transcribed using the first strand cdna synthesis kit for rt - pcr ( roche , bucharest romania ) .
for the gene amplification we used taqman universal pcr master mix , in a 20 l volume in a 96-well plate using the roche lightcycler 480 system .
the qrt - pcr reaction amplification program was as follows : 10 minutes at 95c for enzyme activation followed by 45 cycles of 15 seconds at 95c and 1 minute at 60c for the amplification step .
the data analysis was carried out to compare the gene expression values for the treated and untreated groups using ct method . as housekeeping gene
the evaluation of the vegf protein expression at 48 hours post treatment was done using human vegf quantikine elisa kit ( r&d , catalog no .
xcelligence system is an innovative device that allows the scanning of cellular response via an impedance - based technology in real time , lacking any exogenous labels .
the cim - plate 16 furnishes a kinetic cell - response profile to p53sirna throughout an investigation , specifying the commencement and ratio of invasion and migration of hela cells .
this data can facilitate to comprehend the response to treatment in dynamic . in figure 1
we can observe a delay and a reduction of the cell migration after the p53sirna treatment .
taqman qrt - pcr assay was used to examine the effect of p53sirna on a panel of 8 genes related to apoptosis and angiogenesis .
relative gene expression quantification using ct method leads to the downregulation of the selected gene , presented in the figure 2 .
after 48h post transfection with p53 sirna inhibition in hela cell line , vegf protein was found dowregulated in the culture medium than in the control group ( figure 3 ) .
xcelligence system is an innovative device that allows the scanning of cellular response via an impedance - based technology in real time , lacking any exogenous labels .
the cim - plate 16 furnishes a kinetic cell - response profile to p53sirna throughout an investigation , specifying the commencement and ratio of invasion and migration of hela cells .
this data can facilitate to comprehend the response to treatment in dynamic . in figure 1
we can observe a delay and a reduction of the cell migration after the p53sirna treatment .
taqman qrt - pcr assay was used to examine the effect of p53sirna on a panel of 8 genes related to apoptosis and angiogenesis . relative gene expression quantification using ct method leads to the downregulation of the selected gene , presented in the figure 2 .
after 48h post transfection with p53 sirna inhibition in hela cell line , vegf protein was found dowregulated in the culture medium than in the control group ( figure 3 ) .
although at this moment cervical cancer is considered as a preventable disorder there is a significant risk of disease recurrence causing a persuasive necessity to research new therapeutic targets for this disease management .
it is now well recognized that the tumour progression of all cancers is characterized by intensified proliferation and invasion rate and diminished in apoptosis . at the same time
the apoptosis and angiogenesis are interconnected as can be observed from figure 4 , using string.9 database .
the idea of this study is in agreement with the previous studies which are based on the hypothesis that once mutated , p53 exercised oncogenic role . by using sirna
the role of the present study is to emphasize the cooperation between oncogenic mechanisms , confirming the crosstalk between apoptotic and angiogenic mechanisms .
this has a significant therapeutic relevance based on the fact that mutated p53 is related to cancer aggressiveness   or to promoting metastasis . in a similar study
was observed that , by using sirna targeting p53/p73 , tumoral cells were sensitized to chemotherapy . in a recent study , pinx1 was displayed as a novel target gene of p53 , proposing the suppression of p53/pinx1 pathway as a novel mechanism to enhance the telomerase activity in cervical cancer cells , and leading to reduction of cell proliferation .
targeting oncogenic signals such as the mutated p53 gene that gains oncogenic role , we observed a downregulation of specific genes involved in apoptosis but also in angiogenesis , connected with a reduction of the invasion rate in the case of p53sirna therapy .
after 24 hours of treatment with p53sirna we can observe downregulations of important genes involved in apoptosis and angiogenesis , as displayed in figure 2 .
this study confirmed our hypothesis that p53 plays a central role in carcinogenesis as displayed in figure 3 , as important therapeutic target .
p53 could promote the convergence of the apoptotic and angiogenic pathways in cervical cancer cells .
it has been proved that p53 is able to indirectly downregulate vegf gene expression , via the retinoblastoma ( rb ) , in a p21-dependent mode , confirming its function in cell - cycle regulation as displayed by recent studies .
the complete comprehension of the cross - talk between p53 and angiogenic pathways represents a challenge in cancer therapy [ 2426 ] .
another study reveals that mutant p53 correlates with overexpression of vegf . in a similar study
the inhibition of mutant p53 represents a significant therapeutic target , a fact emphasized by walerych as  rebel angel .
p53 gene knockdown in hela cells plays an important role not only in apoptosis but also in angiogenesis through various mechanisms that regulate tumour cell development and migration .
the angiogenic phenotype related to p53 targeted by sirna in human cancers is frequently associated with resistance to conventional genotoxic anticancer agents .
therefore , p53 inhibitions become dependent and rely on angiogenesis for growth and metastasis . the results in the current research
may also have an important significance outside the context of cervical cancer , by using specific inhibitors for p53 for increasing therapeutic response in a wide range of tumoral pathology .
 background and aimssirnas represent an encouraging novel alternative in cancer therapy as a result of targeting the mutated tumour suppressor genes or activated oncogenes . 
 targeting oncogenic signals , as the mutated p53 gene that gains oncogenic role , we observed inhibition of migration , a downregulation of specific genes involved in apoptosis but also in angiogenesis , connected with a reduction in invasion rate in the case of p53sirna therapy.methodsthe study was designed to assess the role of p53 by using rnai ( rna interference ) in hela in vitro cell culture model . therefore cell migration rate was assessed by using xcelligence systems , gene expression for a panel of genes involved in apoptosis and angiogenesis , and validation of gene expression data at protein level.resultson the selected in vitro model p53 sirna therapy was correlated with the reduction of cell migration . 
 the downregulation of p53 , pten , tnf , nfkb , bcl-2 , icam-2 , vegf , and fgfb was evidenced as response to p53 inhibition.conclusionrnai may be a valuable technology in order to restore the normal cellular phenotype . 
 the results in the current research may also have an important significance outside the context of cervical cancer , by using specific inhibitors for p53 for increasing the therapeutic response in a wide range of tumoral pathology . 
Background and aims
Materials and methods
Cell culture and treatment
Dynamic monitoring of invasion of HeLa cells using the xCELLigence System
RNA extraction quality control and qRT-PCR
qRT-PCR data analysis
VEGF protein quantification
Results
Inhibition of HeLa cell migration after p53 gene knockdown
qRT-PCR results for main genes involved in apoptosis and angiogenesis
VEGF protein expression
Discussion
Conclusions