Expression Of HIF-1α In Hepatocellular Carcinoma And Inhibitory Effect Of HIF-1α AS-ODN On Proliferation Of HepG2 Cells

Part 1 Expression and significance of HIF-1α, VEGF and MVD in hepatocellular carcinomaIntroductionHepatocellular carcinoma(HCC)is one of the most common malignancies in China.Owing to the improvement of surgical technique and early diagnostic methods,the resection rate of HCC has increased. However,the postoperative relapse and metastatic rate remains high. Neovascularization may play important roles in the relapse and metastasis of HCC.Hypoxia-inducible factor 1(HIF-1)is a heterodimeric transcriptional factor composed of theαandβsubunits.HIF-1αis the O₂-regulated subunit that determines HIF-1 activity.A series of genes and proteins that may increase the survival of tumor cells under hypoxia conditions,including vascular endothelial growth factor(VEGF),are regulated by HIF-1α.Thus,HIF-1αmay play important roles in tumor progression and neovascularization.The objective of this clinical study is to investigate the expression of HIF-1α,VEGF and microvessel density (MVD)in HCC and to explore their correlation with clinical pathological features.Materials and MethodsPatientsForty-five patients with HCC who underwent hepatic resection between Dec 2005 and Dec 2006 are included in this study.Paraneoplastic liver tissue is taken from non-cancerous tissue 1cm away from the tumor margin. Ten samples of normal liver tissue were also included.All the sections are re-evaluated and the diagnosis are confirmed.ImmunohistochemistryFormalin-fixed,paraffin-embedded tissue specimens are obtained. Serial 4μmol/L sections are prepared,and one is stained with H&E. HIF-1α,VEGF and CD34 were detected by PV9000 immunohistochemistry according to the manufacturer's instruction.Expression of HIF-1αwas determined by assessing semiquantitatively the percentage of stained tumor cells and the staining intensity.The percentage of positive cells was rated as follows:2 points,11-50% positive cells;3 points,51-80%positive cells;4 points,＞81% positive cells.The staining intensity was rated as follows:1 points, weak intensity;2 points,moderate intensity;3 points,strong intensity; points for expression and percentage of positive cells were added.(-), regardless of intensity,≤10%of cells stained positive;(+),3 points; (++),4-5 points;(+++),6-7 points.The results for VEGF are classified as follows:(-),≤10%positive cells;(+),＞10%positive cells. Microvessel density,assessed by immunostaining for CD34,is determined by counting all vessels at a total magnification of×200.Statistical analysisAll statistics were calculated through SPSS 13.0 software. Chi-square test,t test,One-way ANOVA and Spearman coefficient of correlation are used as appropriate.P＜0.05 was considered statistically significant.ResultsExpression of HIF-1α,VEGF and MVD in liver tissueThe expression of HIF-1αin HCC was significantly higher than that in paraneoplastic tissue and normal liver tissue.The positive rate and strong positive rate of HIF-1αin HCC were 91.1%and 68.9%respectively, whereas the positive rate and strong positive rate of HIF-1αin paraneoplastic tissue are 20.0%and 6.7%respectively,and only 1 mild positive staining was observed in normal liver tissue.Compared with paraneoplastic tissue and normal liver tissue,the expression of VEGF in HCC is significantly higher.The positive rate of VEGF in HCC and paraneoplastic tissue is 53.3%and 13.3%respectively, whereas no positive staining observed in normal liver tissue.Compared with paraneoplastic tissue and normal liver tissue,the MVD value in HCC is significantly higher.The MVD value in HCC, paraneoplastic tissue and normal liver tissue is 52.2±18.3,23.2±9.8 and 16.5±9.7 respectively. Association of HIF-1α,VEGF and MVD in HCC with clinicopathological dataSignificant association is found between HIF-1αand HCC differentiation degree,complication with portal venous tumor thrombus (PVTT)and tumor size.The strong positive rate of HIF-1αin poor differentiated,moderate differentiated and well differentiated HCC is 100.0%,62.5%and 16.7%,in HCC with PVTT and without PVTT is 100.0 %and 60.0%,and in tumor size more than 5cm and smaller than 5cm is 86.3%and 52.2%respectively.The expression of HIF-1αin HCC with extrahepatic metastasis showed increase tendency,but had no statistical significance.The expression of VEGF in HCC with PVTT are significantly higher than those in HCC without PVTT,the positive rate is 90.0%and 42.9% respectively.The expression of VEGF in HCC with extrahepatic metastasis, poor differentiation and enhancing tumor size also showed increase tendency,but had no statistical significance.The MVD value in poor differentiated HCC(61.0±17.2)was significant higher than that in well differentiated HCC(40.5±14.8).Compared with the MVD value in HCC without PVTT(46.8±16.0)and HCC without extrahepatic metastasis(49.7±18.5),those in HCC with PVTT(71.3±12.7) and in HCC with extrahepatic metastasis(64.1±12.2)were significant increased.The MVDvalue showed increase tendency with enhancing tumor size,but had no statistical significance.Association of HIF-1α,VEGF and MVD in paraneoplastic tissue with clinicopathological dataIn paraneoplastic tissue,significant difference of HIF-1α expression is found between HCC complicated with PVTT and without PVTT, the positive rate is 50.0%and 11.4%respectively.The expression of HIF-1αin paraneoplastic tissue complicated with extrahepatic metastasis,liver cirrhosis or decreased differentiation degree showed increase tendency,but had no statistical significance.The expression of YEGFin paraneoplastic tissue complicated with PVTT, extrahepatic metastasis,liver cirrhosis or decreased differentiation degree also showed increase tendency,but had no statistical significance.The MVDvalue in paraneoplastic tissue with poor differentiated HCC (28.5±10.6)was significant higher than those with well differentiated HCC(16.6±7.1)and moderate differentiated HCC(21.6±8.4).Compared with the MVD value in paraneoplastic tissue complicated without PVTT(20.2±8.6),without extrahepatic metastasis(21.4±8.7)and without liver cirrhosis(20.1±7.6),those with PVTT(33.7±5.6), extrahepatic metastasis(31.5±9.5)and liver cirrhosis(26.2±10.8) were significant increased.Correlation of HIF-1αexpression with VEGF expression andSpearman correlation analysis showed the expression of HIF-1αin HCC was correlated with the expression of VEGF(r=0.545),and the expression of HIF-1αand VEGF in HCC as correlated with the value of MVD(r=0.705 and 0.710,respectively).The MVD value in HCC with HIF-1αexpression determined as(+),(++)and(+++)were 36.5±7.9,53.8±14.3 and 75.3±9.1 respectively.MVD value in HCC with VEGF expression determined as(-)and(+)were 38.7±10.9 and 64.1±15.9 respectively. In paraneoplastic tissue,the expression of HIF-1αwas correlated with the expression of VEGF(r=0.784),and the expression of HIF-1αand VEGF in paraneoplastic tissue as correlated with the value of MVD(r=0.608 and 0.554,respectively).MVD value in paraneoplastic tissue with HIF-1αexpression determined as negative and positive was 20.0±7.6 and 36.0±6.2 respectively.MVD value in paraneoplastic tissue with VEGF expression determined as(-)and(+)was 20.9±7.9 and 38.5±6.0 respectively.Conclusions1.The expression of HIF-1αand VEGF and the value of MVD in HCC was significantly higher than those in paraneoplastic tissue and normal liver tissue,suggest HIF-1αand VEGF may play important roles in HCC progression and neovascularization.2.Significant association was found between the expression of HIF-1αand HCC differentiation degree,complication with PVTT and tumor size; and was found between the expression of VEGF and HCC complication with PVTT.Significant association was also found between the value of MVD and HCC differentiation degree,complication with PVTT and extrahepatic metastasis.3.In HCC and paraneoplastic tissue,the expression of HIF-1αand VEGF had closely correlation with the value of MVD. Part 2 Inhibitory effect of HIF-1αAS-ODN on proliferation of HepG2 cellsIntroductionHepatocellular carcinoma(HCC)is one of the most common malignancies worldwide,especially in Asia and Afria.Patients with HCC usually present very poor prognosis inspire of improvement of diagnostic modalities and treatment in the recent years.The hypoxia-inducible factor-1(HIF-1),an oxygen-sensitive transcriptional activator,is a key regulator responsible for the induction of genes that facilitate adaptation and survival of cells and the whole organism from normoxia to hypoxia.And the role of HIF-1αin tumor angiogenesis and cellular adaptation to the hypoxic microenvironment has been well established.Antisense oligodeoxynucleotide(AS-ODN)is a single-strand DNA that is complementary to specific regions of mRNA and capable of inhibiting expression of that gene.AS-ODNs are widely used for the elucidation of gene and protein function and as therapeutic agents in clinical trials.In the present study,we designed 6 AS-ODNs targeted against the HIF-1αgene to identify the most potent AS-ODN sequence that specifically inhibit HepG2 cells proliferation.And then the most potent AS-ODN sequence of HIF-1αwith various concentrations were employed respectively to inhibit the HIF-1αmRNA and protein in the HepG2 cells, and their inhibitory effect was examined. Materials and MethodsPreparation of HIF-1αAS-ODNAS-ODNs to the human sequence of HIF-1αmRNA were synthesized by using an automated DNA synthesizer.After deprotection,AS-ODNs were purified by HPLC.Oligodeoxynucleotide TransfectionHepG2 cells at exponential phases of growth were inoculated into 96-well plates(4x10³/100μl)in the presence of 10%fetal bovin serum(FBS). When cells grown to 40-60%confluence,the AS-ODNs were transfected into HepG2 cells with lipofectin reagent(Invitrogen,USA)according to the manufacturer's instructions.AS-ODNs with various concentrations, Liposomeand cell controls weredesigned,each group had fourwells.After transfectedfor 6h,the culture fluid were discarded,each well was added with 100μl DMEM supplement with 10%FBS,and incubated at hypoxic culture condition in humidified 5%CO₂ and air mixture at 37℃.MTS analysisThe cells were harvested at 72h after transfection,each well was added with 20μl MTS(5mg/ml)and incubated in humidified 5%CO₂ and air mixture at 37℃for another 90min.The optical density(OD)values of the slides were read on enzyme-labeled reader at the wavelength of 492 nm. The proliferation inhibition rate were calculated. RT-PCR analysis48h after transfection,total RNA was isolated from cells of each group by Trizol Reagent(GIBCO).Reverse transcription was carried out on 2μg of total RNA,20μl reaction system contained 0.5μl /mL oligodT 1μl,5×RT buffer 4μl,10mM dNTP 1μl,RNase 0.5μl,25mM MgCl₂ 4μl,AMV 1.5μl,8μl ddH₂O,incubated at 42℃for 1h,heated to 72℃for 5 min to inactivate AMV reverse transcriptase.PCR reaction system contained 1μL RT products,15μl PCR mixture,0.5μl 20μmol/L forward primer and 0.5μl 20μmol/L reverse primer(forward primer:5'- CATTACCCA CCGCTGAAACGCC-3', reverse peimer:5'-CTGGGACTATTAGGCTCAGGTG-3'),0.5μl Taq DNA polymerase and 1.5μl ddH₂O.PCR amplification was conducted in following condition: pre-denaturation at 94℃for 4 min,denaturation at 94℃for 30 second, annealing at 56℃for 30 second,and extension at 72℃for 30s.After 30 amplification cycles the products were extended at 72℃for 5 min. The RT-PCR products were visualized byβ-actin and analyzed by 2%agarose gel electrophroesis.Western Blot Analysis48h after transfection,total protein was isolated and quantified by Lowry methods.Proteins(6Oμg/10μl)were boiled at 100℃in equal volume 2×SDS loading buffer for 5 min.Each sample was electrophoresed on a 8%of SDS-polyacriylamide gel,and the fractionated proteins were then transferred to PVDF membranes(Amersham Bioscience)by semi-dry transfer apparatus.The membranes were blocked with 1%skim milk in TBST for 1h at room temperature,and then incubated with primary antibodies against HIF-1αandβ-actin(1:400 dilution)for 1h at room temperature. After washing with TBST three times,the membranes were incubated for 1h with secondary antibody against HIF-1a andβ-actin(1:2000 dilution) and finally visualized using a chemiluminescence detection kit ECL.Statistical analysisSignificance of statistical difference was analyzed by using ANOVA test,a value of P＜0.05 was considered as significant.All statistics are calculated through SPSS 13.0 software.ResultsInhibition of cell proliferation by HIF-1αAS-ODN_1-6HepG2 cell growth was inhibited by 6 HIF-1αAS-ODNs at various concentrations significantly.At the concentration of 0.8μmol/L,the proliferation inhibitory rate were 57.2±2.4%,61.8±1.9%,57.4±2.6%, 49.1±3.4%,53.3±3.1%and 53.7±2.7%respectively.Among them,HIF-1αAS-ODN₂ showed the most effective proliferation inhibition ability(P＜0.01),the proliferation inhibitory rate went up to 37.8±2.0%,49.2±1.7%,61.8±1.9%at the concentration of 0.2μmol/L,0.4μmol/L and 0.8μmol/L respectively.Inhibition of cell proliferation by HIF-1αAS-ODN₂Then the HIF-1αAS-ODN₂ with various concentrations(0.2umol/L, 0.4umol/L,0.8umol/L,1.0umol/L)were transfected into HepG2 cells.The inhibition effect of HIF-1αAS-ODN₂ in cell proliferation showed increase tendency with increased concentration.At concentration of 1.0μmol/L,the proliferation inhibitory rate was 89.7±3.8%.Compared with the liposome control group,there was no inhibitory effect of S-ODN in HepG2 cell proliferation(P＞0.01). Inhibitory effect of AS-ODN₂ on HIF-1αmRNA expressionCompared with the cell control and the S-ODN(0.4umol/L)control, HIF-1αmRNA expression were down-regulated by AS-ODN₂ 48h after transfection at both concentration of 0.4μmol/L and 0.8μmol/L.And there was no significant difference between 0.4μmol/L and 0.8μmol/L concentration on the inhibitory effect of HIP-1αAS-ODN₂.Inhibitory effect of AS-ODN₂ on HIF-1αprotein expressionCompared with the cell control and the S-ODN(0.4umol/L)control, HIF-1αprotein synthesis were decreased by AS-ODN₂ 48h after transfection at both concentration of 0.4μmol/L and 0.8μmol/L.And the inhibitory effect of AS-ODN₂ at concentration of 0.8μmol/L was higher than 0.4μmol/L.Conclusions1.The HepG2 cells proliferation were significantly inhibited by HIF-1αAS-ODN_1-6at different degree.At the concentration of 0.8μmol/L,the proliferation inhibitory rate were 57.2±2.4%,61.8±1.9%,57.4±2.6%, 49.1±3.4%,53.3±3.1%and 53.7±2.7%respectively.2.Among them,HIF-1αAS-ODN₂ showed the most effective proliferation inhibition ability.At the concentration of 1.0μmol/L,the proliferation inhibitory rate was up to 89.7±3.8%.3.The expression of HIF-1αmRNA and synthesis of protein were reduced by HIF-1αAS-ODN₂.suggested that the mechanism of inhibition action on HepG2 cells proliferation is through the inhibition of transcription and translation of HIF-1αmRNA.