The Expression Of Wnt-1 And Nm23-H₁ Gene In Adenocarcinoma And The Effect Of SiRNA Transfection In Human Lung Adenocarcinoma Cell Line A2

Compared with lung squamous cell carcinoma,the ability of metastasis of adenocarcinoma was more stronger and the prognosis was more bader,even in the early stage,the sequence of lymph or blood metastasis was more frequent.So,to study the difference of gene expression in adenocarcinoma would be helpful to understand the mechanisms of promotion or inhibition metastasis,and it was possible to find out some way that could inhibit metastasis of adenocarcinoma and increase the total survival rate of lung cancer.During the embry development,embryogenes joined in the regulation of proliferation,differenciation and apoptosis.Constantly,expression and silence were acted in a programmed way.When this balance was broken,it would lead to some diceases including carcinoma.Yet,the most basic face of cancer cells was lost of regulation of proliferation and differenciation,the cells grew limitedless and survived forever.Most recently,some studies showed that a few of embryogenes had taken part in the process of carcinoma development,and some oncogenes or cancer surppressor genes themselves were embryogenes.So,some people thinked that the genes which were linked with metastasis,proliferation and apoptosis were the same one during the embry development.Both oncogene Wnt-1 and cancer surppressor genes nm23-H₁ were.belong to embryogene family,and their produces all played a key role in cells proliferation,differenciation and apoptosis.Recently,a study showed that nm23-H₁ could inhibit metastasis by broken Wnt/β-catenin,but Wnt-1 could influence the expression of nm23-H₁.At present,studies about materstasis and prognosis of the two genes were a lots,but the outcome was not the same one and had no report which tested the two genes at the same time.For these resones,wo explored the method through which RAN form paraffin embedded tissues were extracted and detected by RT-PCR.At the same time,immunohistochemical method was taken to test the relationship between the expression of the two genes and to explore the relationship with clinical factors.We also evaluated the effect of siRNA on Wnt-1 gene expression in human lung adenocarcinoma cell line A2,so as to asses the role of Wnt-1 in A2 cells'proliferation and apoptosis..Materials and methods1.Materials48 samples during Membray 1998 to September 2000 were collected in the department of thoracic surgery,First Affiliated Hospital China Medical University. There were 28 male,20 female patients with an average age of 57.7(from 35 to 76)years.As to the positive and the negative lymph node metastasis,the cases were 25 and 23,respectively.According to 1997 UICC staging criteria,17 cases were classified as stageâ… ,13 casesâ…¡,18 casesâ…¢.No cases were received rediotheropy or chemiotheropy before operation.2.Methods(1)Immunohistochemisty for Wnt-land nm23-H₁Immunohistochemical tests were performed by SP method.The intensity of primary antibodies was 1;50.Both of the positive stain of Wnt-1 and nm23-H₁ were defined as brown to yellow staining in cytoplasm.The stain of Wnt-1 was classified into 4 degrees according to the number and color intensity of the cells.Negative 0,faint yellow l light yellow 2,brown yellow3,intense brown 4.Those which color intensity was above or equoe to 3 or color intensity was 2,but positive cells≥10%,were defined as positive.The positive stain of nm23-H₁ was defined as positive cells≥31%.The negative control was prepared in the PBS.(2)RT-PCR for Wnt-1 and nm23-Hl in adenocarcinomaTotal RAN form paraffin embedded sample(contain 100mg tissue)were isolated by the way of using Trizol one-stage method,then the concentration and purity of RNA PCR Buffer 1μl,dNTP 1μl,MgCl₂ 2μl,Random 9mers 0.5μl,RNA 1μl,RNAse inhibitor 0.25μl,Reverse Transcriptase 0.5μl,ddH₂O 3.75μl。Followed by the condition;10min at 42℃30min,99℃5min,5℃5min.The gene-specific primers;Wnt-1 (a;5'-TACCTCCAGTCACACTCCCC-3';b;5'-CCATGGCAGGAGAATAGGAA-3', 348bp),13-actin 318bp,nm23-H₁(a;5'-GCAGCCGGAGTTCAAACCTA-3';b; 5'-CTGGGAGGAAGCATTTTATC-3',273bp),β-actin 512bp。PCR amplification was performed on a PCR thermal cycler.2.5μof cDNA mixture was subject to amplification in 12.5μl solution containing the following;ddH₂O 7.16μl, 20mmol/L each of 5' and 3' primers 0.15μl,Taq Hs 0.04μl and 5×Buffer 2.5μl,PCR conditions were initial elenaturation for 2min at 94℃,followed by 30 cycles of denaturation of 94℃for 40S and annedaled at 57℃for 40S,then extended at 72℃for 10min.(3)Wnt- 1 siRNA transfectionThe cells(1×10⁶)were cultured in 12 plastic flasks,with RPMI1640 medium,without 10%fetal calf serum,at 37℃in a humidified atmosphere(5%CO₂ and 95%air)for 24 hours.Then,0.8μg siRNA,98μL RPMI 1640 and 21μL Lipofectamine were added in test group(Wnt-1 group),respectively.After transfection,followed by the condition;5%CO₂ and 95%air for 4 hours,new RPMI1640 with 10%fetal calf serum was added in and cultured continuely.(4)Western-blot analysisEach samples contained 1×10⁶cells.The protein concentration was measured according to the Lowry method;polyacrylamide gel eletrophoresis;After 2h of transferring to PVDF membrane,the blot was probed with Wnt- 1 antibody(1;400) and murine monoclonal anti-β-actin antibody(1;400),incubated 2h in room temperature.The secondary antibody was goat anti-rabbit and horse anti- murine monoclonal antibody,challenged with the appropriate second antibody conjugated with alkaline phosphatase for 2h at room temperature,stained with alkaline phosphatase 15min.The positive blot were scanned.(5)MTT methodAfter transfection,the liquid containing single cell of each test groups was regulated by PBS into 2×10⁵/mL cell/ml.The cells were cultured in 96 plastic flasks respectively,with RPMI 1640 medium containing 10%fetal calf serum,2g/L NaHCO₂ at 37℃in a humidified atmosphere(5%CO₂ and 95%air)for 24 hours.At the time of 24h,48h,72h,new RPMI1640(80μL)and MTT(20μL,Smg/mL)were added in,respectively.Cultured 4h and then added in DMSO(100μL),the each groups were tested(OD,570nm).(6)Flow cytometry to detect apoptosisAfter transfection 48h,the liquid containing single cell of each test groups was regulated by PBS into 1×10⁶cell/ml.PI liquid(PI 5mg.Rnase 2mg.TritonX-100 1 ml, normal saline 65ml,sodium citrate 100mg,add distilled water to 100ml)lml was added to one,30min at 4℃in dark.Stained cells were analyzed by means of a FACSCalibu(B.D,USA).10000 cells were counted in every specimen.Before detection,the CV was regulated under 3.0%. PI could reflect activity of apoptosis.The early apoptosis cells were counted on two-dimensional lattice picture.(7)Statistical analysisThe data were analyzed by the SPSS 11.5 software.Chi-squared test and Fisher test were used in datas of quantity.One-way ANOVA test was used in data of quality.Cox regression was used to analyse prognosis. Results1.The quality of total RNA extracted from paraffin embedded tissues was favorable.2.The expression of Wnt-1 mRNA and its protein were shown in 39.6%and 43.7%of all cases respectively.The expression rate of Wnt-1 mRNA or its protein in the group of which patients survived more than 3 years(29.0%;32.3%)was lower when compared with the less than 3 years group(58.8%;64.7%),P＜0.05.3.The expression rate of nm23-Hl mRNA or its protein was 62.5%and 68.8% respectively.The expression rate of nm23-H₁ mRNA or its protein in the positive lymph node metastasis group(48.0%;52.0%)was lower than the negative group(78.3%;87.0%),P＜0.05.The expression rate of nm23-H₁ mRNA or its protein in the group of which patients survived more than 3 years(74.2%;83.9%)was higher than those survived less than 3 years(41.2%;41.2%),P＜0.05.4.In the group of Wnt-lmRNA expression,the expression rate of nm23-H₁ mRNA was 52.6%;as to the Wnt-1 protein,the rates was 61.9%.The expression of Wnt-1 gene was not correlated to the expression of nm23-H₁ gene,P＞0.05.5.Multivariable Cox regression analysis revealed that the expression of nm23-H₁ mRNA and TNM were two important and independent prognostic factors for adenocarcinoma;the hazard ratios of nm23-H₁ mRNA expression and TNM were 0.313 and 2.015,respectively.6.Compared with control group,both the expression of mRNA and protein of Wnt- 1 in A2 cells were obviously decreased after siRNA transfected cells,respectively.7.The cell growth and viability of siRNA transfected group(Wnt-1 group)were significantly inhibited(P＜0.05)and the apoptosis of A2 cells increased,too.Conclusion1.The expression of Wnt-1 gene had negative correlation with patient's survival time.2.The expression of nm23-H₁ gene had negative correlation with lymph node metastasis and had positive correlation with the patient's survival time. 3.The expression of Wnt-1 gene was not correlated to the expression of nm23-H₁ gene.4.The expression of nm23-H₁ mRNA and TNM served as independent prognostic indices of adenocarcinoma.Detection of nm23-H₁ mRNA would be helpful in screening the cases with high risk of matastasis,based on TNM staging.5.Inhibitted expression of Wnt-1 gene could promot the apoptosis and inhibit the proliferation of the A2 cells.