| Recently, the mortality of lung cancer is the first in human cancers. All of the world, the scientists have been trying kinds of methods to treat lung cancer, but the effect is not satisfactory. The total cure rate is less than 14%. With the development of molecular biology, people have known more about the molecular mechanisms of tumorigenesis, progression and metastasis. Gene therapy becomes a new prospective therapy. ASODN is a paragraph of artificial synthesis molecules which specifically bind to mRNA or DNA and suppress their expression. ASODN may specifically suppress the proliferation of tumor cells by blocking or suppressing the key coding gene in tumor cell.Antisense drug is a new type of safe effective drug which based on the antisensenucleic acids techniques and is on the purpose of treatment. Recent years,with the elucidation of antisense mechanism of the antisense oligonucleotides and its constructive ramifications like phosphorothioic acid oligodeoxynucleotide, many antisense drugs which target for the proteins of viruses , oncogene, cytoactive factors and other disease association proteins successively enter clinical trial, which make the research on the antisense drugs reenter rising development period.MMPs is a group of important ECM catabolic enzymes containing zinc ion. There have been more than 20 kinds of MMPs identified. MMP-9 is a important group within the MMPs and expressed in many tumor tissues.It has the key effects on the apoptosis, invasion and metastasis of tumor.Using human lung adenocarcinoma A549 line which has been transfected by MMP-9ASODN with liposome, this study is to detect the effect of MMP-9ASODN transfection on the A549 cell growth by MTT, observe the expression of MMP-9mRNA proteins by RT-PCR, detect the changes of MMP-9 proteins by Westem blot, detect the effect of MMP-9ASODN on the rate of cell proliferation/apoptosis;observe the effect of MMP-9ASODN on the adhesive and migratory ability of cell in vitro, observe the inhibition effect of MMP-9ASODN on the tumor in vivo, which supply a new method for the gene therapy for the lung cancer.The research on the targeted therapy drugs for the lung cancer have entered the exploitated phase. Resently, there have been many types of antisense drugs which have enter clinic trial period. This study demonstrated that antisense oligonucleotides MMP-9 may inhibit the tumor cell growth and induce apoptosis by transinfecting the A549 cell, which supply reliable experimental evidence for the antisense gene therapy for the lung cancer.Methods1. Liposome mediate the oligonucleotide transfeetion of A549 cell.Human lung adenocarcinoma A549 cell is conventional cultured in 1640 complete medium, and logarithmic-phase growth cells were inoculated to 96 well plates. The cells were divided into 4 groups: MMP-9ASODN, MMP-9SODN, liposome and control. And they were observed by fluorescence microscope after transfection 24hrs, select 5 high power fields randomly, count all cell population and green fluorescence cell population, and calculate transfection efficiency. 2. Detect the effect of MMP-9ASODN transfection on the A549 cell growth by MTTASODN transfection group is divided into different concentration groups: 200nmol/L, 400nmol/L, 600nmol/L and 800nmol/L. There are 3 holes in each group. Detect the effect 0h,4h,8h,12h,24h,48h,60h after transinfection by MMT, respectively.the wavelength detected by ELIASA is 570nm, calculate the survival rate ofA549 cell.3. Detect the cell proliferation/apoptosis rate by flow cytometryCollect the cells treated in every group into the centrifuge tube, centrifuge, wash, fix, stain, be laid on the flow cytometer, detect in the excitation wavelength of 488nm, and calculate the cell apoptosis rate4. Detect the expression of MMP-9mRNA by RT-PCR.Collect the cells in each group after transinfection, extract the RNA, synthetize the cDNA by reverse transcription with the template after quantitation, then amplification by PCR with primer, detect the products by agarose gel electrophoresis.5. Detect the changes of MMP-9 protein by Western blotCollect the cells after 24h transinfection and extract the total proteins within the cells, detect the protein content in the sample, then detect the proteins by discontinuation PGE, stain, trarsmembrane and observe the result.6. Dectet the effect of MMP-9ASODN transinfection on the adhesive and migratory ability of A549 cell in vitroCoat and hydrate the basal membrane, inoculate and culture cell, then detect by MMT chromatometry, calculate the adhesive rate of cell. Likewise, coat and hydrate the basal membrane, prepare culture cell monolayer, select 5 field of vision of 100×randomly in contrast phase microscope by artificial scratch method and calculate the total cellular score migrating into the scratch space. 7. Observe the inhibition rate of tumor by athymic mouse modelConstuct the transplantation tumor athymic mouse model, then inject the MMP-9ASODN into the transplantation tumor, observe the growth effect of anti-transplantation tumor including general state, the detection of growth inhibiting rate of regional solid tumor and pathohistology.Results1. The transinfection rate of MMP-9ASDN, MMP-9SODN and Lip group is respectively (65.85±9.61) %, (56.38±9.82) %, (53.62±6.08) %。2. Among certain range, the inhibiton of A549 cell survival rate by MMP-9ASODN is concentration and time dependent (inhibitory effect is most obvious when the antisense oligonucleotides concentration is 600nmol/L and the action time is 48h.)3. When the antisense oligonucleotides concentration is 600nmol/L and the action time is 48h, by RT-PCR amplification, the relative expression level of MMP-9mRNA is 0.573±0.071 which is obviously less than other 3 groups(0.924±0.044, 0.863±0.054, 0.765±0.072)4. When the antisense oligonucleotides concentration is 600nmol/L and the action time is 48h, by Westem detection, the expression level of MMP-9 protein is 0.53±0.068 which is obviously less than other 3 groups(0.76±0.052, 0.79±0.075, 0.81±0.126).5. After the A549 cell have been transinfected by MMP-9ASODN, the cell scores in G1 phase obviously increase, the cell scores in S phase obviously decrease, there is no obvious changes of cell scores in G2 phase., which suggested that MMP-9ASODN may delay the G1-S transition of tumor cell. after the A549 cell have been transinfected by MMP-9ASODN, the apoptosis percentage of A549 cell is much more than other 3 groups. 6. Adhesive experiment suggested that MMP-9ASODN transinfection may decrease the adhesive abilitiy of A549 cell, the adhesive rate is (15.21±0.81)% which is less than other 3 groups (38.92±1.23) %, (36.55±2.32) % and (32.61±1.41 ) %,the difference have significance(p＜0.01).Protraction experiment by scratch method7. Suggested that MMP-9ASODN transinfection may decrease the migratory ability of A549 cell, the migratory cell scores is 74.09±4.48 which is less than other 3 groups124.18±9.77, 120.14±8.50 and 112.82±9.87(p＜0.05).8. Tumor growth index in MMP-9ASODN group is 3.20±0.14 which is less than other 3 groups 5.93±0.20, 5.87±0.02 and 6.40±0.33 (P＜0.01).The tumor weight in MMP-9ASODN group is 1.25±0.03 which is less than other 3 groups 2.15±0.07, 2.31±0.04 and 2.43±0.04 (P＜0.01).The inhibition rate of tumor in MMP-9ASODN group is (33.41±1.18)% ,and the heart , liver and kidney's tissue and construct are normal on the whole, there are no obvious tissue damage changes.Conclusions1. MMP-9ASODN may effectively inhibit the lung cancer A549 cell proliferation,the mode of action is concentration and time dependent.2. MMP-9ASODN may down regulate the expression of MMP-9mRNA and MMP-9 proteins33. MMP-9ASODN may inhibit the A549 cell proliferation by influencing lung cancer A549 cell generation cycle distribution and promoting the A549 cell apoptosis.4. MMP-9ASODN may depress the adhesive and migratory ability of A549 cell.5. MMP-9ASODN may obviously inhibit athymic mouse transplantation tumor growth, which is save and no adverse effect.
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