Effect Of5-Aza-CdR On The Proliferation And Invasion Of Lung Adenocarcinoma A549Cells And Expression Of TFPI-2Gene

BackgroundTissue factor pathway inhibitor-2(TFPI-2) was a serine protease inhibitor, and played important roles in maintaining structural integrity of extracellular matrix and suppression of invasion and metastasis of the tumor cells. It inhibited growth and angiogenesis of tumor, and induced apoptosis of the tumor cells. So, TFPI-2was considered as a potential tumor suppressor gene. With the advent of post-genomic era, epigenetics draws more and more attention. The reseach of DNA methylation had opened up a new area of tumorous research of epigenetic. The abnormal methylation of the promoter region of tumor suppressor gene was generally considered as the reason for deactivation of tumor suppressor genes. In recent years, it was found that methylation of the CpG island of TFPI-2gene in common malignant tumors such as breast cancer, nasopharyngeal carcinoma, pancreatic cancer, esophagus cancer, which caused to the expression of TFPI-2gene was down or lost that was associated with growth, invasion and metastasis of the tumor. Rollin and colleague found that deactivation of the methylated TFPI-2gene obviously increased invasive ability of non-small cell lung cancer (NSCLC), a further study found that it was significant to study relationship between the methylation status of TFPI-2gene and the occurrence, development and poor prognosis of lung cancer. Wu and colleague found that methylation degree of TFPI-2gene in patients with NSCLC was higher, the prognosis was worse. Our initial study found that the aberrant methylation of TFPI-2gene was found in NSCLC, which might be closely associated with the clinical stage, tumor size, lymph node metastasis. Methylation of TFPI-2gene in NSCLC was closely associated with growth, infiltration and metastasis of tumor.5-aza-2’-deoxycytidine (5-Aza-CdR) was a kind of DNA methyltransferase inhibitors which resulted in loss of DNA methylation with cells division, that inhibited the activity of the DNA methyltransferase by covalent bond, reversed the methylation of tumor cells, reactivated the tumor suppressor gene, or induced or improved the expression of tumor suppressor gene. Nowadays, the researchs of5-Aza-CdR in the majority of tumors were still at the experimental research stage, clinical application of5-Aza-CdR mainly applied in second-line chemotherapy for hematological malignancy such as myelodysplastic syndrome, which obtained a certain effect.ObjectiveThe present study employed5-Aza-CdR to treat lung adenocarcinoma A549cells to observe whether5-Aza-CdR recover expression of TFPI-2gene, and inhibit the proliferation and invasive ability of the lung adenocarcinoma A549cells by detection of the proliferation and invasive ability of A549cells and detection of methylation status and expression of TFPI-2gene, and to observe whether the inhibitory effect is closely related with drug concentrations.Materials and Methods1. Cell culture and experimental groupThe lung adenocarcinoma A549cells were grown in RPMI1640medium containing 10%fetal bovine serum,100U/ml penicillin and100μg/ml streptomycin, cultivated at37℃in a humidified incubator with5%CO2. At the logarithmic phase of the cells growth, the cells which were trypsinized with0.25%trypsin were passaged. Those were divided into four groups according to different concentrations of5-Aza-CdR: Control group:OμM group, joined into the same amount of common culture liquid. Experimental group:①1μM group, A549cells were treaed with1×10-6mol/1of5-Aza-CdR.②5μM group, A549cells were treaed with5×10-6mol/1of5-Aza-CdR group.③10μM group, A549cells were treaed with10≤10-6mol/l of5-Aza-CdR group.2. Detection of cell proliferation activity of A549cellsAt the logarithmic phase of the cells growth, A549cells were trypsinized and seeded in96-well plates at a density of1×104cells/well and cultured for24h. And then,0,1,5,10μmol/l5-Aza-CdR were added and the plates were incubated at37℃,5%CO2for24,48,72h. At the end of incubation,20μL of10mg/ml MTT was added into each well followed by incubation for another4h. The medium was then aspirated and150μL dimethylsulfoxide(DMSO) was added into each well. The plates were mixed gently by rocking back and forth until the blue sedimentation was completely dissolved. optical density (OD) was measured at490nm using an ELI AS A and the rate of growth inhibition was calculated using the following formula:Inhibition rate(%)=(Average OD value of experimental group-Average OD value of control group)/Average OD value of control group×100%. Each experiment was repeated three times.3. Detection of invasive ability of A549cellsThere were three membrane filter in each group, the lower transwell chambers were filled with RPMI-1640medium containing10%fetal bovine serum, the upper transwell chambers were filled with different concentrations of5-Aza-CdR. The filters were removed and RPMI-1640medium was washed away with phosphate buffer solution(PBS) after transwell chambers were placed in the incubator for24h, membranes were stained with crystal violet for10min; Cells on the upper side of the membrane filter were removed gently with a cotton swab; The cells on the lower side of the membrane filter were counted under a inverted microscope (200X)and take pictures, the cell invasive ability was used by cells count that penetrate membrane. Each test group was assayed in triplicate.4. Detection of the A549cell cycles distributionThe distribution of cell cycle was analyzed using propidium iodide staining. Cells were seeded at1×106cells/well in6-well plates, and exposed to0,1,5,10×10-6μmol/L5-Aza-CdR for72h. The cells were harvested and washed twice with PBS, then fixed with ice-cold70%ethanol. The sample was concentrated by removing ethanol and re-suspended in a PBS solution containing propidium iodide (0.05g/L) and RNaseA(100mg/ml) for3h at37℃in the dark. The samples were then measured by flow cytometry. The of activity cell division was measured by proliferation index(PI), PI=(S+G2/M)/(G0/G1+S+G2/M)。Each sample was repeated three times.5. Detection of methylation status of TFPI-2gene of A549cellsGenomic DNA was obtained from the cultured cells using the standard phenol-chloroform protocols. The DNAs were treated with sodium bisulfite. MSPCR primers were designed using Primer Premier5.0software. Methylation upstream primer:5’-TTTATGTTTTTAAGAGGTGGATTTC-3’, methylation downstream primer:5’-AACTTTCTCCTATAATCCAAACGAA-3’, unmethylation upstream primer for:5’-TTATGTTTTTAAGAGGTGGATTTTG-3’, unmethylation downstream primers for:5’-CAAACTTTCTCCTATAATCCAAACAA-31. Total reaction mixture volume of25μL, containing2μL DNA,2.5μL10×PCR buffer,0.5μL each of the sense and antisense primers,2μL dNTPs,0.2μL Taq enzyme, and17.3uL double-distilled water. Reaction condition:pre-denatured at94℃for4min, followed by35cycles of94℃for30s,56℃for30s, and72℃for30s, and with a final extension at72℃for5min. The methyltransferase SssI-treated and untreated placenta DNA were used as the positive and negative controls respectively, while the double-distilled water as the blank control. The1000bp DL2000was used as the molecular weight markers. The PCR products were separated by electrophoresis on the2%agarosegel at4V for1min. The gels were photographed using a laser density scanner for analysis. Each experiment was repeated three times.6. Detection of expression of TFPI-2gene mRNA of A549cellsThe expression of TFPI-2gene mRNA was determined with real-time PCR. The total RNA was extracted from A549cells using Trizol method according to the manufacturer’s instruction. The cDNAs were synthesized from the templates in presence of reverse transcriptase and oligo (dT)20primers, As the internal control, P-actin transcripts were amplified from the same cDNA samples.25μL PCR reaction mixture was pre-denatured at95℃for1min, and40cycles of94℃for15s,58℃for20s, and72℃for20s were performed with a final extension at72℃for5min. SYBR green was used for the each sample which was repeated for3times. Comparative delta-delta Ct method was used for final result calculation:Δ Ct=CtTFPI-2-Ctβ-actin,ΔΔCt=ΔCtexperiment group-Δtcontrol group,the relative mRNA level=2-ΔΔCt.Each experiment was repeated three times.7. Detection of TFPI-2protein of A549cells100μL of cell lysis buffer was mixed with40μL harvested cells to isolate the total cellular protein, and then quantitated by the Bradford method,100μl of the total protein was denaturated for10min, and25μL was taken for the10%polyacrylamide gel electrophoresis.The proteins separated by the electrophoresis were then transferred onto the PVDF membrane, and the10%skim milk used to block the PVDF membrane at room temperature for1h. The PVDF membranes were incubated in the1:200dilution of the primary antibody at4℃overnight, and then in the1:2000diluted alkaline phosphatase-labeled secondary antibody solution at37℃for2h, followed by the DAB coloration. The PVDF membranes were photographed using a ChemiImager5500AlPhalnn Ch, and the subsequent data acquisition and mining were conducted with the Fluorchem V2.0system. Taking the expression level of β-actin protein as the internal control, the ratio of scanning density of TFPI-2protein to that of β-actin was finally calculated and used as the analyzing index to further investigate the changes in protein expression of TFPI-2and compared them between different experimental groups. Each experiment was repeated three times.8. Statistical analysisStatistical analyses were performed using the SPSS13.0software package. The results were expressed as mean±standard deviation (SD). Homogeneity test of variance were performed using Levene method. If variance is homogeneous, continuous variables were compared by the analysis of variance (ANOVA), and multiple comparisons were analyzed by the LSD. If variance is not homogeneous, continuous variables were compared by Games-Howell.A P-value of0.05or less was considered significantly.Result1. Effects of5-Aza-CdR on the proliferation of A549cellsA dose and time dependent inhibition of cell growth was observed after A549cells were treated with0,1,5,10μmol/15-Aza-CdR for24,48and72h. When the processing times were same, the inhibition rates of proliferation of A549cells were significantly increased with increasing the concentrations of5-Aza-CdR, the difference is statistically significant(P<0.05).When the concentrations of 5-Aza-CdR were same, inhibition rates of proliferation of A549cells were significantly increased with delaying treatment time, the difference is statistically significant(P<0.05).2. Effects of5-Aza-CdR on the cell cycle of A549cellsAfter A549cells were treated with0,1,5,10μmol/l5-Aza-CdR for72h, In0,1,5,10μM group, the percentage of Go/G1was69.57±0.99%,76.11±0.83%,83.80±0.35%,95.51±0.55%respectively(P<0.05), suggesting the G0/G1arrest of cell cycle;the percentage of S was29.77±0.43%,23.65±0.96%,15.67±0.75%,1.73±0.45%respectively(P<0.05); the percentage of cycling cells was30.43±0.99%,23.89±0.83%,16.19±0.34%,6.49±0.55%respectively(P<0.05). The results suggested that5-Aza-CdR had inhibited proliferation of A549cells with increasing concentration of5-Aza-CdR.3. Effects of5-Aza-CdR on the invasive ability of A549cellsEffect of5-Aza-CdR on the invasive ability of A549cells was detected by Transwell chambers model after A549cells were treated with5-Aza-CdR for24h. After A549cells were treated with0,1,5,10μmol/L5-Aza-CdR concentrations for24h,the average counts of cells throught membrane at per each high magnification were316.15±18.7,84.15±12.14,28.85±7.13,14.35±3.33respectively(P<0.05).4. The methylation status of TFPI-2gene after A549cells were treated with5-Aza-CdRMSPCR showed that hypermethylation in the promoter region of TFPI-2gene was detected in OμM group, and only partial demethylation of the TFPI-2gene promoter were detected in1,5μM group, and unmethylation of the TFPI-2gene promoter was detected in10μM group.The results suggested that5-Aza-CdR treatment induced the demethylation of hypermathylated TFPI-2gene with the concentrations of5-Aza-CdR increasing 5. The expression of TFPI-2mRNA after A549cells were treated with5-Aza-CdRReal-time PCR showed that the expression of TFPI-2gene mRNA was1±0,1.49±0.14,1.86±0.09,5.80±0.15(P<0.05) respectively in0,1,5,10μM group(P<0.05). The results suggest that5-Aza-CdR increased the expression of TFPI-2gene mRNA in A549cells with increasing concentrations of5-Aza-CdR.6. The expression of TFPI-2proteins after A549cells were treated with5-Aza-CdRWestern blotting analysis showed there was low expression of TFPI-2protein in0μM group, and there were obviously expressions of TFPI-2protein in the experimental group. Beta actin was no obvious change before and after5-Aza-CdR treatment. The relative expression levels of TFPI-2protein were0.12±0.01,0.23±0.02,0.31±0.02,0.62±0.03respectively in0,1,5,10μM group(P<0.05)Conclusion1.5-Aza-CdR treatment induced the demethylation of hypermathylated TFPI-2gene and restored expression of TFPI-2gene in the lung adenocarcinoma cell line A549cells, with the concentrations of5-Aza-CdR increasing.2.5-Aza-CdR obviously inhibited the proliferation and invasion ability of the lung adenocarcinoma A549cells, with increasing the concentrations of5-Aza-CdR. The inhibition may be related to the recovery expression of TFPI-2gene.