The Study On Effect Of Decitabine Combined With Paclitaxel On NSCLC Cell Lines

DNA methylation is known as the most commonest manner of epigeneticmodification in mammal. It can be attributed to directly involvement of controlling thegene expression and function, which has close relation with lots of vital movement, suchas organism aging, cell differentiation and development, gene imprinting and activating.Therefore, the dysfunction of gene methylation will induce tumorigenesis, which isespecially the hypermethylation of some particular area in tumor suppressor gene. Thiskind of hypermethylation inactivates the expression of tumor suppressor gene intranscriptional level and promote the transmission of cell proliferation signal. The affectcell also lose the function of negative controlling the cell cycle.According to recent research, miR-200c has been observed to act as tumorsuppressors. Well differentiated cancer always has more expression of miR-200c thanpoorly one. The related mechanism include that loss of miR-200c expression will induce cancer cell to have EMT, called epithelial to mesenchymal transition. It is suggested thatEMT take a important role in early tumor invasion and metastasis by several ways andspeed progress of malignant tumor. The hypermethylation of promoter is considered themajor reason of miR-200c gene inactivation. TGF-β signaling pathway has beenimplicated in such kind of reversible methylation. It is reported TUBB3known as tubulinbeta-3, of which3’untranslated region is able to match the core sequence of miR-200c, asone of important target of miR-200c, can be degraded by RISC. Moreover, TUBB3ispaclitaxel microtubules efficacy of related important targets, and its expression in thetumor cells is inversely proportional to the degree with the curative effect of taxol drug.Thus, we are interested in whether hypomethylating agents such as decitabine combinedwith paclitaxel can strengthen the effect on the proliferation and apotosis of NSCLC cellline and whether this method has the influence on the expression of TUBB3and EMTfunction by up-regulated activity of miR-200c. And it would be discussed and testified byour following study.In order to prove this topic, NSCLC cell line H1299and H1975are treated andrandomly divided into4group, control group, paclitaxel therapy group, decitabine therapygroup, and decitabine combined with paclitaxel therapy group, respectively. Firstly, theworking concentration of decitabine and paclitaxel are tested by MTT assay. Thesubsequent experiments are followed as decitabine15.2umol/L(H1299),18.7umol/L(H1975); paclitaxel64.9nmol/L(H1299),11.2nmol/L(H1975). MTT assay andflow cytometric study are used to detect cell proliferation inhibition and apotosis. The cellproliferation inhibition of each group except control group are (32.97±2.76),(55.23±2.20)and (82.51±2.18) for H1299;(43.11±2.23),(47.71±4.74) and (74.67±3.70) for H1975. Theapotosis rate of those are (1.87±0.52),(19.33±0.57),(37.03±1.81) and (71.8±1.84) forH1299;(2.43±0.53),(16.5±2.61),(44.13±2.50) and (68.2±2.02)for H1975. The resultshowed that decitabine combined with paclitaxel significantly inhibited the proliferationand apotosis of H1299and H1975.Realtime PCR is used to further test the expression of miR-200c in each group. It isrevealed that decitabine group and decitabine combined with paclitaxel group have higher expression of miR-200c than control group and paclitaxel group. E-cadherin, the hallmarkof EMT, has the same trend as miR-200c in the experiment by western blot whileVimentin is down-regulated in decitabine treated group. Moreover, TUBB3mRNA indecitabine group and decitabine combined with paclitaxel group have less expression thancontrol group and paclitaxel group by Luminex liquichip. The result revealed thatdecitabine combined with paclitaxel may induce NSCLC cell to reverse EMT anddown-regulated expression of TUBB3mRNA by up-regulated miR-200c.H1975cell line is treated with different concentration of decitabine, observed the cellmorphological changes and detected the expression of miR-200c, TUBB3, E-cadherin andVimentin in each group after96h. As a typical kind of interstitial cell phenotype, the shapeof control group takes spindle. Control group cell grow in good condition. When theconcentration of decitabine is up to20μmol/L, the different concentrations of decitabineintervention group present the change of the spindle, with the phenotype of epithelioid cell.The number of cells is less than that of control group. The expression level of miR-200cand E-cadherin increase with the increasing of the concentration of decitabine, whereasTUBB3mRNA and Vimentin have the opposite trend.In summary, we discussed the synergy effect of decitabine combined with paclitaxelon the proliferation, apotosis in NSCLC cell lines. Moreover, by up-regulated theexpression of miR-200c, this kind of combination affect the level of TUBB3andstrengthen paclitaxel in the NSCLC cells. The up-regulated miR-200c by decitabineindirectly inducing the tumor cell to occur MET is related to the concentration level ofdecitabine. Due to MET associated with the efficacy of antitumor drug, we think that thiscombination may also realize the synergy of non-small cell lung cancer through thismechanism.