| Objective：To preliminary investigate the effect of lysine-specific demethylase1(LSD1)inhibitors tranylcypromine proliferation, migration and invasion of renal clear cellcarcinoma786-O cells in vitro.Methods：1．Cell viability experiments: After cell cultivate success, carrying out MTTassay in order test tranylcypromine(TCP)’s growth inhibition ratio to786-O celllogarithmic phase at2h、48h&72h, which at different concentration gradient; atsame time, matched group&zero setting are also settled, calculate50%lethalconcentration(IC50), and the curve of concentration-inhibition ration drawn out; thisexperiment have been redo3times.2．Using FCM(Flow Cytometry)(Annexin V/PI staining) analyze the cellapoptosis rate.3．Using Trizol method extract experiment group&matched group cell RNA,with method of spectrophotometer test RNA concentration&purity, and usingmethod of sepharose gel electrophoresis test the RNA integrity, using real-timequantification RT-PCR test the expression of LSD1gene’s mRNA.4．With method of Western-blot test TCP with different concentration gradientaffect on kidney cancer cell-786-O, the change of LSD1protein expression in thecell.5．Scratch wound migration assay experiment testing cell external transfer ability,using TCP of different concentration effect on786-O cell of logarithmic phase growth,take photos&observing cell transfer distance.6．Transwell cell external attack experiment testing the number of differentconcentration gradient TCP’s affect on786-O cell with growth of24h attackTranswell chamber, at same time, settle matched group, take photos with0.1%ofcrystal violet staining. This experiment redo3times.7．Data statistic analysis method: All the data shows with MeAN±standard deviation(x±s), using SPSS19.0statistical package One-way ANOVA, settled thatP<0.01means have significant statistical significance, P<0.05means have statisticalsignificance, P>0.05means have no statistical significance.Results1．Different concentration gradient (0、10、20、40、80、160&320uM) TCP’saffect on786-O cell after24h、48h&72h, MTT method testing result shows thatDifferent concentration gradient (0、10、20、40、80、160&320uM) TCP’s affect on786-O cell after24h、48h&72h, MTT method testing result shows that1‰DMSOsolution has no impact on the786-O cells and TCP could inhibit proliferation of the786-O cells, with the increased drug concentration(＞10uM、extension of acting time,the inhibiting effect also increased with obviously time-dose effect relationship(P＜0.01). Calculate the IC50values of TCP at24h,48h and72h were133.76uM,67.66uM and36.02uM;2．FCM(Flow cytometry) Annexin V-FITC/PI staining methods testing apoptosisinduced by drug analysis shows that TCP affect kidney cancer786-O cell after48h,0uM、DMSO&10uM are compared in pairs, apoptotic rate have no statisticalsignificance(P＞0.05); with the increased concentration of40uM to160uM, the ratioof early apoptosis&end-stage apoptosis are also increased gradually, havingsignificance compared with the matched group. At the same time, the necrobiosis alsoincreased synchronization, which shows that TCP have cytotoxic effect, the toxicityincreased with the drug concentration increased.3．The analysis of LSD1mRNA expression quantity of TCP conduct786-O cellafter48h, the expression of40uM&80uM lower than1‰DMSO&0uM group(P＜0.01), but1‰DMSO group compared with0uM group, the results have nosignificance difference(P＞0.05);4．The expression quantity of LSD1protein in786-O cell after TCP conduct48hby Western-blot analysis, the LSD1protein expression lower than1‰DMSO&0uMgroup(P＜0.01); but1‰DMSO group compared with0uM group, the results have nosignificance difference(P＞0.05);5．With scratch wound migration assay experiment observing TCP’s effect on786-O cell invasion, the results show that: the migration index of0uM786-O after24 h&48respectively are35.14%±3.91%、59.46%±4.36%;1‰DMSO786-O after24h&48h respectively are38.12%±3.86%、56.52%±4.68%; the migration indexof40uM after24h is28.64%±3.15%、after48h is39.72%±4.11%; the migrationindex of80uM after24h is21.11%±4.18%、after48h is30.81%±3.74%. After TCPconduct786-O cell, the cell transfer ability obviously lower than0uM&1‰DMSOgroup, with statistical significance;0uM compared with1‰DMSO, the transferability has no obviously difference(P>0.05), which shows that the cell transfer abilitydecline remarkable after conducted by TCP, and have positive relationship with time&concentration,1‰DMSO have no influence to the transfer ability of786-O cell.6．With Transwell cell testing the influence of TCP attack ability to kidneycancer786-0cell, with the effect of40、80uM, TCP conduct on kidney cancer786-Ocell after24h, the results shows that with0uM、1‰DMSO compared in pairs, the cellattack into Transwell the tiling matrigel have no obviously difference, separately are286.25±25.81&294.82±24.36, which shows that the cell attack ability have noobviously dropped. The number of attack into under-cell of40uM&80uM group are195.86±19.43&115.76±18.56, shows that the cell across transwell cell membranegradually reduced with the increased concentration, the attack ability graduallyreduced(P<0.05), having significance compared with the matched group, Thoseindicate the the786-O cell attack number inhibited by TCP have characteristic ofconcentration-independent.Conclusions1．TCP can restrain the multiplication of external cultivate kidney cancer cell,and have time-concentration independent characteristic;2．TCP can induce the apoptosis of external cultivate kidney cancer cell786-O,and have concentration-independent;3．LSD1inhibitor TCP can restrain LSD1mRNA&the expression of protein;4．TCP can induce the migration&invasion ability of renal cancer cell786-Oeffectivity, and have concentration-independent. |
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