| OBJECTIVE To explore whether PI3 K inhibitor YY-20394 have anti-tumor effect on diffuse large B-cell lymphoma(DLBCL)and whether their effects on DLBCL of different cell origins are different,and to further explore the mechanism of YY-20394,so as to provide theoretical basis for clinical treatment of DLBCL with YY-20394.METHODS1.To detect the inhibitory effect of YY-20394 on the proliferation of SUDHL-4 cells and U2932 cells by cell counting kit-8(CCK-8);Draw the cells growth curve,and calculating the IC25、IC50、IC75 at 24 hours.2.To detect the apoptosis of SUDHL-4 cells and U2932 cells treated with YY-20394 by Flow cytometry.3.High-throughput sequencing was used to analyze the difference of gene expression,in order to explore the effect of YY-20394 on gene expression in DLBCL cells.4.KEGG signal pathway and GO enrichment analysis of differential genes were performed to explore the effect of YY-20394 on signal pathway in DLBCL cells.5.GSEA enrichment analysis of tumor-related signal pathways was performed on the cell gene expression data to further explore the effect of YY-20394 on tumor-related signal pathways in DLBCL cells.RESULTS1.The proliferation inhibition rates of SUDHL-4 cells treated with different concentrations of YY-20394(0.875,1.75,3.5,7.14μmol/L)at 24 hous were(29.19±0.75)%,(42.99±2.51)%,(52.37±2.98)%,(62.56±2.51)%,(72.7±2.53)%respectively;the proliferation inhibition rates at 48 hours were(42.33±3.21)%,(54.55±4.01)%,(73.7±3.86)%,(82.83±3.32)% and(90.3±1.53)%,respectively.The rate of cell proliferation inhibitor increased with the increase of drug concentration and action time,and the difference was statistically significant(P < 0.05).The IC25,IC50 and IC75 of 24 hours were calculated to be(0.69±0.71)μmol/L,(3.30±0.46)μmol/L and(15.79±2.31)μmol/L,respectively.2.The proliferation inhibition rates of U2932 cells treated with different concentrations of YY-20394(0.875,1.75,3.5,7.14μmol/L)at 24 hous were(2.33±0.09)%,(2.95±0.15)%,(2.85±0.12)%,(2.52±0.06)%,(3.17±0.20)%respectively;the proliferation inhibition rates at 48 hours were(2.56±0.21)%,(2.96±0.12)%,(2.85±0.55)%,(2.77±0.29)%,(2.93±0.34)%,respectively.The rate of cell proliferation inhibitor did not increase with the increase of drug concentration and action time,and the difference was not statistically significant(P > 0.05).3.When SUDHL-4 cells were treated with IC25(0.69±0.71)μmol/L,IC50(3.30±0.46)μmol/L and IC75(15.79±2.31)μmol/L calculated in the proliferation inhibition experiment for 24 hours,the apoptosis rates were(6.87 ±0.37)%,(14.95±0.25)% and(40.58±0.52)%,respectively;the apoptosis rates were(11.38±0.19)%,(21.84±0.31)% and(47.94±0.26)%,respectively.There were significant differences in the rate of apoptosis among different concentrations and different time(P < 0.05).4.When U2932 cells were treated with different concentrations of YY-20394(0.875,1.75,3.5,14μmol/L)for 24 hours,the apoptosis rates were(4.42±0.15)%,(4.36±0.21)%,(4.31±0.22)%,(4.29±0.11)% and(4.32 ±0.14)%,respectively.there was no significant difference in apoptosis rate between U2932 cells and the control group(4.28±0.22)%,P> 0.05.5.After treatment with YY-20394,there were 259 up-regulated genes and341 down-regulated genes in SUDHL-4 cells.KEGG enrichment analysis showed that the anti-tumor effect of YY-20394 on SUDHL-4 cells involved m TOR signal pathway,adenosine monophosphate activated protein kinase(AMPK)signal pathway,PI3K-AKT signal pathway,Ras signal pathway,NF-κB signal pathway and BCR signal pathway.GO analysis showed that PI3 K inhibitors exert anti-tumor effects by affecting important cellular biological functions,such as alcohol synthesis,m RNA decomposition,cell amino acid metabolism,cell cycle and apoptosis.6.GESA enrichment analysis showed that YY-20394 down-regulated the activities of Hedgehog(Hh)signal pathway,JAK-STAT signal pathway,MAPK signal pathway,NF-κB signal pathway,NOTCH signal pathway,PI3K-AKT signal pathway and Wnt signal pathway in GCB subtype DLBCL cells.7.After treatment with YY-20394,there were 12 up-regulated genes and 3down-regulated genes in U2932 cells.KEGG enrichment analysis was not enriched to signal pathways with significant levels.GO analysis showed that Cellular component are located in signal recognition granules,endoplasmic reticulum;in terms of biological processes,they are mainly related to the biological processes of co-translated proteins,proteins targeted by the endo plas mic network and other proteins;in molecular function,it is mainly related to the regulation of transmembrane transporter activity and sodium transporter activity of heterologous organisms.8.The results of GSEA enrichment analysis showed that the activation level of PI3K-AKT signaling pathway in U2932 cells was significantly lower than SUDHL-4 cells.CONCLUSION1.PI3 K inhibitor YY-20394 could inhibit proliferation and induce apoptosis in GCB-DLBCL SUDHL-4 cells in a dose-dependent and time-dependent manner.2.YY-20394 exert anti-tumor effect on SUDHL-4 cells mainly by inhibiting the activation of tumor-related signal pathways,such as PI3K-AKT signal pathway,NF-κB signal pathway,MAPK signal pathway,JAK-STAT signal pathway,Notch signal pathway,Wnt signal pathway,Hedgehog signal pathway and so on.3.YY-20394 has no obvious effect on cell proliferation inhibition and apoptosis induction in ABC-DLBCL U2932 cells,the results of GSEA enrichment analysis showed that the activation level of PI3K-AKT signaling pathway in U2932 cells was significantly lower than SUDHL-4 cells. |
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