The Correlation Study On Rapamycin Inhibition Of Cell Proliferation And Angiogenesis On Raji Cells

Purpose:We observed Raji cells on Rapamycin in different concentration and treatment with different time:Vascular endothelial growth factor(VEGF),proteinHypoxia-inducible factor-1?(HIF-1?)and its messenger RNA(mRNA),mammalian target of rapamycin(mTOR)and the expression level of mRNA.Discussing whether RAPA in different concentrations and different time would inhibit proliferation,induce apoptosis and inhibiting mTOR,VEGF,HIF-1? genes and its effect on the protein expression,discussing the related mechanism of action,provide theoretical basis for clinical treatments for application of rapamycin in Burkitt lymphoma.Methods:1 Cell experiment group: select lymphoma Raji cells in logarithmic phase.Experiments are grouped into: RAPA treatment group,blank control group.Only the control group containing 10% newborn calf serum of RP-MI1640 culture medium.Final concentration of RAPA is respectively10 nM,50nM,100 nM,250nM,500 nM.RAPA processing time is respectively:24h,48 h,72h.2 Using theCell Counting Kit-8(CCK-8)method,we observed proliferation inhibition effect to Raji cells deal with RAPA at different concentrations and duration for different time.3 Using the Flow Cytometer(FCM)to observe RAPA of different concentrations and Raji cells apoptosisafter treatment with different time and the influence of installment.4 Raji cellswas determinedafter treated with RAPA byWestern blot(WB)technology,the protein expression of mTOR,VEGF and HIF-1? at different concentrations and continued for different time.5 Raji cells was examined after treated with RAPA by Reverse transcription(RT-PCR)method,the mRNAexpression of mTOR,VEGF and HIF-1? at different concentrations and continued for different time.Results:1 The results of CCK 8 shows: Different RAPA concentrations(10nM,50 nM,100nM,250 n M,500nM)acting on Raji cells for 24 h,the inhibition rate were(0.237±0.042),(0.344±0.035),(0.437±0.043),(0.488±0.028),(0.517 ± 0.037)respectively;after 48 h,the inhibition rate were(0.216 ±0.051),(0.331 ± 0.046),(0.439 ± 0.047),(0.545 ± 0.039),(0.670 ± 0.032)respectively;after 72 h,the inhibition rate was(0.287 ± 0.048),(0.429 ±0.034),(0.589±0.049),(0.652±0.034),(0.751±0.041)respectively.Alsowith theRAPA action timeextension and the increasing of drug dose,the inhibition rate was alsoincreased,they are showing a concentration and time dependence(P<0.01).After Raji cells treated with RAPA for 24 h,48h and 72 h,the IC50were(317.064 ± 2.739)n M,(156.276 ± 1.523)nM,(64.762 ± 1.102)nM respectively.2 The results of FCM test shows: different concentrations(10nM,50 nM,100nM,250 nM,500nM)of RAPA treated on Raji cells:(1)The results of treated for 24 h shows: the cell apoptosis rate of blank control group was(4.88± 1.93)%,RAPA processing each cell apoptosis raterespectively(12.54 ±2.25)%,(16.04±1.39)%,(16.22±1.29)%,(19.31±2.04)%,(20.49±1.54)%.Compared with the blank control group,RAPA Raji cells apoptosis rate of treatment group was obviously increased,the difference had statistical significance(P < 0.01),and with the increase of RAPA concentration,Raji cells apoptosis rate also rose(P < 0.05).(2)The results of treated for 48 h shows: the blank control group in cell apoptosis rate was(6.34 ± 2.01)%,RAPA processing each cell apoptosis rate(15.47±1.83)%,(18.82±1.52)%,(19.82±2.07)%,(20.56±1.81)%,(22.56±1.74)%.Compared with the blank control group,the apoptosis rate of RAPA treatment group in Raji cells was obviously increased,the difference was significantly(P<0.01),and with the increase of RAPA concentration,the apoptosis rate of Raji cells also increased,and there was no significant difference between the treatment group(P>0.05).Raji cell cycle G1 phase increased with RAPA concentration increased from37.54% to 65.20%,compared with the blank control group was statistically significant(P < 0.01),there is no statistical significance in the statistical between RAPA treatment groups(P>0.05).3 The results of WB tests shows: After Raji cells treated with RAPA,the gray ratio between blank control group and different RAPA concentrations(10nM,50 nM,100nM,250 n M,500 n M)treatment group in Raji cells: The results of treated for 48 h shows:(1)the relative expression level of p-mTOR(stripe relative grayscale)were(2.804 ± 0.156),(2.691 ± 0.109),(2.577±0.121),(2.332±0.137),(2.203±0.107),(2.032±0.114)respectively;the relative gray of VEGF protein bands were(2.798 ± 0.149),(2.621 ±0.103),(2.509 ± 0.102),(2.391 ± 0.105),(2.269 ± 0.097),(2.025 ± 0.117)respectively;the relative gray of HIF-1? protein bands were respectively(2.864±0.148),(2.701±0.129),(2.607±0.101),(2.442±0.127),(2.303±0.108),(2.162±0.124).There is no difference between each group of protein?-actin expression level(P=0.87)obviously,and compared with blank control group,the expression of p-mTOR and VEGF and HIF-1? proteins in RAPA treatment group are suppressed(P<0.05).(2)The results of treated for 72 h shows:the relative expression level(stripe relative grayscale)of p-mTOR were respectively(2.924±0.149),(2.781±0.099),(2.647±0.111),(2.493±0.112),(2.023±0.114),(1.833±0.104);the relative stripe relative grayscale of VEGF protein were respectively(2.932 ± 0.156),(2.721 ± 0.169),(2.507 ± 0.141),(2.303±0.132),(1.903±0.134),(1.733±0.124);the relative stripe relative grayscale of HIF-1? protein were respectively(2.912±0.148),(2.711±0.158),(2.518 ± 0.139),(2.343 ± 0.140),(1.943 ± 0.144),(1.763 ± 0.144).There wasno obvious difference(P=0.91)between in each group of ?-actinprotein expression,and compared with blank control group,the expression of p-mTOR,VEGF and HIF-1? proteins in RAPA treatment group are suppressed(P < 0.05).And with the increase of drug concentration,three related protein expression has a decreasing trend,the difference wasstatistically significant(P<0.05).The results show that the RAPA can reduce the expression levels of p-mTOR,VEGF and HIF-1? proteinsin Raji cells.4 The results of RT-PCR tests shows: the relative expression level of different concentrations(10nM,50 nM,100nM,250 nM,500nM)of RAPA treatment group and the blank group in Raji cells: The results of treated for24 h shows:(1)mRNA expression level of mTOR were respectively(0.94830±0.00986),(0.81728±0.00714),(0.73398±0.00962),(0.62370±0.00875),(0.52999±0.00227),(0.52999±0.00227);the relatively expression level of mRNA of VEGF were respectively(0.95162 ± 0.01304),(0.83276 ±0.00759),(0.72035±0.00566),(0.63304±0.04036),(0.52864±0.00425);the relatively expression level of mRNA of HIF-1?were respectively(0.85592±0.00949),(0.71467±0.00931),(0.61719±0.00651),(0.52009±0.00998),(0.34456 ± 0.01020).(2)The results of treated for 48 h shows: mRNA expression level of mTOR were respectively(0.90621±0.00666),(0.75909±0.00624),(0.68925±0.00792),(0.55675±0.01345),(0.41874±0.01487);the relatively expression level of mRNA of VEGF were respectively(0.92884±0.00435),(0.78852±0.00619),(0.67400±0.01092),(0.53642±0.00842),(0.41374±0.01164);the relatively HIF-1? expression level of mRNA were respectively(0.79486±0.01025),(0.69360±0.00717),(0.59055±0.00917),(0.48146±0.00545),(0.26718±0.00938).(3)The results of treated for 72 h shows: the expression level of mRNA of mTOR were respectively(0.84789±0.00563),(0.69022 ± 0.00870),(0.60367 ± 0.00396),(0.49472 ± 0.00814),(0.36469 ± 0.01018);the expression level of mRNA of VEGF were respectively(0.84789±0.00563),(0.69022±0.00870),(0.60367±0.00396),(0.49472±0.00814),(0.36469±0.01018);the expression level of mRNA of HIF-1?were respectively(0.70228 ± 0.00695),(0.59731 ± 0.01057),(0.48719 ± 0.00457),(0.38253 ± 0.01126),(0.16590 ± 0.00932).?-actin mRNA expression level in each group has no obvious difference(P=0.93).Compared with the blank control group,after RAPA processing Raji cells(24h,48 h,72h),the mRNA expression of mTOR,VEGF and HIF-1? were significantly decreased(P < 0.01).With the increase of concentration andaction time of RAPA,the related protein mRNA expression declined obviously.The difference was statistically significant(P<0.05).Conclusion:1 RAPA can obviously inhibiting Raji cells proliferation and induce its apoptosis.With the increasing of RAPA concentration and action time,the inhibition rate and apoptosis rate of Raji cells are increased,presents an obviously dependencies between time and dose.2 RAPA can stagnation Raji cell cycle,make Raji cells mainly stop in G1 phase,but with the increase of RAPA concentration and action time,Raji cells stagnation G1 phase change is not obviously.3 The protein expression of mTOR,VEGF and HIF-1? of in RAPA treatment group are significantly lower than the blank control group,mTOR RAPA can significantly decreased theprotein expression level of p-mTOR?VEGF and HIF-1? of Raji cells.4 Compared with the blank control group,in RAPA treatment group,RAPA can significantly reduce the mRNA expression level of mTOR?VEGF and HIF-1? of Raji cells in RAPA treatment group,at the same time there was a positive correlation between the expression of reduced HIF-1? and VEGF,tip of lower HIF-1 alpha can reduce VEGF transcription and translation.Tips the reduced HIF-1? can reduce the transcription and translation of VEGF.5 We can found that throughthe inhibition of mRNA of VEGF andHIF-1?in Raji cells,RAPA can inhibit the protein expression of VEGF and HIF-1?,and inhibit the angiogenesis of Raji cells.