The Role Of Rapamycin On Inhibiting Metastasis And The Underlying Mechanisms Via Downregulation Of Metal Matrix Proteins In Human Burkitt Lymphoma

Objective:Mammalian target of rapamycin(mTOR)is the hub of many important signaling pathways in cells,involved in many physiological and pathological processes,such as cell proliferation,cell cycle regulation,cell migration,angiogenesis,etc.Abnormal function of mTOR is closely associated with the occurrence and development of tumor.To study the effect of rapamycin(RAPA)on the expression of mTOR、MMP-2 and MMP-9 in burkitt lymphoma cell line Raji,and to discuss the influence of RAPA on the migration of burkitt lymphoma cell line Raji in vitro,and Provides theoretical basis for the application of RAPA in the clinical treatment of Burkitt lymphoma.Methods:Use the human burkitt lymphoma cell line Raji as the experimental object.1.Using transwell model to study the effect of rapamycin(RAPA)on the migration ability of Raji after 18 hour;2.Using western blot to examine the protein expression of mTOR、MMP-2 and MMP-9 after taking action of RAPA for 72 hours in raji cells;3.Using qPCR to examine the mRNA expression of mTOR、MMP-2 and MMP-9 after taking action of RAPA for 24、48、72 hours in Raji cells.Results:1.transwell model was used to observe the effect of RAPA on migration ability of Raji cells after 18 hours: The number of Raji cells treated with 0.1 nmol / L RAPA was significantly lower than the group without RAPA after 18 hours(7.4±2.3 vs 9.7±3.1,P<0.05).The number of Raji cells treated with 1 nmol / L RAPA was less than the group treated with 0.1 nmol / L RAPA(3.7±0.36 对 7.4±2.3,P<0.05);2.The results of Western blot: Raji cells were treated with RAPA at different concentrations(0 nmol/l,10 nmol/L,50 nmol/L,100 nmol/L,250 nmol/L,and 500 nmol/L)for 72 h.,the relative expression level of mTOR were(1.49±0.12)、(1.21.±0.11)、(1.12±0.07)、(0.99±0.05)、(0.44±0.03)、(0.15±0.02).The relative expression levels of MMP-2 protein were(0.53±0.03),(0.41±0.02),(0.30±0.02),(0.25±0.02),(0.23±0.03),(0.18±0.03).The relative expression levels of MMP-9 protein were(0.78±0.05),(0.66±0.03),(0.55±0.04),(0.32±0.02),(0.26±0.03),(0.23±0.02).There was no significant difference in the expression of β-actin protein between the groups.Compared with the blank control group,the expression of mTOR,MMP-2 and MMP-9 in the RAPA treatment group was inhibited(P<0.05).With the increase of RAPA concentration,the expression of three proteins decreased.There was a statistical difference(P<0.05)..3.The results of real-time PCR: The mRNA expression was observed in Raji cells treated with RAPA at different concentrations(0 nmol/l,10 nmol/L,50 nmol/L,100 nmol/L,250 nmol/L,500 nmol/L)for 24 h.,the mRNA expression of mTOR respectively was(0.9±0.03),(0.82±0.02),(0.73±0.01),(0.62±0.05),(0.53±0.02),;the mRNA expression of MMP-2 respectively was(0.44±0.02),(0.34±0.06),(0.31±0.05),(0.24±0.04),(0.16±0.01);the mRNA expression of MMP-9 respectively was(0.93±0.03),(0.89±0.05),(0.81±0.04),(0.72±0.07),(0.45±0.03);The mRNA expression was observed in Raji cells treated with RAPA at different concentrations(0 nmol/l,10 nmol/L,50 nmol/L,100 nmol/L,250 nmol/L,500 nmol/L)for 48 h.the mRNA expression of mTOR respectively was(0.85±0.05),(0.69±0.08),(0.60±0.03),(0.49±0.04),(0.36±0.01);the mRNA expression of MMP-2 respectively was(0.26±0.03),(0.11±0.01),(0.09±0.01),(0.08±0.01),(0.07±0.01);the mRNA expression of MMP-9 respectively was(0.60±0.02),(0.39±0.01),(0.30±0.05),(0.22±0.01),(0.17±0.01);The mRNA expression was observed in Raji cells treated with RAPA at different concentrations(0 nmol/l,10 nmol/L,50 nmol/L,100 nmol/L,250 nmol/L,500 nmol/L)for 72 h,the mRNA expression of mTOR respectively was(0.91±0.06),(0.76±0.06),(0.67±0.05),(0.56±0.01),(0.42±0.03);the mRNA expression of mmp-2 respectively was(0.57±0.03),(0.48±0.05),(0.43±0.03),(0.40±0.02),(0.10±0.01);the mRNA expression of MMP-9 respectively was(0.84±0.03),(0.71±0.02),(0.44±0.04),(0.40±0.03),(0.27±0.01).The mRNA expression of mTOR,MMP-2,and MMP-9 was decreased along with the increase of RAPA concentration.(P<0.05).Conclusion:Rapamycin significantly inhibit the transfer ability of Raji,and reduce the expression of protein and mRNA of mTOR 、 mmp-2 and mmp-9 in raji cells.RAPA as an inhibitor of mammalian rapamycin target protein(mTOR).It suggested that there may be some signal pathways where mTOR can regulate the expression of MMP-2 and MMP-9 to affect the migration of burkitt lymphoma cell line Raji.