The Effects Of MTOR Inhibitors On The Proliferation Of The Thyroid Cancer Cell And The Study Of The Mechanism

Objective:We investigate the expression of autophagy related genes LC3,II,P62 and ubiquitin proteasome family member UCH-L1 in thyroid tissue and PBMC of thyroid cancer patients.We study the effects of metformin and rapamycin on the growth and proliferation of thyroid carcinoma BCPAP cells.We analyze the change that the expression of LC3 ?,P62 and UCH-L1 in BCPAP cells which are treatment with metformin and rapamycin.It is in order to provide new targets and new ideas for thyroid cancer.Methods:1.We detection the growth and proliferation of BCPAP cells which are treatment with different concentrations(0.5,1,2.5,5,10mM)metformin and different concentrations(1,5,10,15,20 M)rapamycin by CCK-8.2.The m RNA and protein expression of LC3?,P62 and UCH-L1 were detected by real time PCR and Western blot in the control group,metformin group,rapamycin group,metformin + 3-MA group and rapamycin + 3-MA group With protein expression.3.The changes of autophagic bodies were observed by transmission electron microscopy(TEM)in BCPAP cells of control group,metformin group,rapamycin group,metformin + 3-MA group and rapamycin + 3-MA group.4.We detect the LC3?,P62 and UCH-L1 mRNA expression in PBMC of 60 cases thyroid carcinoma patients by real time PCR,which is compare with 30 cases of healthy person.We use the same method to detect the LC3?,P62 and UCH-L1 mRNA expression in tissues of 10 thyroid carcinoma patients.Results:1.The cell survival rate of the 1?M rapamycin treatment group and 0.5 mM metformin treatment group decreased slightly than control group,but there were not significant difference(p> 0.05).The cell survival rate of the 5,10,15,20?M rapamycin treatment group and 1,2.5,5,10 m M metformin treatment group was significantly lower than that the control group(p <0.01).The concentration of rapamycin or metformin has a concentration-dependent relationship with cell survival rate.2.The expression of LC3?mRNA in rapamycin group was quantity up-regulated than the control group(p < 0.01).The expression of P62 and UCH-L1 m RNA in rapamycin group was quantity down-regulated(p < 0.01).The expression of LC3?mRNA in rapamycin + 3-MA group was quantity down-regulated than the rapamycin group(p < 0.05).The expression of P62 and UCH-L1 m RNA in rapamycin + 3-MA group was quantity up-regulated(p < 0.01,p < 0.05).The expression of LC3?mRNA in metformin group was quantity up-regulated than the control group(p < 0.01).The expression of P62 and UCH-L1 mRNA in metformin group was down-regulated(p <0.01).The expression of LC3?mRNA in metformin + 3-MA group was quantity down-regulated than the metformin group(p < 0.01).The expression of P62 and UCH-L1 m RNA in metformin + 3-MA group was quantity up-regulated(p < 0.01).3.The expression of LC3?protein in rapamycin group was quantity up-regulated than the control group(p<0.01).The expression of P62 and UCH-L1 protein in rapamycin group was quantity down-regulated(p<0.01,p<0.05).The expression of LC3?protein in rapamycin + 3-MA group was quantity down-regulated than the rapamycin group(p<0.05).The expression of P62 and UCH-L1 protein in rapamycin+ 3-MA group was quantity up-regulated(p<0.01,p<0.05).The expression of LC3?protein in metformin group was quantity up-regulated than the control group(p < 0.01).The expression of P62 and UCH-L1 protein in metformin group was quantitydown-regulated(p < 0.01).The expression of LC3?protein in metformin + 3-MA group was quantity down-regulated than the metformin group(p < 0.05).The expression of P62 and UCH-L1 protein in metformin + 3-MA group was quantity up-regulated(p<0.01,p<0.05).4.Compared with the control group,the structure of autophagy bilayer in the rapamycin group and the metformin group was significantly higher than that in the control group(P <0.05).Compared with the control group,rapamycin + 3-MA group,metformin + 3-MA group intracellular autophagic body was significantly reduced.5.The expression of LC3? mRNA in lymphatic metastasis group and lymph node metastasis group was lower than in control group(p <0.01).Compared with the control group,the expression of P62 m RNA in the lymphatic metastasis group was significantly higher than the control group(p <0.05).While the expression of P62 was no significant difference between the control group and the non-lymphatic metastasis group.There was no significant difference in the expression of UCH-L1 mRNA between control group,lymphatic metastasis group and non-lymphatic metastasis group.In the thyroid tissue,the expression of LC3? mRNA was down-regulated compared with the adjacent tissues,while the expression of P62 and UCH-L1 mRNA was up-regulated(p <0.01).Conclusions:1.Metformin and rapamycin can effectively inhibit the proliferation of BCPAP cell.This inhibition was connected with autophagy.Metformin or rapamycin induce autophagy and cause proliferation inhibition can provide new ideas and new ways to treat thyroid cancer.2.The incidence of thyroid cancer may be related to autophagy levels.3.UCH-L1 may be involved in the formation of thyroid cancer as a cancer-causing factor,and UCH-L1 may be involved in the autophagy process of thyroid cancer cells.