The Study On The Change Of Autophagy Regulated By Insulin Like Growth Factor-1(IGF1) In Human Colorectal Carcinoma Resistant Strains And Its Mechanism Of Drug Sensitivity

Backgrouds: The incidence of colorectal cancer in human digestive tract tumor is ranked the second place,and the age of onset has a tendency towards younger.Meanwhile,the incidence of colorectal cancer in China has increased year by year.Therefore,improving survival is of great significance for the delay of colorectal cancer patients in the effective anti-tumor therapy.At present,a wide variety of main chemotherapy drugs,cisplatin and fluorouracil in the control of human colorectal cancer play an important role in the treatment of colorectal cancer.However,many types of malignant tumors acquire drug resistance after treatment.How to increase the sensitivity of malignant tumors is a long-term topic of cancer.The previous study shows that insulin growth factor-1(IGF-1)plays a key role in cell survival,proliferation,differentiation and metabolism,while the effect of IGF-1 on tumor autophagy and signal pathways in the process remains unclear.Therefore,this study has been carried out on the effect of IGF-1 on the autophagy of human colorectal cancer cell line which is involved in protein kinase / m TOR pathway in vitro,resulting in providing a new way of thought and increasing the sensitivity of the drug to provide experimental basis.Objective: To investigate the effect of IGF-1 on the autophagy of human colorectal cancer cell line by protein kinase / m TOR pathway and the possible mechanisms in vitro.Methods: Human colorectal cancer cell lines HCT-8 and HCT-8 / R(Human colorectal cancer fluorouracil resistant cell line)were used in this study.Autophagy gene and fluorouracil resistance gene overlap were detected by gene detection kit,a total of 13 overlapping genes.It has been shown that IGF-1 is associated with autophagy,but how to affect IGF-1,especially in human colorectal cancer cells.Moreover the mechanism is not clear.We inserted human LC3(also known as ATG8)complementary deoxyribonucleic acid into Ds Red,so that the Ds Red protein fused to the N-terminus of the LC3 protein.Production of continuous and stable cell lines reported autophagy activity,transfection of target cells and production of recombinant retrovirus expression by using Ds Red-LC3 reporter gene.cells were seeded in 5 x 105 / ml in triplicate(10 or 50 n M)and IGF-1,MK-2206(10 n M)or 3-MA(100 n M)with 5-FU(10 ug/ml)in a petri dish,after 24 h collection is analyzed with flow cytometry instrument cells and dyeing membrane associated protein V-fluorescein isothiocyanate(FITC)and propyl-iodide organism(PI).The effect of IGF-1 on the expression of autophagy in three stages was verified by Real-time PCR.The expression of LC3 B was measured by western blotting treatment,indicating the variety of autophagy and the status of AKT.Results: 1.After the serum free culture of 24 h,the autophagy of the drug resistant cells increased significantly,and the autophagy bodies decreased after IGF-1 treatment.After the addition of AKT,the phenomenon was reversed.2.In non resistant cells,apoptosis was significantly increased under 5-FU treatment,while the resistant strains of HCT revealed less apoptosis(24h).In serum free culture of 24 h,with different concentrations(10 n M or 50 n M)of IGF1 treatment on drug resistant strains(HCT-8R),apoptosis increased significantly.With addition of AKT inhibitor MK-2206(10 n M)or autophagy inhibitors 3-MA(100 n M),the apoptosis reverse significantly.3.Real-time q PCR results validated this process and its impact on the three stages of autophagy gene expression levels.In the initiation stage,IGF-1 decreased theautophagy related genes(including ULK1,beclin-1 and Vps34)of HCT.With the addition of IGF-1 and AKTi,the expression levels of autophagy related genes(including ULK1,Becn-1 and Vps34)were significantly increased.However,no changes were observed with the addition of IGF-1 and 3-MA.In the elongation and expansion stage,the similar trend was observed with the addition of IGF-1.4.The expression of LC3b-I was measured by western blotting treatment,indicating the elevation of autophagy and the activation of AKT.The microtubule-associated protein light chain 3 increased with the addition of IGF-1,and decreased with the addition of IGF-1 and AKTi.Similarly,the p-AKT expression was increased with the addition of IGF-1(50 n M),and decreased with the addition of IGF-1(10 n M or 50 n M)and AKTi.Conclusion: The autophagy-related gene IGF-1 was screened by the chip of the resistant strain,IGF1 activated AKT and inhibited autophagy through the AKT/m TOR signal pathway.After autophagy was inhibited,the drug resistant strains become sensitive to the 5-Fu.5-Fu,in combination with IGF-1 could increase the apoptosis.