The Difference Of The MiRNAs Secreted By Exosomes Of Lung Cancer Cells Analysis

Objective: Through extracting mi RNA from exosomes which is secreted by different lung cancer cells and normal lung epithelial cells,analysis the difference of mi RNAs.Thereby screening mi RNA targets,determine what kind of mi RNAs in exosomes have a relationship with the recurrence and metastasis of lung cancer.This research will help the clinical diagnosis and treatment of lung cancer.Methods: Firstly,experiment purchased three types of lung cancer cells 95 d high metastasis of lung cancer cells(people),HCC827(non-small cell carcinoma),the NCI-H1395 lung adenocarcinoma(people)and HBE(normal lung epithelial cells)from Shanghai life science research institute of Chinese academy of sciences cell resource center.Then culture different types of lung cancer cells and normal cells cultured cells by attatchment after the centrifugal method.instruments and equipment for the lung cancer cell culture Gibco company RPMI-1640 medium and fetal bovine serum FBS(10%),double,penicillin and streptomycin resistance(1%),the Sigma company pancreatic enzyme-0.02% EDTA and 0.25% of Japanese SANYO company MCO-20 aic CO2 constant temperature incubator.Normal cell culture of instruments and equipment for the American Gibco company DMEM diet culture medium,fetal bovine serum FBS(10%)and double(penicillin and streptomycin resistance(1%),the Sigma company,pancreatic enzyme-0.02% EDTA and 0.25% of Japanese SANYO company MCO-20 aic co2 constant temperature incubator.Then,carries on the cell subculture,extend the method is as follows: suck out the cultivation of the old;PBS washing 2 times,each time 2 ml;Digest: with 1 ml0.25% of pancreatic enzyme-0.02% EDTA digestion;Termination of digestion: add 4 ml medium termination of digestion;Percussion;1000g5mincentrifugal;Abandon supernatant,add new culture medium,separate administration in the new culture bottle.After subculture,collect different types of culture medium,guarantee the sterile pollution-free collection process.Make sure that will be collected in the conditioned medium of cryopreserved-80 ℃ refrigerator(< 1 month),when the collection get 500 ml,unified thaw(4 ℃).Then extract and separation exosomes by ultracentrifugation.Finally,analyze the exosomes concentration with BCA kit.Results: After ultracentrifuges,transmission electron microscopy(sem)observed that the vesicle structure is full of solution.The vesicle structure’s diameter is 30-100 nm which is at least 38 nm and at most 100 nm,the average diameter of 95% confidence interal(72.613±3.845nm).Compared with extraction exosome composition,normal lung subcutaneous cell and three types of lung cancer cells exosomes show differences.Get the following conclusion: HCC827(human non-small cell lung cancer cells-adenocarcinoma)compared with HBE(normal lung epithelial cells),increase number of mi RNAs is 36,decrease number of mi RNAs is 26;95-d(high metastasis of lung cancer cells)compared with HBE(normal lung epithelial cells),the increase number of mi RNAs is 60,decrease number of mi RNAs is 22;NCI-H1395(human lung adenocarcinoma cells)compared with HBE(normal lung epithelial cells),the increase number of mi RNAs is 295,decrease number of mi RNAs is 25;Three kinds of mi RNAs in lung cancer cells exosomes compared to normal cells raises more than 10 times are four kinds of mi RNAs(hsa-mi R-3960,hsa-mi R-6729-5p,hsa-mi R-6090);Compared to normal cells raises more than 5 times are four kinds of mi RNAs(mi R-6803-5p,hsa-mi R-6087,hsa-mi R-638,hsa-mi R-6089);Compared to normal cells,there is only one kind of mi RNA decreases more than 10 times,which is mi R-155-5p.Conclusion: Human lung cancer cells and normal lung epithelial cells can secrete exosomes,minimum diameter is exosomes 38 nm,maximum diameter is 100 nm,the average diameter of 95% confidence interal(72.613 ± 3.845 nm).And these exosomes can be absorbed by human lung cancer cells and normal lung epithelial cells and concentrated enrichment in the cytoplasm.