Application Of Exosomes Gene Detection In Colorectal Cancer

The individual treatment of tumor is a model based on the pharmacogenomics of a single patient to make rational chemotherapy regimens,and to reach a maximal of clinical effects and a minimum of toxic side effects.For individual treatment,genic detection of tumor histology is a precondition.However,due to the difficulty of finding,sampling and the heterogeneity of early tumors,the diagnosis and following-up treatment of patients are restricted by various factors.It is urgent to find a substitution that is easily obtained and shares the same genetic features with the tumor tissues from the same patient.Exosomes,spherical to cup shaped nanoparticles under electronic microscope with a diameter between 30 to 150 nm,are originated from the endocytic system of cells,and have stable double membrane structures.Under exocytosis,exosomes can be excreted out of cells,enter the bloodstream and other fluid systems.Since most of the cells are able to secrete exosomes,we can see exosomes are spreading over most fluid systems,and building a bridge among cells.Researches have shown that exosomes in the bodies of cancer patients are far more than those who are healthy.What's more,exosomes contain double-stranded DNA,which carries a kind of gene information that to some extent has a consistency with the DNA of tumor cell.This study is intend to explore the relationship between DNA mutation in serum exosomes and gene mutation among tumor tissues in the patients with colorectal cancer,and then demonstrate the feasibility of exosomes being a new gene detecting means to guide individual treatments.In addition to the convenience and richness,it is necessary for exosomes to reach extreme stability to be ideal material for gene detection.This is the precondition of the whole work,and now we are focusing on the stability of exosomes in the first chapter.We stored fresh serum samples at different condition of 4°C(24 hours,72 hours,168hours);room temperature(6 hours,12 hours,24 hours,48 hours);-80°C with different freeze-thaw cycles(1 time,3 times,5 times).In contrast to the fresh segregated serum,we then extracted the serum exosomes under different conditions separately,and kept the remaining serum.The next step was to extract the protein and DNA in the exosomes and the remaining serum.By the Western blot test of TSG101 and CD63 protein,PCR amplification and Sanger sequencing of DNA,we validated the stability of exosomes and the DNA of it.The results showed that exoDNA was the main source of serum DNA,and always remained stable regardless of the change of outer environment.The duration of exoDNA stability changed with storage conditions.ExoDNA extracted from serum being stored at as the most stable one,and could keep this state for a minimum of 1 week,the one being extracted from serum at room temperature could keep a stability of 1 day,and freeze-thaw cycles had a worst damage to exosomes.After 3 times of freeze-thaw,exoDNA from serum would reach a great damage.However,exoDNA could keep amplifying and detecting a kind of gene mutation which was the same as fresh serum exosomes.All the results above fully testified the strong stability as well as the duration of it that exosomes and exoDNA held,and provided a reliable guarantee for exosomes being a new detecting means of tumor gene.To test if the gene status of exosomes were the same as the result of tumor tissue gene test,we randomly selected the serum exosomes and blood cell DNA from 96 rectal cancer patients under the gene test of KRAS histology.Then we selected 8 genetic loci from 6 genes(KRAS G12 G13,KRAS Q61,BRAF V600,NRAS Q61,PIK3 CA E542 E545,PIK3 CA H1047)which were closely related to rectal cancer to start an PCR amplification and electrophoresis test of exosomes and blood cell DNA,and a high-flux sequencing analysis of the amplification products.At the same time,we randomly selected the blood sample from 30 healthy volunteers to conduct a genetic loci amplification(KRAS G12 G13,KRAS Q61,BRAF V600)and second generation sequencing of exosomes and blood cells.As a result,we found that the wild types of KRAS gene in exoDNA were the same when in tumor tissues.All the patients who had been tested through histology to have KRAS gene mutation were able to be tested as mutate type in exoDNA,well those who were wild type could also partly show as mutate in exosomes.The situation above was also reflected in NRAS gene.For PIK3 CA gene,all the three loci had a proportion of mutation in exoDNA,especially the E545 gene locus,which could reach a mutation rate of 92.7% in exosomes and a relatively high mutation rate in blood cell DNA.On the contrary,the other 7 genetic loci could barely test out a blood cell DNA mutation.Differ from PIK3 CA gene,the mutation rates of BRAF V600,KRAS Q61,NRAS Q61 in exosomes were relatively low which had been reported before,and we didn't find any mutation in the blood samples of those healthy volunteers.What we had acquired from the above research was that,to some extent,gene testing result of exosomes had a consistency with it of tumor tissues,and may chose as a potential biomarker in clinical test.Since gene mutation is able to exist in exosomes and has a relatively high rate in some of them,then is it possible to have other genes with such high mutation rate? Will the mutation rate of exosomes change begin to change during the clinical treatment? Will this kind of change predict the reaction of patients? Carrying these questions,we collected 37 blood samples which were right in the medication course of the advanced colorectal cancer patients who had experienced complete treatments,and extracted the serum exoDNA and blood cell DNA.Under the guide of colorectal cancer gene mutation in literature reports and COSMIC database,we selected 30 genetic loci from 24 genes,which were closely related to colorectal cancer,to do the PCR amplification and high high-flux sequencing analysis of exosomes and blood cell DNA.Based on the sequencing data,we drew the following conclusions.First of all,some patients,whose KRAS gene has been clearly defined as the wild type by histopathological gene detection occurred gene mutation in exosomes DNA during the course of the treatment.Secondly,besides the relevant genetic loci in chapter 2(PIK3CA E545,PIK3 CA H1047,KRAS G12 G13),MAP2K1 K57 also had the same character of hyper mutation.Moreover,the mutation rate of gene we mentioned above would change constantly during the treatment,and this kind of change would occur before the image examination,even earlier than the change of tumor marker during the treatment.Therefore,some specific gene mutation of exosomes would help to do a prediction of treatment effect,and this kind of change could lead us to do the clinic treatment better,adjust the therapeutic regimen quicker,and set a more effective individual-based treatment for the whole-body tumor of patients.