The Enrichment Of Circulating Tumor DNA And Exosomal MiR-122 As A Biomarker Of Colorectal Cancer With Liver Metastasis

Background and objective:Cancer is a serious public burden with an estimation of 18.1 million new cancer cases and 9.6 million cancer deaths for 36 cancers in 185 countries[1].To reduce cancer-related morbidity and mortality,more effective approaches in diagnosis and treatment are urgently needed.It is well known that genomic abnormalities are in evolution and hallmarks of cancers in different stages of progression.Cancer is characteristic of genetic abnormalities including chromosomal gain/loss and point mutations[2].With the development of modern molecular biology,molecular detection has been widely used,but conventional molecular detection still regards cancer tissue as the "gold standard",using primary or single metastatic tumor tissue to obtain DNA or RNA to detect gene mutation.Due to intratumor heterogeneity,however,genomic sequencing from a single tumor biopsy may not fully capture the genomic profile of tumors[3].Moreover,tissue biopsy is limited on tissue availability,sampling frequency,and may increase patients' risk of complication because of the invasive procedure[4].Compared with molecular detection of tumor tissue,liquid biopsy has many advantages,such as rapidity,convenience and less damage.In the long run,liquid biopsy can be used to detect the response of tumors to treatment,predict the recurrence of tumors,and even help doctors detect early tumors without any symptoms,even achieve early diagnosis.Genomic information obtained from free DNA fragments in the blood can even indicate where tumors originate in the body.This blood-based approach has been referred to as liquid biopsy and has demonstrated great potential in cancer diagnosis,treatment and prognosis.In recent years,many researches are focus on liquid biopsy technologies,such as circulating tumor DNA(ct DNA)and exosome.Free DNA fragments in blood or other body fluids are called cell free DNA(cf DNA),and ct DNA is the part of cf DNA fragments from tumors.Circulating tumor DNA in plasma has been used as a biomarker for cancer detection and outcome prediction.To sensitively detect the level of ct DNA,multiple experimental and computational approaches have been evaluated,including size selection and molecular barcoding[5,6].Recent literatures have reported the discovery of ct DNA in extracellular vesicles(EVs),which can enrich ct DNA[7-9].Exosome is an important subtype of EVs and also an important detection in the field of liquid biopsy of tumors.The extraction methods of exosome include traditional differential centrifugation,density gradient centrifugation,ultrafiltration and reagent extraction.So far,the study of ct DNA has been limited to the whole plasma or some part of the plasma(e.g.EVs).However,few studies have combined the enrichment of ct DNA with the extraction of exosomes,have systematically examined the effect of variable processing of plasma to generate fractionated plasma samples for ct DNA content comparison to establish a method for isolation and enrichment of ct DNA.Circulating exosomes are small membrane vesicles(30–150 nm)that are released into the extracellular environment upon fusion of multivesicular bodies with cellular membrane.The lipid membranes on the surface of exosomes make the proteins and RNA in the exosomes free from degradation by various enzymes and have high stability[10].It has been found that many kinds of biologically active molecules are differentially expressed in the exosomes of disease origin.Among them,exosomal micro RNA has been extensively studied.Some specific micro RNAs in serum exosomes have been reported as biomarkers for the diagnosis,prognosis and even treatment of various malignant tumors[11-13].Our previous study showed that circulating exosomal mi R-27 a and mi R-130 a were novel diagnostic and prognostic biomarkers of CRC[14].However,specific mi RNAs in serum exosome associated with LM have not been adequately investigated in CRC.Based on the above research background,the first part of this project is to separate different components of plasma,combining the isolation of cf DNA with the extraction of exosomes,further library building,sequencing and bioinformatics analysis were carried out to establish a method for isolation and enrichment of ct DNA.The second part is to extract the peripheral blood exosomes of CRC patients,select specific micro RNAs based on database analysis,detect the expression of specific micro RNAs in exosomes,and search for the diagnostic and prognostic markers of CRC patients with liver metastasis.Part ? The enrichment of circulating tumor DNA indifferent fractions from Peripheral Blood Methods:1.We performed a five consecutive physical and chemical precipitations ofplasma samples from 9 pre-selected small cell lung cancer(SCLC)patientswith high ct DNA.2.Fraction 3 and fraction 6 isolated from plasma were estimated and identified bytransmission electron microscopy(TEM)and flow cytometry of characteristiprotein.3.We quantified the DNA concentration and investigate DNA size distributionafter isolating DNA from each fraction.4.After library preparation,whole genome sequencing and bioinformaticsanalysis,we calculated ct DNA content in each of these fractions using a novelcopy number-based algorithm and compared ct DNA contents between thesefractions and conventional cf DNA from the same patients.Results:1.We successfully isolated six fractions from 1 milliliter plasma(fraction 1: cellsand large debris,fraction 2: debris and large vesicles,fraction 3: largemicrovesicles,fraction 4: fibrin,fraction 5: exosomes,fraction 6: leftoversupernatant)for a total of 54 fractionated specimens.2.Different DNA yields: Although the average DNA yields in fractions 1,5 and 6were among top three and accounted for 79.9% of all DNA yields,theirvariations were also among the top three(Figure 2a).In contrast,fractions 2-4showed relatively low but stable DNA yield.3.Different DNA size distribution: the larger fragment(?10,000 bp)was slowlydiminished from fraction 1 to 3 while smaller fragment(?160 bp)was graduallyincreased from fraction 4 to 6.In some samples,the fraction 6 also showed peaksize at ?140 bp.4.Successful library and sequencing: we received approximately 20 million(range 9.4-42.1 million)mappable reads per sample,and 88.0%(range71.9%-91.0%)mappable ratio.5.Detectable copy number variations(CNVs)and ct DNA content in fraction 6(leftover supernatant)was the highest among the six components.The fraction5(exosome fraction)and fraction 3(large microvesicle fraction)showed the2nd and 3rd highest ct DNA content,respectively.6.We also compared the fractionated plasma DNA to whole plasma DNA for theirct DNA content differences,the fraction 6 clearly had more ct DNA content thanwhole plasma.Conclusions: Our study provided a new insight into fragment size and ct DNA content variations in different fractions of plasma.DNA from supernatant after sequential centrifugation contains more ct DNA than total plasma cf DNA.Part? Circulating exosomal mi R-122 as a potential diagnostic and prognostic biomarker of colorectal cancer with liver metastasisMethods: 1.Candidate mi RNA was selected through integrated analysis of Gene Expression Omnibus(GEO)database as well as clinical samples.2.Exosomes isolated from serum and cultured media were identified by using transmission electron microscopy(TEM)and western blot.3.QRT-PCR was used to detect the expression level of selected micro RNAs in cells,tissues,blood and exosomes,and validated in CRC.At the same time,clinical pathological information and prognosis follow-up information were collected.4.The diagnostic value of candidate mi RNA was judged through receiver operating characteristic curve(ROC)analysis.5.The association of candidate exosomal mi RNA expressions with clinicopathological parameters and prognosis of patients was analyzed with Cox regression model and Kaplan-Meier survival curve.Results: 1.After integrated analysis of three GEO datasets and clinical samples,mi R-122 was discovered to be remarkably overexpressed in tissues of CRC patients,especially in CRC with LM.2.Mi R-122 was discovered to be remarkably overexpressed in tissues of CRC patients and various CRC cell lines.3.The expressions of serum mi R-122 were significantly upregulated in CRC patients,and elevated serum mi R-122 was tumor-derived by being packaged into exosomes.4.The expressions of serum exosomal mi R-122 were significantly upregulated in CRC patients,especially in those with LM.5.Serum exosomal mi R-122 expressions could differentiate CRC patients with LM from healthy controls and patients without LM with area under the ROC curve(AUC)of 0.89 and 0.81.6.CRC patients,especially those with high expression of serum exosome mi R-122 and liver metastasis,have short overall survival time and poor prognosis.7.Uni-and multivariate logistic regression showed that the expression level of serum exosomal mi R-122 and liver metastasis was an independent prognostic indicator of overall survival CRC patients.Conclusion: Serum exosomal mi R-122 was a novel potential diagnostic and prognostic biomarker in CRC patients with LM.