Screening And Identification Of Gastric Cancer Serum Aptamers

Gastric cancer is one of the most common malignancies and it has a great threat to human health.Currently,most of cancers in clinic are diagosed in the middle or terminal stage,which delays the most treatable time.Therefore,the current therapy of cancer is not ideal.Given this,finding an early diagnosis of cancer is in urgent,since early diagnosis may be the key to achieve a better therapeutic effect.Detecting serum markers in gastric cancer is one of the main methods of early diagnosis in clinic.And here are the common gastric cancer serum markers: carcinoembryonic antigen(CEA),sugar chain antigen 19-9(CA19-9),125(CA125),50(CA50),etc.However,because of its low sensitivity and specificity,a variety of tumor markers are usually used combined together in order to detect more accurately.So it is very important to find new molecular probes to detect early gastric cancer.Collected 50 cases of preoperative serum of patients with pathologically confirmed gastric cancer and 50 cases of normal serum without abnormality,and mixed them together respectively to eliminate individual differences.Treated the sera of gastric cancer with acetonitrile for three-rounds positive selection,then developed and separated the secondary ss DNA library by streptavidin magnetic bead method.Conducted normal serum treated with acetonitrile as negative-selection target.Combined the serum of gastric cancer treated with acetonitrile with the secondary ss DNA library,and prepared the single chain product by streptavidin magnetic beads as the library for the following nine-round selection.Then used high throughput sequencing to isolate a single aptamer and analyze its structure.Selected four high-abundance aptamer sequences and synthesized by an biotech company,then operated them on clinic trial with the 50 cases of gastric cancer serum treated by acetonitrile and the 50 cases of normal serum treated by acetonitrile,respectively to a further identification of these four aptamers.The main results are as follows:1.Collected gastric cancer serum,normal serum,according to the serum: water:acetonitrile = 1: 2: 0.5 treatment.the amount of LAP that is stored is relatively high.2.Make the serum of gastric cancer as the positive target,the normal serum as negative target,and use subtracting SELEX technique to do screen.After 12-rounds screen,we choose the 12 th library which shows the highest affinity to do high-throughput sequence.The sequences of them were immobilized at the same time as the sequence of ss DNA library.Sequence of the two sequences was different,Indicating successful selection of a group of gastric cancer serum aptamers.3.We get 4 high-abundant nucleic acid aptamer sequences by software.The structure of 10 representative nucleic acid aptamers was analyzed by NUPACK software.The results indicate that aptamers are mainly composed of stem-stem and bulge.Homology sequences were found to have multiple conserved motifs.Indicating that the nucleic acid aptamer may target a variety of different targets and binding sites in serum.4.Collect 50 cases of normal serum treated with acetonitrile and 50 cases of normal serum treated with acetonitrile,bind them with mentioned 4 aptamers,respectively,and detect them by real-time quantitative PCR.The results showed that the ?Ct values of the four high abundance nucleic acid aptamers were about 4-5 cycles.The results shows that the four high-abundant nucleic acid aptamers have a strong specificity for targeting gastric cancer serum.