Screening Of Nucleic Acid Aptamers In The Serum Of The Lung Cancer By Bidirectiona Thermal Circulation Subtractive SELEX

Background and objective:Lung cancer is the most lethal malignant tumor in the world,and the majority of lung cancer patients are in the middle and late stages of clinical treatment.Nowadays,general radiotherapy and chemotherapy are commonly used in the treatment of lung cancer.Although there are some curative effects,it is inevitable that the treatment cycle is long,the side effects are large and the cost is expensive.Early diagnosis and early treatment can greatly improve the 5-year survival rate of patients with lung cancer and become the key to improve the prognosis of lung cancer.The serum tumor markers of lung cancer which are known and used at present can not be used as clinical markers because of their low specificity and sensitivity.Therefore,the selection of a known or unknown serum tumor marker nucleic acid aptamers from a lung cancer serum using a SELEX technique has become a hot spot in the early diagnosis and treatment of lung cancer.Aptamers are a kind of small oligonucleotide(DNA or RNA),which have large specific surface area and rich three-dimensional(3D)space structure.They can coupled to the corresponding target materials with high affinity and strong specificity.Compared with the antibody,the aptamers can be synthesized in a large amount in vitro,has strong repeatability,high stability and has been stored.Therefore,the specific aptamers of non-cancerous and lung cancer serum were obtained,which the thermal circulation subtractive SELEX technique,and to establish a detection method for lung cancer,so as to improve the efficiency of diagnosis and treatment.Methods:(1)Selected of SELEX medium The results of enzyme linked immunosorbent assay(ELISA)were as follows:(1)To compare the coupled protein ability of carboxyl magnetic microspheres and epoxy magnetic microspheres;(2)After the magnetic microspheres were treated with acid and alkali,compare the coupled protein ability of carboxyl magnetic microspheres and Epoxy magnetic microspheres;(3)Optimized Epoxy magnetic microspheres storage medium and storage time.(2)Screening of serum nucleic acid aptamers Through the thermal circulation subtractive SELEX technique:(1)Preparation of serum target and target magnetic microspheres: bloody were collected from 20 patients with lung cancer and 20 normal subjects,and the same amount of serum was drawn from each tube for mixing.The mixed serum target of lung cancer(+)and non-cancer mixed serum(-)were prepared after handling.The purpose of the mixed serum preparation was to eliminate individual differences.The targets were coupled with epoxy magnetic microspheres to prepared the target magnetic microspheres.(2)The negative target magnetic microspheres is used for subtracted the ss DNA primary library: using the same amount of negative target magnetic microspheres for 3 times to subtraction SELEX screening,and the purpose is to remove the nucleic acid sequence which is specifically combined with the negative target;(3)Screening of nucleic acid aptamers in the serum of the lung cancer by bidirectional thermal circulation subtractive SELEX: Using the positive target magnetic microspheres as the forward screening target and the negative target as the subtractive screening target,the primary ss DNA library in the positive screening was by the subtractive screening with negative target magnetic microspheres,the positive ss DNA was obtained.Using the negative target magnetic microspheres as the forward screening target and the positive target as the subtractive screening target,the negative primary ss DNA library used the primary ss DNA library,the negative ss DNA was obtained.The secondary ss DNA library was prepared by the streptavidin magnetic microspheres separation method.The screening steps were repeated and 19 rounds.(3)Sequencing and structural analysis of aptamers41 aptamers in lung cancer serum and 40 aptamers in non-cancer serum were obtained by high-throughput sequencing.4 high abundance sequences were selected from serum-specific aptamers sequences of lung cancer and non-cancer to analyze the structure of the aptamers.(4)Make test reagent and carry out clinical verification4 lung cancer aptamers high abundance sequences and 4 non-cancer aptamers high abundance sequences were synthesized and the detection reagent A,B was prepared.50 lung cancer serum samples and 50 non-cancer serum samples were collected and combined with detection reagent A,B,respectively.The positive rate by calculating the difference ?Ct values which the detection reagents A,B are respectively combined with two serum samples.Results:(1)An epoxy magnetic microspheres is used as a screening medium,and the epoxy magnetic microspheres are preferably stored in the epoxy chloropropane and are acidified when used.(2)Through 19 th rounds of screening,the most specific negative,positive ss DNA was sequenced by high-throughput sequencing,and 4 high-abundance sequences were selected from each of them.The structure of aptamers was analyzed by RNA structure software.The main aptamers structure were loop-stem.(3)The positive rate of lung cancer was 90%,the non-cancer positive rate was 82%,and the total positive rate was 86%.Conclusion:The high-specific nucleic acid aptamers of lung cancer and non-cancer serum is screened out by the bidirectional thermal circulation subtractive SELEX technique,and the clinical test method is designed for verification.The results show that the detection sensitivity is high,the specificity is strong,and a new method is provided for the early diagnosis and treatment of the lung cancer.