| Purpose: Malignant tumor is a kind of disease with high morbidity and mortality,which seriously endangers human health,so overcoming and curing tumor is the primary task of human scientific research,and the origin of tumor has always been an important issue that people pay attention to.At present,there have been a large number of studies that believe that tumors originate from stem cells.It is the theory of stem cell maturation that puts forward this point of view.In the early stage of this laboratory,an experimental model of stem cell differentiation arrest has been established to explore the effects of stem cell differentiation inhibition on stem cell differentiation and proliferation.It has been found that stem cell differentiation inhibition can form heterogeneous cell clusters and then form tumors,which provides basis support for stem cell maturation arrest theory.However,the hypothesis that stem cell differentiation is blocked to form tumors still needs to explore and tap more scientific evidence.In this study,the bidirectional thermal cycle reduction SELEX technology was used to screen nucleic acid aptamers with high specificity from liver cancer,gastric cancer,lung cancer and healthy human sera;the nucleic acid aptamers obtained from screening were used to screen liver and stomach cancer,lung cancer and health Proteins specifically bound by nucleic acid aptamers were extracted from human serum,and then bioinformatics and proteomics were used to study the serum proteome of tumor patients and healthy people to explore the relationship between tumor formation and stem cell differentiation and evolution Sex.Method:1.Using agar magnetic beads as a carrier,use two-way thermal cycling subtractive-SELEX technology to screen tumor patient and healthy human serumspecific nucleic acid aptamers;sequence the aptamers obtained by high-throughput sequencing technology;collect liver cancer,Gastric cancer,lung cancer patients,and healthy human serum were collected from 50 patients each,and specific verification studies were performed.2.Select high-abundance serum-specific binding aptamers and retrieve aptamerspecific binding proteome from the serum of liver cancer,gastric cancer,and lung cancer patients,respectively;use LC-MS/MS mass spectrometry to perform unlabeled quantification of the proteome Proteomics analysis and relative quantification.3.Differential protein analysis,GO functional annotation,and KEGG pathway analysis of the target proteome through bioinformatics methods to construct a tumor target proteome interaction network to find key proteins in the tumor pathogenesis process.4.Use public databases to verify key proteins related to tumor pathogenesis.Soft agar clone formation experiments were used to verify the role of key proteins in human liver cancer(HepG2)cells and lung cancer(PC-9)cell clones.Results and Conclusions:1.Screening nucleic acid aptamers by two-way thermal cycling subtractive SELEX-technology,and obtained 8,14,8,and 10 sequences of high-abundance nucleic acid aptamer sequences from liver cancer,gastric cancer,lung cancer,and healthy humans,respectively.Five syntheses were selected from the high-abundance aptamer sequences and tested for positive detection rate.The results were 92%,90%,90%,and 88%,respectively.The results showed that serum-specific nucleic acid aptamers were obtained for liver cancer,gastric cancer,lung cancer,and healthy humans,respectively.2.Utilizing synthetic nucleic acid aptamers to extract aptamer-specific binding proteome from liver cancer,gastric cancer,lung cancer and healthy human serum,respectively.A total of 390,490,358,and 474 serum-specific proteins were obtained from liver cancer,gastric cancer,lung cancer,and healthy human sera by Lable-free relative detection technology.Through differential protein bioinformatics analysis,it was found that the functions of up-regulated proteins of three tumors are mainly lipid transport,microcapsules and inflammatory responses;the functions of down-regulated proteins are mainly damage repair and defense systems.The results show that when tumors occur,tumor tissues need a large number of cells to produce and clear apoptotic cells.Lipid transporters and microcapsules are required to transport the cell membrane and cytoplasmic constituents.At the same time,the body clears apoptosis through inflammatory reactions.The waste produced by cells;similarly,down-regulated proteins reflect the tumor patient’s body due to damage repair and weakened defense system,which makes tumorigenesis possible.3.Through the analysis of specific protein bioinformatics,we obtained the key proteins(such as P36955 and Q8N474 proteins)during tumorigenesis and development.The key protein is that when the cells proliferate to contact each other,the feedback produced inhibits the mediator protein,thereby preventing further division and differentiation of the stem cells and restoring the state of silence.The key protein in the serum of tumor patients is a down-regulated state,which reduces the feedback signal that mediates stem cell proliferation,resulting in stem cell proliferation and differentiation that cannot be terminated,resulting in abnormal differentiation and infinite proliferation of stem cells,forming tumors.4.Two methods were used to verify the above results:(1)The public database was used to verify the key proteins related to tumor pathogenesis,and the results showed that the expression of Serpinf1 gene and sFRP1 gene had statistical differences in the survival of patients with liver and gastric cancer(p<0.05),there was no statistical difference in the survival of lung cancer patients(p>0.05).(2)The key proteins were verified using soft agar clone formation experiments,and the effects of P36955 protein,Q8N474 protein and Way-316606 protein(Q8N474 protein inhibitor)on the colony formation of liver cancer HepG2 cells and lung cancer PC-9 cells were performed.The results showed that both P36955 and Q8N474 proteins inhibited the formation of cell clones(p<0.05),and Way-316606 protein promoted the formation of cell clones(p<0.05),thus proving that P36955 and Q8N474 proteins are involved in feedback regulation of stem cell termination differentiation.Combined with proteomics analysis,it has been further proved that abnormalities in normal human body damage and repair cause tumors. |
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