Research On The Role Of ITGB1 In The Development Of Gastric Cancer

Background:Gastric cancer is one of the most common malignancies and ranks second in global cancer mortality.Although medicine has made some progress in the treatment of gastric cancer,the survival rate of gastric cancer patients has not been significantly improved due to the high morbidity and mortality of gastric cancer.Early gastric cancer can be surgically removed,but due to the lack of specific symptoms in early gastric cancer,most patients have reached the advanced stage of diagnosis,lost the opportunity for surgery,and can only be treated conservatively.In recent years,have been developed some new therapeutic techniques for the treatment of gastric cancer,such as gene therapy and molecular therapy,which provide a new outlet for the treatment of gastric cancer.Studies have shown that the occurrence of gastric cancer is the result of the accumulation of genetic mutations,so it is important to find the target molecular genes that play important roles in gastric cancer.ITGB1(integrin?1),also known as CD29,FNRB,MDF2,VLAB,GPIIA,MSK12,VLA-BETA,is a human protein-coding gene with a full length of 58048 bp,located on chromosome 10 of the human body,containing 18 exons and 3 transcriptional variants.ITGB1 is a member of the family of integrins,an important class of cell adhesion molecules that promote cancer survival,proliferation and motility through signaling to downstream targets.Therefore,it is important to study the function of the ITGB1 gene in tumors.CRISPR/Cas9 is derived from the self-defense mechanism of S.pyogenes and has now evolved into a novel gene editing technique that is widely used in genetic engineering.The principle is achieveing accurate cleavage of the target gene by the Cas9 protein through the specific sgRNA,to cause DNA burst of the target gene,realizing gene editing.This method is convenient to operate and low in cost,so it is favored by more and more researchers.Objective:The ITGB1-/-SGC7901 cell line was established by CRISPR/Cas9 technology.Based on this,the regulation of ITGB1 on the malignant behavior of gastric cancer cells was studied,which provided new ideas and materials for the development of new strategies and methods for gastric cancer prevention and treatment.Methods:1.Use the NCBI website to find the genetic sequence of ITGB1.Find the ORF regions of all transcript variants of ITGB1 and find common parts.Three pairs of sgRNAs targeting ITGB1 were designed in the common ORF region at exons 3,4 and 6 of ITGB1,respectively.Designed websites are:(1)http://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design(2)http://crispr.mit.edu(3)http://www.e-crisp.org/E-CRISP/designcrispr.html2.Synthesis and annealing of the designed sgRNAs.The Cas9 vector PX459 was extracted,and the vector was digested with Bbs I,and the vector was recovered by using a gel recovery kit.Finally,the recovered vector fragment is ligated to the annealed sgDNA.3.The constructed CRISPR/Cas9 recombinant plasmid was transfected into SGC-7901 cells(transfection efficiency should be above 80%),and the activity of sgRNA was detected by T7E1 enzyme(activity should be greater than 25%).Then,the puromycin resistance screening was carried out,the screened cells were monoclonalized and finally several monoclonal cells were obtained.The ITGBI+/-SGC7901 and ITGB1-/-SGC7901 cell lines were confirmed by sequencing and Western Blot,and then the cells were expanded and frozen.4.Detection of the biological behavior of SGC7901 cells after knockout of ITGBI.The cell motility was detected by woud healing assay and Transwell.The cell proliferation ability was determined by MTT and plate cloning assay.The cell surface microstructure was observed by Coomassie blue and scanning electron microscopy.Cell apoptosis was observed by DAPI staining.5.A series of analyses of ITGB1 bioinformatics using the cBio Cancer Genomics Portal database.Results:1.A CRISPR/Cas9 recombinant vector targeting ITGB1 was successfully constructed.2.The ITGB1+/-SGC7901 and ITGB1-/-SGC7901 cell lines were successfully constructed.3.The woud healing assay and Transwell results show that the migration ability of cells is significantly decreased after ITGB1 knockout;MTT and plate colony formation assay showed that the ability of cells was significantly attenuated after ITGB1 knockout;Coomassie blue staining and scanning electron microscopy showed that after ITGB1 knockout,the microfilament pseudopods of the cells were significantly reduced,and the degree of malignancy was significantly reduced;DAPI staining experiments showed that after ITGB1 knockdown,SGC7901 cells had a tendency to apoptosis,but there was no statistical difference.4.Bioinformatics results indicate that the ITGB1 gene has been mutated in many tumors,and mutations have occurred in the genome and mRNA levels of gastric cancer.The ITGB1 mutation has significantly reduced the survival rate of gastric cancer patients;the genes associated with ITGB1 and their interactions are predicted.The network diagram laid the theoretical foundation for the further study of ITGB1.Conclusion:Using CRISPR/Cas9 technology,the relevant behavior of gastric cancer cells was affected after successful knockdown of the ITGB1 gene.ITGB1 can significantly promote the malignant behavior of gastric cancer cells such as Viability,Proliferation,Motility,etc.ITGB1 can significantly enhance the development of motor-related micro-structures of gastric cancer cells;ITGB1 is associated with apoptosis of gastric cancer cells but has little effect on apoptosis.Bioinformatics results indicate that ITGB1 has variability in genomic and mRNA levels in gastric cancer;ITGB1 mutations significantly reduce survival in patients with gastric cancer.In summary,ITGB1 plays an important role in the role of cancer-promoting genes in the development and progression of gastric cancer,and participates in the process of proliferation,migration and apoptosis of-gastric cancer cells,and can be used as a new therapeutic target for gastric cancer.