Use CRISPR Technology To Study The Function Of Rgnef In The Occurrence And Development Of Gastric Cancer

Background and Objective:Gastric cancer is one of the most common malignant tumors of the digestive system.The incidence and mortality rates of gastric cancer is higher in China,and due to the lack of specific clinical manifestations in early,and it is difficult to detect,So,gastric cancer diagnosed usually have entered the middle-later period,with the invasion and metastasis.At present,surgery,chemotherapy,radiotherapy were the main treating methods of gastric cancer,but the overall effect is not ideal,the metastasis rate and the recurrence rate is very high,the 5 years survival rate as low as 20%?30%.pathogenesis of gastric cancer is one of the many long-term gene mutation accumulation,leading to a series of gene expression and signal transduction pathway become deferentially regulated.Therefore,study the function of the associated genes in oncogenesis and oncodevelopment in gastric cancer is still an important part of the basic theory of the pathogenesis of gastric cancer,which has important significance for developing a new technology in diagnosis and treatment of gastric cancer in the future.ARHGEF28(Rho guanine nucleotide exchange factor 28),also known as:Rgnef,p190RHOGEF,RIP2,is a protein encoding gene,located on chromosome 5q13.2315836bp,contains 44 exons and 45 introns.Due to different splicing of genes,there are fourteen transcription variants totally,the longest one's mRNA is 6369bp,ORF(open reading frame)is 5196bp in length,containing 36 exons.Rgnef gene is a member of Rho Rho guanine nucleotide exchange factors(RhoGEFs).It has a binding site with FAK(Focal Adhesion Kinase)in its molecular structure.FAK could phosphorylate Rgnef and regulate the activity of RhoGTPase,which may be closely related to the occurrence and development of cancer.In recent years,the rise of CRISPR(Clustered Regularly Interspaced Short Palindromic Repeas)gene editing system,was developed from the bacteria and archaea defense system.The Cas9(CRISPR-associated nuclease 9)technology is the most commonly using and its principle is editing gene accurately in target site by using Cas9 enzyme guided with sgRNA.It has the advantages of low cost,simplicity,and more specificity and efficiency.It will achieve the target gene knockout.So,it become the most popular technique in the life science research since it was founded.In order to study the function of Rgnef during the oncogenesis and tumor development accurately,we used CRISPR/Cas9 system to establish Rgnef-/-SGC7901 cell line,and then evaluat the role of the gene in the oncogenesis and development of gastric cancer through a series of methods of cell behavior and fate detection.Method:1.Analyse the relevant information of Rgnef gene in NCBI database,find common ORF from the different transcriptional variants,analyze the ORF and select the appropriate exons.Design sgRNA online.(Website 1:http://crispr.mit.edu,Website 2:http://www.broad institute.org/rnaive/publicanalysis-tools/sgrna-desig)2.Anneal and phosphorylate the sgRNAs,digest the PX459 plasmid with Bbs I and purified by gel,then the Cas9 vector for Rgnef gene was constructed.3.The vector was transfected into SGC7901,and identify the activity more than 25%of the vector by T7E1.The monoclonal cell line was isolated through puromycin screening and cell dilution cloning.Selected the Rgnef +/-SGC7901 and Rgnef-/-SGC7901 cell lines after sequencing.Lastly,confirmed the success by immunoblotting.Detected by Western blot and extended culture and cryopreservation.4.The behavior and morphology of WT?Rgnef +/-SGC7901 and Rgnef-/-SGC7901 cell lines was investigated using MTT assay,Plate cloning assay,the wound healing assay,Transwell assay,Coonmassie blu staining assay,scanning electron microscope,flow cytometry,and DAPI fluorescence detection.At the last,appraising the function of Rgnef.Result:1.The sgRNA-Cas9 vector targeting Rgnef was successfully constructed,and the activity of the recombinant vector was more than 25%by T7E1 assay.2.The Rgnef +/-SGC7901 and Rgnef-/-SGC7901 cell lines were successfully constructed.3.The results from MTT assay,Plate cloning assay,the wound healing assay,Transwell assay,Coonmassie blu staining assay,scanning electron microscope,flow cytometry,and DAPI fluorescence detection showed that the proliferation,migration,invasion and motility of the cells were significantly inhibited after Rgnef gene knockout,and the cells were blocked in G1 phase,but no obvious apoptosis was observed.Conclusion:The Rgnef +/-SGC7901 and Rgnef-/-SGC7901 cell lines successfully constructed by using CRISPR/Cas9 system compared with WT cells beyond the experimental results show that Rgnef can significantly promote tumor cell's malignant behavior such as proliferation,migration,invasion and motility,and it can reinforce the development of related micro structure,Rgnef gene can induce cancer cell cycle in G1,but no obvious apoptosis effect.The results support the role of Rgnef in gastric cancer as a oncogene.The possible signaling pathway of this gene is also preliminarily analyzed,and the regulation of its specific pathway remains to be further studied.