Prokaryotic Expression Of Cold Shock Protein DbpA And The Correlation Of Its Regulatory Gene GPR126 In Colorectal Cancer

Objective: The recombinant protein of dbpA(DNA binding protein A)was prepared by prokaryotic expression method,and the monoclonal antibody against dbpA was further prepared,which lays a foundation for the study of dbpA transformation.The regulatory gene of dbpA adhering to G protein coupled receptor(GPR126)was screened by bioinformatics analysis,and its expression trend in colorectal cancer(CRC)and its correlation with dbpA expression were detected,so as to find the related signal pathway and explore its mechanism,so as to provide new ideas for early diagnosis and drug targeted therapy of colorectal cancer.Methods:1.The specific sequence of dbpA was extracted from Uniport database,and the recombinant plasmid dbpA was synthesized.the recombinant dbpA protein was expressed in prokaryotic expression system and its expression conditions were optimized.2.After dbpA silencing using DNA microarray technology,the team reintegrated the data of differential genes obtained from human colorectal cancer SW620 cells,drew a volcanic map,screened out the genes expressed as membrane proteins for follow-up experiments,and analyzed the differential genes by GO enrichment analysis and KEGG enrichment analysis.3.Specimens of cancer tissues and paracancerous tissues after colorectal cancer surgery in Shaanxi Provincial people’s Hospital from February 2017 to April 2017 and from January 2021 to January 2022 were collected.UALCAN-TCGA database,real-time fluorescence quantitative PCR(RT-q PCR),Western blot and immunohistochemistry(IHC)were used to detect the expression of GPR126 in colorectal cancer tissues,and the experimental data were statistically analyzed.Results:1.The recombinant dbpA protein about 19 KD was successfully expressed by prokaryotic expression system,and the best induction conditions were 25℃,180 rpm,IPTG concentration: 0.4m M,overnight induction.2.Among the 575 differentially expressed genes in SW620 cells,GPR126,RBSN and PRKD3,which may be regulated by dbpA and expressed as membrane proteins,were screened.GO entry analysis involves cadherin binding involved in cell adhesion.KEGG enrichment analysis included MAPK,Wnt and PI3K/Akt signal pathways.3.(1)UALCAN-TCGA database: the expression of GPR126 at m RNA level was significantly increased in 41 cases of colorectal cancer normal tissues and 286 cases of colorectal cancer tissues.Combined with pathological data,the expression of GPR126 increased gradually with the increase of stage of colorectal cancer,in addition,the expression of GPR126 in colorectal cancer was significantly higher than that in women,and survival analysis showed that the increased expression of GPR126 was related to the poor prognosis of female patients with colorectal cancer.It is suggested that the increased expression of GPR126 may be related to the progression of colorectal cancer and the prognosis of female patients.(2)RT-q PCR and Western blot of GPR126: in 8 cases of fresh frozen tissues of colorectal cancer,87.5%(7 of 8 cases)expressed GPR126 m RNA and protein,and the expression in colorectal cancer was higher than that in adjacent tissues(P = 0.017 and P = 0.018).(3)RT-q PCR and Western blot of dbpA: in 8 fresh frozen tissues of colorectal cancer,87.5%(7 of 8 cases)and 75%(6 of 8 cases)were positive for dbpA m RNA and protein,respectively.Compared with adjacent tissues,the expression of dbpA m RNA and protein in colorectal cancer was higher(P = 0.045 and P= 0.048).(4)IHC of GPR126 and dbpA: in 50 cases of colorectal cancer and 20 cases of paracancerous tissues,GPR126 and dbpA were mainly located in the cytoplasm of colorectal cancer cells,and the positive expression rates of GPR126 in cancer tissues and paracancerous tissues were 68% and 35%,respectively.In terms of clinicopathological data,the expression of GPR126 and dbpA was related to the clinical stage of TNM,and the expression of dbpA was also related to the depth of invasion(P < 0.05),but GPR126 and dbpA were not related to age,sex,tumor size,tumor location,histological grade,lymph node metastasis and distant metastasis.It is suggested that the increased expression of GPR126 and dbpA is related to the occurrence and progression of colorectal cancer.(5)Correlation analysis: Spearman correlation analysis showed that the m RNA and protein expression of GPR126 and dbpA were positively correlated(r =0.905,P = 0.002 and r = 0.341,P = 0.015),suggesting that the expression of GPR126 and dbpA is related to the development of colorectal cancer.Conclusion:1.The recombinant dbpA protein about 19 KD was successfully prepared by prokaryotic expression system,which provides conditions for the preparation of dbpA monoclonal antibodies in the later stage.2.The genes GPR126,RBSN and PRKD3 which are suitable as drug targets,which are expressed as membrane proteins,were screened.According to the results of GO enrichment analysis and KEGG enrichment analysis,it is speculated that the effect of dbpA on colorectal cancer is related to PI3K/Akt and EMT signal pathway.3.The expression of GPR126 and dbpA in colorectal cancer is significantly increased and is related to the progression of colorectal cancer.And the two are related in the occurrence and development of colorectal cancer.