Screening Of MDCK Cell Tumorigenesis-related Proteins And Researching The Role Of The Protein Mechanism

The MDCK cell line was established by Madin and Darby in 1958 from the kidney of the American Cocker Spaniel female spaniel.It is usually an epithelioid cell that grows in an adherent manner.Due to its high viral infection efficiency,adaptation and serum-free growth Due to the conditions,rapid proliferation,and resistance to mutation,the MDCK cell line is recognized as one of the most suitable cell lines for influenza virus vaccine production.Due to the tumorigenicity of MDCK cells,the safety of using them to produce vaccines has always been controversial.In recent years,our research group has obtained several MDCK cell lines with low tumorigenicity through single-cell clonal culture and screening,which were combined with the existing high tumorigenic cell lines in the laboratory for high-throughput analysis.Some classic tumor pathways are activated by target genes.Among them,lnc RNA MSTRG.1056.2directly regulates ERBB3 to activate the PI3K-Akt pathway,which promotes the occurrence of tumors.In the long run,in order to ensure the safety of the vaccine,the development of a low tumorigenic MDCK cell line can not only reduce the difficulty of the downstream purification process,but also fundamentally reduce the risk of vaccination after it is used to produce influenza vaccine.The use of genetic engineering methods to specifically transform the tumorigenicity of MDCK cells to construct a stable tumor-free MDCK cell line is more in line with the industrialization needs of modern vaccines.Finding the main tumorigenic proteins of MDCK cells is the key to the problem.At present,there are few reports on the protein and tumorigenesis mechanism that effectively exert the tumorigenesis function on this cell.In this study,the quantitative proteomics analysis technology of DIA(Data-independent Acquisition)data-independent acquisition technology is used to combine the MDCK adherent cells introduced by ATCC in our laboratory and the low tumorigenic MDCK monoclonal that have been prepared and screened by the limiting dilution method.The protein expression of the cell line was compared,and the proteins on the cell that may be related to tumor formation were screened,and then the DCLK1 and TFPI2 proteins,which have a large difference in multiples and may play an important role in cell tumor formation,were selected by RNA interference technology A cell line with low expression of DCLK1 and a cell line with overexpression of DCLK1 were constructed,and the tumor-forming function of different tumor-forming MDCK cells in vitro was analyzed,and the role of DCLK1 in the tumor-forming process of MDCK cells was studied,in order to establish genetic engineering without tumor formation.The MDCK cell line was screened for effective target genes.The results of the study found that:1.Taking high tumorigenic MDCK cells as a control,there are 163 significantly different proteins in low tumorigenic cells,of which 93 are up-regulated and 70 are down-regulated.2.Using highly tumorigenic MDCK cells as a control,classify the differential proteins according to the molecular function of the gene,the location of the cell and the biological process that the gene participates in,so as to predict the biological function they may have,and clarify The main signal transduction pathways and metabolic pathways that the protein participates in are different proteins.At the same time,the analysis of different proteins in the living body coordinated with each other to perform biological behaviors,and obtained the following 7proteins on MDCK cells that may be related to tumorigenesis: DCLK1,TFPI2,and TFPI2.ATXN1,COL1A2,EPHB2,DNAJC6,UPK3 A,the differential expression of the above seven proteins in different tumorigenic MDCK cells was preliminarily verified by RT-PCR,and the results were consistent with the DIA sequencing results.The Western blot verified that DCLK1 and TFPI2 are in The differences in different tumorigenic MDCK cells showed that the differential expression results of DCLK1 and TFPI2 in different tumorigenic MDCK cells were consistent with the results of DIA sequencing.3.Using RNA interference technology,through lentivirus infection,construct MDCK cells with low DCLK1 expression and DCLK1 overexpressing cell lines.RT-PCR and Western blot results confirmed the successful establishment of MDCK cell lines with DCLK1 interference expression.4.Observe the cell morphology and analyze the effect of interfering DCLK1 on the proliferation of MDCK cells by CCK8 method.It is found that interfering with DCLK1 does not change the morphology of MDCK cells and does not significantly change the viability of MDCK cells.5.Plate clone formation experiment showed that when DCLK1 was knocked down,the clone formation rate of high tumorigenic MDCK cells decreased,and the clone formation rate of non-tumorigenic MDCK cells increased when DCLK1 was overexpressed.6.The results of scratch healing experiment and Transwell experiment showed that knocking down DCLK1 significantly reduced the migration and invasion ability of high tumorigenic MDCK cells.On the contrary,overexpression of DCLK1 significantly promoted the migration and invasion ability of low tumorigenic MDCK cells.7.RT-PCR results show that knocking down DCLK1 will change the regulation of ATXN1,EPHB2 and UPK3 A in the tumorigenesis signal pathway of MDCK cells,thereby determining the positive regulation of DCLK1 on the tumorigenesis of MDCK cells.