The Effect Of ?-ENaC On Type ? Alveolar Epithelium Cells Of Rat Proliferation And Migration

BackgroundEpithelial sodium channel?ENaC?,is a member of the amiloride-sensitive,non-voltage gated ion channel degradation protein family,heterologous polymeric proteins composed of four subunits??,?,?and?subunit?,which main physiological function is to transfer epithelial sodium ions uniaxially across epithelial cells connected closely,regulating water and ion transport.Type?alveolar epithelial cells is considered as stem cells in adult lung,which plays an important role in lung injury repair and?subunit is widely distributed in type?alveolar epithelial cells.Studies have shown that?subunit participate in regulating the development of the organism and the occurrence of diseases,such as cell proliferation,cell differentiation and the occurrence of pulmonary edema.But the effect and molecular mechanism of?subunit on type?alveolar epithelial cells is still unclear.Objective?-ENaC gene knockout rat type?alveolar epithelial cell line?L2 cell line?was generated by CRISPR/Cas9 gene engineering technology combined with SSA-RPG report vector and the changes of cell proliferation vitality,migration ability and gene expression profile was detected.To illuminate the effect and function of?subunit on L2 cell line.Methods1.Three groups of guide RNA targeting the first exon of?-ENaC gene were designed and synthesized using online analysis software?http://benchling.com?,and cloned into the CRISPR/Cas9 expression vector skeleton,respectively.At the same time,three groups of matched SSA-RPG report vectors containing target sites were designed and constructed,which were used as the target site simulation system free from the cell genome to evaluate the positive rate of cells and screen positive cells by repairing the purinomycin resistance gene on the report vector.The Lipofectamine^TM 2000 Reagent was used to co-transfect 3groups of CRISPR/Cas9 expression vector and SSA-RPG report vector into HEK293T cells,respectively.The most efficient CRISPR/Cas9 expression vector and SSA-RPG report vector were selected by flow cytometry.2.Then the Lipofectamine^TM 2000 Reagent was used to co-transfect the best efficient group of CRISPR/Cas9 expression vector and SSA-RPG report vector into L2 cells.After48 h,purinomycin was added into the cells with the concentration of 3 ug/mL and monoclonal cell lines with purinomycin resistance were obtained after 5 days.Western Blot was used to detect the expression level of?-ENaC protein in the monoclonal cell lines with purinomycin resistance.Furthermore,?-ENaC gene knockout mutant cell lines were detected through the sequencing.3.The proliferation vitality of the mutant cell line was detected by cck-8 and EdU staining,and the migration ability of the mutant cell line was detected by scratch test.4.Finally,the effect of?-ENaC gene knockout on the gene expression profile was determined by RNA sequencing.Results1.Three groups of CRISPR/Cas9 expression vectors targeting the first exon of?-ENaC gene and the matched SSA-RPG report vectors were successfully generated.The activity of 3 groups of CRISPR/Cas9 system was detected on HEK293T cells.The highest active?-ENaC-3/Cas9 expression vector and the macthed RPG/?-ENaC-3 reporting vector were transfected into L2 cells.2.8 single cell clones were obtained after purinomycin screening.Western Blot results showed that the expression level of?-ENaC protein was decreased in 2 single cell clones,while the expression of?-ENaC protein was completely absent in one single cell clone.Sequencing results showed that 2 single allele mutant cell lines and 1 biallele mutant cell line were obtained after purinomycin screening.3.CCK-8 test and EdU staining test showed that the proliferation vitality of mutant cell lines was decreased?P<0.05?,and the biallelic mutant cell lines was decreased more significantly than single allele mutant cell lines?P<0.05?.The results of the scratch test showed that the migration ability of the mutant cell line was decreased?P<0.05?,and the migration ability of the biallele mutant cell line was decreased more significantly single allele mutant cell lines?P<0.05?.4.RNA sequencing results between the double-allele mutant L2 cell line and wild L2cell line showed that a total of 514 genes were altered after?-ENaC gene knockout,among which 168 genes were up-regulated and 346 genes were down-regulated.Functional analysis showed that differential genes affect biological processes such as inflammatory response,cell adhesion,signal transduction,cell differentiation and cell proliferation through signaling pathways such as cytokine-cytokine receptor interaction,systemic lupus erythematosus,ECM receptor interaction,and nerve ligand-receptor interaction.And further analysis indicated that Cxcl1,Cxcl12,Tlr2,Tlr4,Il33,Bmp6 and Mmp9 may play a key role in the progress of cell proliferation and migration mediated by?-ENaC gene.Conclusions?-ENaC gene knockout rat L2 cell line was generated successfully in our study.The proliferation vitality and migration ability of rat L2 cell line were significantly inhibited after?-ENaC gene knockout.The differentially expressed genes caused by?-ENaC gene knockout are mainly involved in biological processes such as inflammatory response,cell adhesion,signal transduction,cell differentiation and cell proliferation,and influence signaling pathways such as cytokine-cytokine receptor interaction,systemic lupus erythematosus,ECM receptor interaction,and nerve ligand-receptor interaction.