ABSTRACT
Objective:RNA interference technology (siRNA) was used to inhibit the expression of DJ-1 gene in lung squamous cell carcinoma HTB-182 cells, then, tandem affinity purification mass spectrometry (TAP-MS) was performed to screen the interacting proteins of DJ-1 in lung cancer cell line of HTB-182.Methods:The siRNA lentivirus vector targeting DJ-1 gene was constructed to infect HTB-182 cells (DJ-1 siRNA group), and the lentivirus vector control group (control siRNA group) and blank control group were established. The expression level of DJ-1 protein was detected by Western blot, and the endogenous DJ-1 protein silenced si-DJ-1-HTB-182 cells were established. The specific primers of DJ-1 were designed, and the DJ-1 expression plasmid pNTAP-DJ-1 with streptomycin binding peptide label (SBP) and calmodulin binding peptide label (CBP) was constructed. The cell line DJ-1 siRNA HTB-182 was stably transfected with liposome, and the positive clones were screened by G418. The positive clones were verified by Western blot, and the interacting proteins of DJ-1 were found by TAP-MS.Results:The protein expression of DJ-1 in DJ-1 siRNA interference group was significantly lower than that in empty plasmid group and blank control group ( P<0.05); HTB182 cell line stably expressing pNTAP-DJ-1 plasmid was successfully constructed; Three proteins interacting with DJ-1 were screened by TAP-MS: cytokeratin 1 (keratin 1), cytokeratin 10 (keratin 10) and NADPH oxidase activating protein P47 (P47 Px). Conclusions:Keratin 1, Keratin l0 and P47 Px protein may be DJ-1 interactions protein.
ABSTRACT
OBJECTIVE@#RNA interference technology (siRNA) was used to inhibit the expression of DJ-1 gene in lung squamous cell carcinoma SK-MES-1 cells, and the cell biological behaviors were investigated to explore the function of DJ-1 gene.@*METHODS@#A targeted DJ-1 siRNA lentiviral vector with a green fluorescent protein (GFP) as a reporter was constructed. The constructed DJ-1 siRNA and control-siRNA vectors were infected into SK-MES-1 cells as experimental (DJ-1 siRNA) and control (Control siRNA) groups, respectively. The DJ-1 protein expression was determined by Western blot. The cell proliferation capability was measured with methyl thiazolyl tetrazolium (MTT). The cell cycle was analyzed by flow cytometry. The capability of cell migration was determined by Transwell method.@*RESULTS@#Compared with control-siRNA and blank-control groups, the protein expression of DJ-1 gene was down-regulated, the capability of cell proliferation was obviously inhibited (P<0.01), the cell cycle was arrested with increased number of G1- and G2-phase cells and reduced number of S-phase cells, and the capability of cell migration was significantly decreased (P<0.01) in the DJ-1 siRNA-infected cells.@*CONCLUSION@#DJ-1 gene might play a role in promoting cell proliferation and cell migration capability in vitro in lung cancer SK-MES-1 cells.