The Mechanism And Significance Of USP7 Stabilizing Ki-67 In NSCLC

Background Usp7 as a deubiquitinating enzyme plays a certain role in the occurrence and development process of tumor; ki67 as a marker of cell proliferation, also participate in the development and metastasis of tumor. In non small cell lung cancer,we found that usp7 can regulate ki-67 to promote the development of cancer.Methods 1. Immunohistochemical method was used to detect the expression of usp7 in normal lung tissues and NSCLC tissues; western blot and real-time PCR methods were used to detect the protein and m RNA expressions of usp7 in normal lung epithelial cell Beas2 B and NSCLC cells; the proliferation and survival state of NSCLC cells was detected by CCk8 and AO-EB double fluorescence staining methods after the application of P22077 and transfection of usp7 si RNA plasmid. 2. Western blot and CIF methods were used to detect mcm2, pcna and ki67 protein expression after the application of P22077 and transfection of usp7 si RNA plasmid; CIF method was used to detect the protein expression of ki67 in normal lung epithelial cell Beas2 B and NSCLC cells; immunohistochemical method was used to detect the expression of ki67 in normal lung tissues and NSCLC tissues; the proliferation of A549 cells was detected by subcutaneous transplantation tumor in nude mice after the transfected usp7 si RNA plasmid. 3. The m RNA expression of ki67 was detected by Real-time PCR in normal lung epithelial cells Beas2 B and NSCLC cells and after the application of P22077 and transfection of usp7 si RNA plasmid; CIF was used to detect the localization of usp7 and ki67 in cells; using MG132 and transfection of usp7 c223 a mutant plasmid tofurther study the regulatory mechanism of usp7 on ki67;through the CCK8 experiment,drug sensitivity was tested after transfection of usp7 si RNA plasmid.Results 1. Usp7 overexpressed in the nucleus of NSCLC tissues, and the lower degree of differentiation of the tissue, the higher usp7 expressed; protein and m RNA expression of usp7 was higher in NSCLC cells than Beas2B;the proliferation decreased but cell still alive after the application of P22077 and transfection of usp7 si RNA plasmid. 2. Using P22077 and transfection of usp7 si RNA plasmid,mcm2 and pcna protein expression almost unchanged, but the expression of ki67 protein decreased; protein expression of ki67 was higher in NSCLC cells than Beas2B; in the NSCLC tissues, ki67 also highly expressed in the nucleus, and the lower degree of differentiation of the tissue, the higher ki67 expressed; after the transfection of USP7 si RNA plasmid,the tumor volume was significantly less than the control group and NC group. 3. Ki67 m RNA remained unchanged in Beas2 B and NSCLC cells,also after the application of P22077 and transfection of usp7 si RNA plasmid; usp7 and ki67 were co-localized in NSCLC cells; after the application of MG132, the inhibition effect of P22077 and transfection of usp7 si RNA plasmid on the protein expression of ki67 disappeared; after usp7 c223 a mutant plasmid overexpressed in A549, ki67 protein expression decreased;after transfection of usp7 si RNA plasmid, drug sensitivity didn’t change.Conclusions 1. Usp7 overexpressed in NSCLC tissues and cells, and inhibiting the activity of usp7 or down-regulation of usp7 expression could slow down the cell proliferation, but cell still remained alive. 2. Usp7 could regulate the expression of ki67 protein, and ki67 overexpressed in NSCLC tissues and cells;down-regulation of usp7 the tumor volume was significantly less than the control group and NC group, and ki67 expression was also down-regulated. 3. In NSCLC cells, usp7 regulated the stability of ki67 through deubiquitination.