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Archive > Volume 23 Issue 2 > 2016,23(2):243-249. DOI:10.3872/j.issn.1007-385X.2016.02.015 Prev Next
Basic Research
Effects of silenced CDK2 gene mediated by lentiviral vector on biological behavior of melanoma B16-F1 cells
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Abstract:
Objective:To study the effect of silencing CDK2 gene induced by the recombinant lentiviral vector pUL-CDK2-shRNA 3 (CDK2-shRNA) on the malignant biological behavior of mouse melanoma B16-F1 cells, and preliminarily investigate its mechanism.Methods: Melanoma B16-F1 cells were transfected by recombinant lentivirus vector CDK2-shRNA, and effect of which on proliferation of the B16-F1 cells was examined by MTT assay. Apoptosis of the cells was detected by DAPI staining and AnnexinV-FITC/ PI staining flow cytometry assay. Cell cycles were evaluated with flow cytometry assay. Cell adhesion assay and transwell chamber experiment were used to detect the changes of adhesion, invasion and migration abilities of the cells, respectively. Expression levels of proteins RB and pRB on signaling pathway, cell cycle related transcription factor E2F1 and migration related protein MMP-2 and MMP-9 were detected with Western blotting assay. Results: Compared with the blank control and negative control groups, relative proliferation and adhesion rates, migration and invasion abilities of the cells in the CDK2-shRNA group significantly decreased (P<0.05); while the apoptosis rate significantly increased (P<0.05); Number of the cells at G1 phase significantly increased, but number of the cells at S and G2 phases significantly decreased (P<0.05); expression of pRB and E2F1 proteins significantly decreased, but expression of RB significantly increased (P<0.05), which were related with cell cycles; and expression of MMP-2 and MMP-9 proteins for invasion and migration of the cells significantly decreased (P<0.05). Conclusions: Silencing CDK2 gene mediated by recombinant lentiviral vector pUL-CDK2-shRNA could significantly inhibit malignant biological behavior of the B16-F1 cells, and its mechanism might associate with the expression changes of the proteins for cell cycles, invasion and migration of the cells.
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Project Supported:
Project supported by the Henan Provincial Since-Technology Program (No.122300410193), and the Henan Provincial Program for Key Members of Young Teacher of the Higher Education Institutions (No.2011GGJS-262)
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