Cyclin D1, CDK4, P16, And Rb Gene Expression During Arsenic-induced Malignant Transformation Of Human HaCaT Cells And Their Significance

Objective: To investigate the expression levels of cyclin D1, CDK4, P16, and Rb gene during arsenic-induced malignant transformation of HaCaT cells and their significance.Methods: Before and after long-term application of low concentration of arsenic (100nM) for the culture of HaCaT cells for 20 weeks, morphological changes of the cells were observed, Soft agar colony formation assay was used to observe the monoclonal formation, and the expression level of MMP-9 mRNA was detected by Real Time-PCR, so as to construct arsenic-induced HaCaT malignant phenotype cells. At different time points (0 and 6 weeks, 15 weeks, 20 weeks) during arsenic-induced malignant transformation of HaCaT cells, the expression levels of cyclin D1, CDK4, P16, and Rb gene were detected by Real Time-PCR.Results:1. Arsenic-induced HaCaT malignant phenotype cells were successfully constructed.Morphological changes were observed in the HaCaT cells cultured with low concentration of arsenic (100nM) for 20 weeks: single cell boundary was unclear, being varied shapes, poor stretching, cells became Small, multinucleated giant cells being seen frequently . The monoclonal formation rate in the 20 weeks arsenic treated group (0.67‰) was significantly higher than that in the control group (0.16‰) by soft agar colony formation assay(P﹤0.05). Expression level of matrix metalloproteinase -9 (MMP-9) mRNA in the 20 weeks arsenic treated group was significantly higher than that in the control group by Real Time-PCR.2.Gene expression changes of CDK4/cyclinD1/p16/Rb pathway.(1) P16: Compared with the control group, P16 mRNA expression levels in the 6 weeks, 15 weeks, 20 weeks arsenic treated group were significantly higher (P﹤0.05). The highest level of P16 mRNA was in the 6 weeks and deceased in 15weeks and 20 weeks.(2) Rb: Compared with the control group, Rb mRNA expression levels in the 6 weeks, 15 weeks, 20 weeks arsenic treated group were significantly higher(P﹤0.05). The highest level of Rb mRNA was in the 6 weeks and deceased in 15weeks and 20 weeks. but still higher than the control group(P﹤0.003,P﹤0.004).(3) CDK4: CDK4 mRNA expression levels in the 6 weeks, 15 weeks, 20 weeks arsenic treated group were all significantly higher than that in the control group(P﹤0.05). CDK4 mRNA expression level increased in the 6 weeks and deceased in 15weeks, but increased again in 20 weeks. The highest level of CDK4 mRNA was in the 20 weeks.(4) cyclin D1: cyclin D1 mRNA expression levels in the 6 weeks, 15 weeks, 20 weeks arsenic treated group were all significantly higher than that in the control group(P﹤0.05). The highest level of cyclin D1 mRNA was in the 6 weeks and deceased in 15weeks and increased again in 20 weeks.Conclusion:1. Treated with low concentration of arsenic (100nM) for 20 weeks, human HaCaT cells can transform into invasive malignant phenotype cells. The single cell boundary is unclear, being varied shapes, poor stretching, multinucleated giant cells being seen frequently . The monoclonal formation rate increases. Expression level of matrix metalloproteinase -9 (MMP-9) mRNA significantly increases.2. Gene expression of p16-cyclin D1/CDK4-pRb pathway changes during arsenic-induced malignant transformation of human HaCaT cells.3. In the process of malignant transformation of HaCaT cells, p16, cyclinD1, CDK4 and Rb mRNA increase significantly in the early stage, Which means the gene expression changes of p16-cyclin D1/CDK4-pRb pathway is an early molecular event.4. At different time points (0 and 6 weeks, 15 weeks, 20 weeks) during arsenic-induced malignant transformation of HaCaT cells, the expression levels of cyclin D1, CDK4, P16, and Rb gene change differently, which suggest their function may be different in cell proliferation, differentiation and malignant transformation. The study provides new clues for the early diagnosis, prevention and screening therapeutic targets of skin cancer.