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The effect of methylated oligonucleotide targeting Ki-67 gene in human 786-0 renal carcinoma cells
Xue-Qin Li1, Dong-Sheng Pei, Guo-Wei Qian
1Jiangsu Key Laboratory of Biological Cancer Therapy, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, 221002, Jiangsu, People's Republic of China.
Abstract:
To investigate the effect of methylated oligonucleotide (MON) targeting Ki-67 promoter on the expression of Ki-67 gene and the proliferation and apoptosis of the human 786-0 renal carcinoma cells, human 786-0 cells were transfected with MON. The activity of Ki-67 promoter was detected by dual-luciferase reporter assay system. Among the five methylated oligonucleotides (MON(1)-MON(5)), MON(4) is the best excellent one in the inhibition of the Ki-67 promoter activity. The activity of Ki-67 promoter is decreased to 77.88% in 40-nM group, 50.07% in 80-nM group, 35.63% in 120-nM group, 26.09% in 160-nM group, and 16.98% in 200-nM group compared with 0-nM group. The activity of Ki-67 promoter in MON group is decreased to 61.96% at 8 h, 48.93% at 12 h, 15.97% at 24 h, 26.00% at 36 h, 35.01% at 48 h, 46.08% at 72 h, and 66.12% at 96 h compared with pGLBK235 group. These results show that the effect of MON is time- and dose-dependent. The activity of Ki-67 in MON group is decreased to 16.73% compared with pGLBK235 group, while the control groups have no significant difference. The expression of Ki-67 gene in 786-0 cells was detected by RT-PCR and immunohistochemistry, respectively. The expression of Ki-67 mRNA is decreased to 61.04% and that of Ki-67 protein is decreased to 32.07% in MON group compared with the blank group. The proliferation of 786-0 cells was determined by WST-8. The cell proliferation in MON group is decreased to 61.02% at 24 h, 73.78% at 48 h, 79.72% at 72 h, and 91.53% at 96 h compared with the blank group. The cell apoptosis was measured by annexin V and propidium iodide. The number of apoptosis cells in MON group is 2.42 times of that in the blank group at earlier period and 2.57 times at mid-anaphase. We detected the effect of MON on the expression of bax and p53 by Western blot. Compared with the blank group, the expression of bax protein in MON group is increased by 66.12%, while the expression of p53 is decreased to 67.31%. Our study demonstrates that the methylated oligonucleotide targeting Ki-67 promoter has a remarkable effect on the inhibition of Ki-67 expression and the proliferation of the human 786-0 renal carcinoma cells and can induce apoptosis of the 786-0 cells.
Insights
Methylated oligonucleotide (MON) targeting the Ki-67 promoter effectively inhibits human renal carcinoma cell proliferation and induces apoptosis. This study demonstrates MON
Area of Science:
- Molecular Biology
- Cancer Research
- Oligonucleotide Therapeutics
Background:
- The Ki-67 gene is a critical marker for cell proliferation and is often overexpressed in various cancers, including renal carcinoma.
- Targeting oncogenic gene promoters with specific oligonucleotides offers a potential therapeutic strategy for cancer treatment.
- Methylated oligonucleotides (MONs) present a novel approach for gene silencing by interfering with promoter activity.
Purpose of the Study:
- To evaluate the efficacy of a methylated oligonucleotide (MON) targeting the Ki-67 promoter in inhibiting Ki-67 gene expression.
- To assess the impact of MON on the proliferation and apoptosis of human 786-0 renal carcinoma cells.
- To determine the dose- and time-dependent effects of MON on Ki-67 promoter activity.
Main Methods:
- Human 786-0 renal carcinoma cells were transfected with various concentrations of MON.
- Dual-luciferase reporter assays were employed to measure Ki-67 promoter activity.
- Quantitative RT-PCR, immunohistochemistry, WST-8 assay, and flow cytometry (Annexin V/propidium iodide staining) were used to assess gene expression, cell proliferation, and apoptosis, respectively.
- Western blot analysis was performed to evaluate the expression of bax and p53 proteins.
Main Results:
- MON significantly inhibited Ki-67 promoter activity in a dose- and time-dependent manner, with MON(4) showing the most potent effect.
- MON treatment led to a substantial decrease in Ki-67 mRNA and protein expression in 786-0 cells.
- Cell proliferation was significantly reduced, while apoptosis was markedly increased in MON-treated cells compared to controls.
- MON treatment modulated the expression of apoptosis-related proteins, increasing bax and decreasing p53.
Conclusions:
- Methylated oligonucleotide targeting the Ki-67 promoter is a promising strategy for inhibiting the proliferation of human 786-0 renal carcinoma cells.
- MON effectively suppresses Ki-67 gene expression and induces apoptosis in renal carcinoma cells.
- The findings support the potential of MON as a therapeutic agent for renal carcinoma.
