[Inhibition of evi1 expression by siRNA in HEL cell line]
Pu Zhang1, Kai-Lin Xu, Bing DU
1Department of Hematology, Xuzhou Medical College Affiliated Hospital, Xuzhou 221002, Jiangsu Province, China.
Abstract:
The aim of study was to investigate the inhibitory effect of small interfering RNA on evi1 gene expression and biological characteristics in HEL cells and its mechanism. 3 siRNA (siRNA-1, siRNA-2, siRNA-3) specific for evi1 gene were synthesized and transfected into HEL cells in vitro. Experiments were divided into test and control groups. MTT method was used to assay the inhibitory effect of siRNA on cell proliferation; semiquantitative RT-PCR was used to detect the expression of evi1 gene mRNA; the cell viability was determined by trypan blue dye test; the change of cell cycle and apoptosis of cells were analyzed by flow cytometry. The results showed that siRNA-1 had strongest effect, and inhibitory effect was most obvious at 48 hours after transfection. When the concentration of siRNA raised to 120 nmol/L, the inhibitory rate reached to the peak. The inhibitory rate of siRNA-1 on proliferation of HEL cells, relative expression level of evi1 mRNA and cell viability at 48 hours after transfection were 72.22 ± 2.80%, 27.31 ± 1.11% and 26.05 ± 2.49%, which had significant difference from other groups (p < 0.001). The siRNA resulted in arrest of cell cycle at G(0)/G(1) phase, the cell amount at S phase obviously decreased, the apoptotic rate of HEL cells obviously increased (p < 0.01). It is concluded that the siRNA specific for evi1 gene can suppress the proliferation of HEL cells, reduce the expression of evi1 mRNA, decrease the cell viability, arrest the cell cycle at G(0)/G(1) phase, suppress cell mitosis, and promote cell apoptosis.
Insights
Small interfering RNA (siRNA) targeting the evi1 gene effectively inhibits HEL cell proliferation and viability. This evi1 gene silencing also induces cell cycle arrest and promotes apoptosis.
Area of Science:
- Molecular Biology
- Cell Biology
- RNA Interference
Background:
- The evi1 gene plays a role in cell proliferation and survival.
- Understanding mechanisms to control evi1 gene expression is crucial for therapeutic interventions.
Purpose of the Study:
- To investigate the inhibitory effect of small interfering RNA (siRNA) on evi1 gene expression and biological characteristics in HEL cells.
- To elucidate the mechanism of evi1 gene silencing in HEL cells.
Main Methods:
- Synthesis and in vitro transfection of three siRNAs specific for the evi1 gene into HEL cells.
- Assay of cell proliferation using MTT method.
- Detection of evi1 gene mRNA expression via semiquantitative RT-PCR.
- Determination of cell viability using trypan blue dye test.
- Analysis of cell cycle and apoptosis by flow cytometry.
Main Results:
- siRNA-1 demonstrated the strongest inhibitory effect on evi1 gene expression and HEL cell proliferation, with optimal results at 48 hours post-transfection.
- The highest inhibitory rate of 72.22% on proliferation was observed at 120 nmol/L siRNA concentration.
- Evi1 gene silencing led to cell cycle arrest at the G(0)/G(1) phase, decreased cell viability, and significantly increased apoptosis.
Conclusions:
- siRNA targeting the evi1 gene is effective in suppressing HEL cell proliferation and reducing cell viability.
- Evi1 gene silencing by siRNA induces cell cycle arrest at G(0)/G(1) phase, inhibits mitosis, and promotes apoptosis in HEL cells.
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