Objective: To investigate the effect of IER3IIP1 gene silence by RNA interference on erythroid differentiation and proliferation of K562 cells.
Methods: The shRNA eukaryotic expression vectors targeting IER3IP1 gene were constructed. Silence effect was detected by semiquantitative RT-PCR after the vectors being transfected into K562 cells. The impact on K562 cells was studied by MTT assay, benzidine staining, light microscope and electron microscopy observation, flow cytometry and RT-PCR, respectively. The expression of erythroid differentiation-related genes Gfi-1B, GPA and gamma-globin and GPA protein expressed on these cells were studied after being exposed to 0.2 micromol/L imatinib for 48 hours.
Results: The expression of IER3IP1 gene was significantly inhibited with a inhibition rate of 76% (P<0.01). Compared with the control group, bcr-abl mRNA level was significantly increased in K562-shRNA-IER3IP1 group (P<0.05). The proliferation capacity was significantly enhanced (P<0.01) and the proportion of cells at G0/G1 phase significantly decreased while at S phase significantly increased (P <0.05) in K562-shRNA-IER3IP1 group. Under the electron microscopy observation, the number of euchromatin was increased while heterochromatin decreased. There were structural abnormalities in endocytoplasmic reticulum and clusters of vesicular. The proportion of benzidine staining positive cells decreased from (44.7 +/- 2.5)% in control group to (22.7 +/- 1.5)% in tested group (P<0.01). mRNA expression of Gfi-1B, GPA and gamma-globin gene and the expression of GPA protein on cell surface were all significantly decreased after being exposed to 0.2 micromol/L imatinib for 48 hours in K562-shRNA-IER3IP1 group (P<0.01). During the erythroid differentiation induced by imatinib, mRNA expression of IER3IP1 obviously increased at the 3 h, and decreased from 6 h to 48 h.
Conclusions: Inhibition of expression of IER3IP1 gene can inhibit erythroid differentiation and elevate the proliferation of K562 cells. The IER3IP1 gene may play a role on erythroid differentiation and proliferation of K562 cells.