Effect On The Proliferation And Erythroid Differentiation Of K562 Cells By IER3IP1-Knockdown

Objective: To investigate the effects on biological behaviour and erythroid differentiation of K562 cells by RNAi targeting at IER3IP1 gene. To form the basis for investigating IER3IP1 gene's function and erythroid differentiation's molecular mechanism in K562 cells.Methods: Two pairs of DNA fragments targeting at IER3IP1 gene and one pair of DNA fragment of negative control were synthesized and cloned into the eukaryotic expression vector pGenesil-1.The inhibitory effect were detected by semiquantitative RT-PCR after the recombined vectors being transfected respectively into K562 cells. The recombined cells with highest inhibitory effect was chosed for the following tests, named as K562/shRNA-IER3IP1 .The impacts on K562 cells by IER3IP1 knockdown were studied by MTT assay , flow cytometry , RT-PCR and observation under electron microscopy . The benzidine-positive rates , the expression of GPA protein and erythroid differentiation correlated genes Gfi-1B,GPA andγ-globin were studied after K562/shRNA-IER3IP1 cells being exposed to 0.2μmol/L imatinib and 30μmol/L hemin respectively for 48 hours .Results: These shRNA eukaryotic expression vectors were successfully constructed . Compared with pGenesil-1empty vector and negative vector , One of shRNA vectors could significantly and specially inhibit the expression of IER3IP1 gene with the inhibition ratio 76%. Compared with the control groups, K562/shRNA-IER3IP1showed higher proliferation ability in MTT experiment(P <0.01). The proportion of cells at G0/G1 phase were decreased from (44.04±2.15)﹪to (34.04±1.83)﹪, but S phase increased from (50.02±2.37)﹪to (61.14±2.20)﹪(P<0.05)in K562/shRNA-IER3IP1 group. Under the electron microscopy, the amount of euchromatin was observed increased but heterochromatin decreased. There were structural abnomalities in endocytoplasmic reticulum and clusters of vesicular. bcr/abl mRNA level was increased in K562/shRNA-IER3IP1 group(P<0.05).After being exposed to 0.2μmol/L imatinib and 30μmol/L hemin for 48 hours, the proportion of benzidine staining positive cells were decreased from (44.67±2.52)﹪to(22.67±1.53)﹪and from(23.00±3.61)﹪to(11.67±2.08)﹪respectively in K562/shRNA-IER3IP1 group(P<0.01). The mRNA expression of Gfi-1B,GPA andγ-globin and the expression of GPA protein on cell surface were all decraesed in K562/shRNA-IER3IP1 group(P<0.01). Conclusion: The combinated eukaryotic expression vector could inhibit the expression of IER3IP1 gene in K562 cell and it formed the basis of investigating IER3IP1 gene's function . After Inhibitig expression of IER3IP1 gene ,the level of K562 cells'proliferation and bcr/abl mRNA expression were elevated . It implys that IER3IP1 gene may impact on the bcr/abl mRNA expression and the proliferation of K562 cells. Inhibition expression of IER3IP1 gene can down-regulate erythroid differentiation of K562 cells induced by imatinib and hemin respectively . The gene may play a role in erythroid differentiation of K562 cells .