PIP4KIIα Gene Silencing by Lentivirus-Mediated Delivery of shRNA in Human K562 Cells,

Phosphatidylinositol-phosphate-kinase type II alpha (PIP4KIIα) belongs to a family of lipid kinases responsible for the production of a variety of lipid second messengers, such as PI4,5P₂ (phosphatidylinositol 4,5-biphosphate), and appears to be implicated in the regulation of gene expression, pre-mRNA processing and mRNA export. In a previous study, two transcripts, PIP4KIIα and β-globin, were found to be overexpressed in reticulocytes from two siblings with Hb H disease, suggesting a possible relationship between this enzyme and the production of globins, particularly β-globin. Recently, we established a gene expression pattern for PIP4KIIα in healthy individuals during in vitro erythropoiesis and observed a gradual increase in the expression of this gene during erythroid differentiation similar to that observed for globin genes, reinforcing the hypothesis of a relationship between PIP4KIIα and globin expression. Aim: To investigate the effects of PIP4KIIα gene silencing on the expression of α- and γ-globin genes in human K562 cells. Methods: Two different human K562 cells cultures were transduced with a lentiviral vector encoding PIP4KIIα-specific shRNA or non-relevant control shRNA. After transduction the positive cells were selected by adding puromycin to the culture and collected 2, 6, 8 and 10 days later to analyze gene and protein expression. PIP4KIIα and α- and γ-globin gene expression was assessed by qRT-PCR and quantified using the equation RQ=2^−ΔΔCt. Western blot analysis was performed to determine PIP4KIIα protein expression. β-actin and GAPDH were used as endogenous controls in the qRT-PCR, and β-actin in the Western blot. Results: Analysis of the results showed that there was a statistically significant reduction in PIP4KIIα mRNA levels in knockdown cells (79%) (0.208 ± 0.048; p<0.0001) compared with the control culture. Western blot analysis corroborated these findings. PIP4KIIα silencing resulted in an 18% (0.927 ± 0.244; p=0.09) and 44% (0.625 ± 0.124; p=0.03) reduction in the expression of α- and γ-globin genes, respectively, compared with the control. Conclusion: Although the reduction in α-globin gene expression did not achieve statistical significance, our results revealed alterations in α- and γ-globin gene expression in PIP4KIIα knockdown cells, suggesting a parallelism between the expression of PIP4KIIα and globin genes and reinforcing the hypothesis that the former may be involved in regulation of the latter.

This work was supported by FAPESP, CNPq and INCTS.

No relevant conflicts of interest to declare.