The Study Of New Functional Genes Involved In Erythroid Differentiation In Human Erythroleukemia K-562Cell Line

Erythroid differentiation is defined as hematopoietic stem cells (HSCs) undergoing differentiation steps to become mature erythrocytes. It is a tightly regulated multi-step process that has not been fully elucidated. RNAi technology is a powerful tool to study gene function, which has been used in RNAi-based genome-wide functional analysis, as well as the study of individual gene function. We previously reported a combinatorial high-throughput screen with a random siRNA library on human erythroleukemia K-562cell line differentiation. Two siRNAs(siRNA clone-60and siRNA clone-67) screened from the random siRNA library were validated to be able to induce erythroid differentiation. We further found some putative target genes for the above two siRNAs and validated the function of some of these genes in K562cells.We hope our study will be helpful to the study of differentiation therapy of cancer and mechanisms involved in erythroid differentiation.We used whole-genome gene expression profiling and affinity purification with labeled siRNA to find the putative target genes of siRNA clone-67and clone-60. CCDC12and SLC30A3are two of the genes that we found by the above methods. CCDC12is one of the proteins that contain coiled-coil domain. SLC30A3is a Zinc transporter-3(ZnT3) protein,which plays an essential role in higher nervous activities such as learning and memory.So far, there is no report yet about their function in erythroid differentiation.We futher demonstrated that both CCDC12and SLC30A3promoted erythroid differentiation in human K562cell. Over-expression of CCDC12and SLC30A3up-regulated the expression level of erythroid differentiation specific marker CD235. For CCDC12, the percentage of CD235(+) cells increased to78.07%compared to that of the negative control which was32.25%. For SLC30A3, the percentage of CD235(+) cells increased to95.7%compared to that of the negative control which was34.25%. For CCDC12, the expression of ε-and γ-globin was up-regulated,whereas for SLC30A3, the expression of ε- γ-and β-globin was remarkably up-regulated. The expression of erythroid differentiation-related transcription factor GATA-2was down-regulated, and the cell proliferation rate was accelerated in response to CCDC12and SLC30A3over-expression.In summary, based on two siRNAs which induced erythroid differentiation in human K562cell, we found some of their putative target genes. Among these genes, CCDC12and SLC30A3were found to be able to induce erythroid differentiation in human K562cell, which has not been reported yet. Our data lay foundation for futher study of CCDC12and SLC30A3in erythroid differentiation in vivo.