Ubiquitous expression of MAKORIN-2 in normal and malignant hematopoietic cells and its growth promoting activity

Abstract

Makorin-2 (MKRN2) is a highly conserved protein and yet its functions are largely unknown. We investigated the expression levels of MKRN2 and RAF1 in normal and malignant hematopoietic cells, and leukemia cell lines. We also attempted to delineate the role of MKRN2 in umbilical cord blood CD34+ stem/progenitor cells and K562 cell line by over-expression and inhibition of MKRN2 through lentivirus transduction and shRNA nucleofection, respectively. Our results provided the first evidence on the ubiquitous expression of MKRN2 in normal hematopoietic cells, embryonic stem cell lines, primary leukemia and leukemic cell lines of myeloid, lymphoid, erythroid and megakaryocytic lineages. The expression levels of MKRN2 were generally higher in primary leukemia samples compared with those in age-matched normal BM cells. In all leukemia subtypes, there was no significant correlation between expression levels of MKRN2 and RAF1. sh-MKRN2-silenced CD34+ cells had a significantly lower proliferation capacity and decreased levels of the early stem/progenitor subpopulation (CFU-GEMM) compared with control cultures. Over-expression of MKRN2 in K562 cells increased cell proliferation. Our results indicated possible roles of MKRN2 in normal and malignant hematopoiesis.

Figure 1. Expression of MKRN2 and RAF1 in primary hematopoietic cells and embryonic stem cell lines.

Expression levels of MKRN2 and RAF1 mRNA were measured in adult and CB hematopoietic cells, enriched CD34⁺ stem/progenitor cells and human embryonic cell lines H9 and H14 by qPCR (n = 2–6). The Y-axis represents the expression level relative to GAPDH.

Figure 2. Expression of MKRN2 and RAF1 in leukemic cell lines.

Expression levels of MKRN2 and RAF1 mRNA were measured by qPCR in leukemic cell lines of specific lineage subtypes. The Y-axis represents the expression level relative to GAPDH.

Figure 3. Expression of MKRN2 and RAF1 in primary human leukemic cells.

Expression levels of MKRN2 and RAF1 mRNA were measured by qPCR in bone marrow cells collected from leukemic patients (Ph–B-ALL, n = 8; Ph+B-ALL, n = 7; T-ALL, n = 5; AML, n = 22 and CML, n = 11). The Y-axis represents the expression level relative to GAPDH. Expression levels of MKRN2 and RAF1 were compared with those of age-matched normal bone marrow cells (n = 9) (* P<0.05, ** P<0.01 and *** P<0.001). Ph  =  Philadelphia chromosome or BCR/ABL translocation.

Figure 4. Ex vivo expansion of CD34⁺ cells overexpressing MKRN2.

CD34⁺ cells were transduced with MKRN2 cDNA subcloned into lentiviral vector (pLEF1α-IG-MKRN2) and cultured in expansion medium for 11 days (n = 3). The kinetics of expansion was not different between the MKRN2-transduced and control cells containing the empty vector (pLEF1α-IG).

Figure 5. Colony forming capacity of ex vivo expanded CD34⁺ cells over-expressing MKRN2.

CD34⁺ cells were transduced with MKRN2 cDNA subcloned into lentiviral vector (pLEF1α-IG-MKRN2) and expanded for 8 days, and subjected to CFU culture for 14 days (n = 3). There was no difference between MKRN2-transduced cells and control cells containing the empty vector (pLEF1α-IG).

Figure 6. ShRNA-silencing of MKRN2 in cord blood CD34⁺ cells.

MKRN2 expression was down-regulated in CD34⁺ cells by nucleofection of shRNA. CD34⁺ cells expressing pGFP-V-RS-MKRN2 had lower expansion capacity in day 7 culture (P = 0.005; n = 4) and a trend of reduced expansion at days 2 and 8, compared with cells transfected with non-effective sh-pGFP-V-RS-NE or empty vector (pGFP-V-RS).

Figure 7. Colony forming capacity of ex vivo expanded CD34⁺ cells with silenced MKRN2.

MKRN2 expression was down-regulated in CD34⁺ cells by nucleofection of shRNA. CD34⁺ cells expressing pGFP-V-RS-MKRN2 had a lower level of CFU-GEMM, compared with cells transfected with non-effective sh-pGFP-V-RS-NE or empty vector (pGFP-V-RS) (** P<0.01; n = 3).

Figure 8. Proliferation capacity of K562 cells over-expressing MKRN2.

K562 cells were transduced with MKRN2 cDNA subcloned into lentiviral vector (pLEF1α-IG-MKRN2). MTT assay of pLEF1α-IG-MKRN2-transduced cells showed a significantly increased proliferation in culture, compared with pLEF1α-IG (empty vector) control cells (*P = 0.05; n = 3).