MicroRNAs 221 and 222 inhibit normal erythropoiesis and erythroleukemic cell growth via kit receptor down-modulation

Abstract

MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression primarily through translational repression. In erythropoietic (E) culture of cord blood CD34+ progenitor cells, the level of miR 221 and 222 is gradually and sharply down-modulated. Hypothetically, this decline could promote erythropoiesis by unblocking expression of key functional proteins. Indeed, (i) bioinformatic analysis suggested that miR 221 and 222 target the 3' UTR of kit mRNA; (ii) the luciferase assay confirmed that both miRs directly interact with the kit mRNA target site; and (iii) in E culture undergoing exponential cell growth, miR down-modulation is inversely related to increasing kit protein expression, whereas the kit mRNA level is relatively stable. Functional studies show that treatment of CD34+ progenitors with miR 221 and 222, via oligonucleotide transfection or lentiviral vector infection, causes impaired proliferation and accelerated differentiation of E cells, coupled with down-modulation of kit protein: this phenomenon, observed in E culture releasing endogenous kit ligand, is magnified in E culture supplemented with kit ligand. Furthermore, transplantation experiments in NOD-SCID mice reveal that miR 221 and 222 treatment of CD34+ cells impairs their engraftment capacity and stem cell activity. Finally, miR 221 and 222 gene transfer impairs proliferation of the kit+ TF-1 erythroleukemic cell line. Altogether, our studies indicate that the decline of miR 221 and 222 during exponential E growth unblocks kit protein production at mRNA level, thus leading to expansion of early erythroblasts. Furthermore, the results on kit+ erythroleukemic cells suggest a potential role of these miRs in cancer therapy.

Fig. 1.

Expression of miR 221 and 222 and kit in unilineage E±KL culture. (A Top) Growth curve and KL release in HPC E culture. Shown are mean values from seven independent experiments. (A Middle) Growth curve of HPC E culture supplemented with KL. Shown are mean values from three separate experiments. (A Bottom) E maturation in E and E+KL culture: percentage of late (polychromatophilic + orthochromatic) erythroblasts is presented. (B) miR 221 and 222 expression in HPC E culture. (Upper) Microarray results, as compared with normalized day 0 level. (Lower) Northern blot results. Representative experiments are presented. (C) kit expression in HPC E culture. (Upper) Representative immunoblotting of kit protein. (Lower) Real-time PCR of kit mRNA level (mean ± SEM values from four separate experiments). (D) miR 221 and 222 expression versus kit protein level in E culture (Upper). (Lower) Inverse correlation of miR 221 and 222 vs. kit (r² = 0.96, P < 0.01 in both cases).

Fig. 2.

kit mRNA 3′ UTR site targeted by miR 221 and 222

Fig. 3.

miR 221 and 222 directly interact with kit 3′ UTR, as evaluated by luciferase targeting assay. Shown are mean ± SEM values from four separate experiments. **, P < 0.01 when compared with control.

Fig. 4.

miR 221 and 222 overexpression impairs cell growth in HPC E+KL culture. Growth curve (Upper) and kit protein expression (Lower) in E+KL culture transfected on day 4 with miR 221 and/or 222 oligonucleotide, as compared with vehicle and control oligonucleotide. A representative experiment of four independent experiments is presented.

Fig. 5.

Inhibition of cell growth in TF-1 cell line and unilineage E+KL culture upon infection with Tween-221 and Tween-222 vectors. (A Top) Growth curve of kit+ TF-1 cells infected with Tween-221 or Tween-222 vectors, as compared with empty Tween control vector. (A Middle) Western blot of kit protein and Northern blot of miR 222 in Tween-222-infected TF-1 cells. Similar results were obtained in Tween-221-infected cells (data not shown). (A Bottom) Growth curve of kit-HL-60 cells, infected with Tween-221 or Tween-222 vectors, as compared with empty Tween control vector. A representative experiment of six independent experiments is presented. (B) Growth curve (Top) and maturation of late erythroblasts (Middle) in E+KL culture of HPCs transduced with Tween-221 or -222 vectors, as compared with Tween control vector. (B Bottom) Western blot histogram of kit protein and Northern blot of miR 222 in Tween-222-infected cells at day 10 of culture, as compared with control value; similar results were obtained for Tween-221-infected cells (data not shown). A representative experiment of four independent experiments is presented.