Limits...
Overexpression of primary microRNA 221/222 in acute myeloid leukemia.

Rommer A, Steinleitner K, Hackl H, Schneckenleithner C, Engelmann M, Scheideler M, Vlatkovic I, Kralovics R, Cerny-Reiterer S, Valent P, Sill H, Wieser R - BMC Cancer (2013)

Bottom Line: Transfections and infections of human cell lines were performed using standard procedures. 64 miRNAs were significantly differentially expressed between AML and controls.Expression of some miRNAs is strongly regulated at the posttranscriptional level in AML.Pri-miR-221/222 represents a novel molecular marker and putative oncogene in this disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria.

ABSTRACT

Background: Acute myeloid leukemia (AML) is a hematopoietic malignancy with a dismal outcome in the majority of cases. A detailed understanding of the genetic alterations and gene expression changes that contribute to its pathogenesis is important to improve prognostication, disease monitoring, and therapy. In this context, leukemia-associated misexpression of microRNAs (miRNAs) has been studied, but no coherent picture has emerged yet, thus warranting further investigations.

Methods: The expression of 636 human miRNAs was compared between samples from 52 patients with AML and 13 healthy individuals by highly specific locked nucleic acid (LNA) based microarray technology. The levels of individual mature miRNAs and of primary miRNAs (pri-miRs) were determined by quantitative reverse transcriptase (qRT) PCR. Transfections and infections of human cell lines were performed using standard procedures.

Results: 64 miRNAs were significantly differentially expressed between AML and controls. Further studies on the clustered miRNAs 221 and 222, already known to act as oncogenes in other tumor types, revealed a deficiency of human myeloid cell lines to process vector derived precursor transcripts. Moreover, endogenous pri-miR-221/222 was overexpressed to a substantially higher extent than its mature products in most primary AML samples, indicating that its transcription was enhanced, but processing was rate limiting, in these cells. Comparison of samples from the times of diagnosis, remission, and relapse of AML demonstrated that pri-miR-221/222 levels faithfully reflected the stage of disease.

Conclusions: Expression of some miRNAs is strongly regulated at the posttranscriptional level in AML. Pri-miR-221/222 represents a novel molecular marker and putative oncogene in this disease.

Show MeSH

Related in: MedlinePlus

Malignant human hematopoietic cells display impaired pri-miR-221/222 processing capacity. Mature miR-221 was measured by qRT-PCR, normalized to RNU6B, and expressed relative to ß-2-microglobulin-normalized pri-miR-221/222. A) Human myeloid cell lines, and HeLa cells as a reference; B) primary AML samples (n = 8; Additional file 1: Table S1B), enriched for blasts through Ficoll gradient purification, and normal control PB (squares; n = 4) and BM (triangles; n = 2) samples (Additional file 1: Table S1A). HeLa cells were used as a calibrator in both experiments. *, p < 0.05; ***, p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3733744&req=5

Figure 2: Malignant human hematopoietic cells display impaired pri-miR-221/222 processing capacity. Mature miR-221 was measured by qRT-PCR, normalized to RNU6B, and expressed relative to ß-2-microglobulin-normalized pri-miR-221/222. A) Human myeloid cell lines, and HeLa cells as a reference; B) primary AML samples (n = 8; Additional file 1: Table S1B), enriched for blasts through Ficoll gradient purification, and normal control PB (squares; n = 4) and BM (triangles; n = 2) samples (Additional file 1: Table S1A). HeLa cells were used as a calibrator in both experiments. *, p < 0.05; ***, p < 0.001.

Mentions: To investigate whether the presumed processing deficiency also affected endogenous miR-221/222, qRT-PCR was performed on a panel of human myeloid cell lines. Because PCR cannot selectively measure pre- versus pri-miRNAs, and efficient reverse transcription of the short pre-miRNA molecules into full-length, PCR amplifiable cDNAs is unlikely based on technical considerations, the RNA species expected to be predominantly measured in these assays is pri-miR-221/222. The miR-221:pri-miR-221/222 ratio was moderately decreased in the myeloid cell lines compared to HeLa cells (Figure 2A), indicating that the capacity of myeloid cells to process endogenous pri-miR-221/222 may be compromised to some extent. More importantly, the miR-221:pri-miR-221/222 ratio was strongly reduced in primary AML cells compared to PB and BM cells from healthy donors (Figure 2B). This could indicate that a pri-miR-221/222 processing defect is acquired during leukemogenesis, or that hematopoietic cells have an a priori limited pri-miR-221/222 processing capacity that becomes saturated when this pri-miRNA is transcriptionally upregulated in the context of leukemia.


Overexpression of primary microRNA 221/222 in acute myeloid leukemia.

Rommer A, Steinleitner K, Hackl H, Schneckenleithner C, Engelmann M, Scheideler M, Vlatkovic I, Kralovics R, Cerny-Reiterer S, Valent P, Sill H, Wieser R - BMC Cancer (2013)

Malignant human hematopoietic cells display impaired pri-miR-221/222 processing capacity. Mature miR-221 was measured by qRT-PCR, normalized to RNU6B, and expressed relative to ß-2-microglobulin-normalized pri-miR-221/222. A) Human myeloid cell lines, and HeLa cells as a reference; B) primary AML samples (n = 8; Additional file 1: Table S1B), enriched for blasts through Ficoll gradient purification, and normal control PB (squares; n = 4) and BM (triangles; n = 2) samples (Additional file 1: Table S1A). HeLa cells were used as a calibrator in both experiments. *, p < 0.05; ***, p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3733744&req=5

Figure 2: Malignant human hematopoietic cells display impaired pri-miR-221/222 processing capacity. Mature miR-221 was measured by qRT-PCR, normalized to RNU6B, and expressed relative to ß-2-microglobulin-normalized pri-miR-221/222. A) Human myeloid cell lines, and HeLa cells as a reference; B) primary AML samples (n = 8; Additional file 1: Table S1B), enriched for blasts through Ficoll gradient purification, and normal control PB (squares; n = 4) and BM (triangles; n = 2) samples (Additional file 1: Table S1A). HeLa cells were used as a calibrator in both experiments. *, p < 0.05; ***, p < 0.001.
Mentions: To investigate whether the presumed processing deficiency also affected endogenous miR-221/222, qRT-PCR was performed on a panel of human myeloid cell lines. Because PCR cannot selectively measure pre- versus pri-miRNAs, and efficient reverse transcription of the short pre-miRNA molecules into full-length, PCR amplifiable cDNAs is unlikely based on technical considerations, the RNA species expected to be predominantly measured in these assays is pri-miR-221/222. The miR-221:pri-miR-221/222 ratio was moderately decreased in the myeloid cell lines compared to HeLa cells (Figure 2A), indicating that the capacity of myeloid cells to process endogenous pri-miR-221/222 may be compromised to some extent. More importantly, the miR-221:pri-miR-221/222 ratio was strongly reduced in primary AML cells compared to PB and BM cells from healthy donors (Figure 2B). This could indicate that a pri-miR-221/222 processing defect is acquired during leukemogenesis, or that hematopoietic cells have an a priori limited pri-miR-221/222 processing capacity that becomes saturated when this pri-miRNA is transcriptionally upregulated in the context of leukemia.

Bottom Line: Transfections and infections of human cell lines were performed using standard procedures. 64 miRNAs were significantly differentially expressed between AML and controls.Expression of some miRNAs is strongly regulated at the posttranscriptional level in AML.Pri-miR-221/222 represents a novel molecular marker and putative oncogene in this disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria.

ABSTRACT

Background: Acute myeloid leukemia (AML) is a hematopoietic malignancy with a dismal outcome in the majority of cases. A detailed understanding of the genetic alterations and gene expression changes that contribute to its pathogenesis is important to improve prognostication, disease monitoring, and therapy. In this context, leukemia-associated misexpression of microRNAs (miRNAs) has been studied, but no coherent picture has emerged yet, thus warranting further investigations.

Methods: The expression of 636 human miRNAs was compared between samples from 52 patients with AML and 13 healthy individuals by highly specific locked nucleic acid (LNA) based microarray technology. The levels of individual mature miRNAs and of primary miRNAs (pri-miRs) were determined by quantitative reverse transcriptase (qRT) PCR. Transfections and infections of human cell lines were performed using standard procedures.

Results: 64 miRNAs were significantly differentially expressed between AML and controls. Further studies on the clustered miRNAs 221 and 222, already known to act as oncogenes in other tumor types, revealed a deficiency of human myeloid cell lines to process vector derived precursor transcripts. Moreover, endogenous pri-miR-221/222 was overexpressed to a substantially higher extent than its mature products in most primary AML samples, indicating that its transcription was enhanced, but processing was rate limiting, in these cells. Comparison of samples from the times of diagnosis, remission, and relapse of AML demonstrated that pri-miR-221/222 levels faithfully reflected the stage of disease.

Conclusions: Expression of some miRNAs is strongly regulated at the posttranscriptional level in AML. Pri-miR-221/222 represents a novel molecular marker and putative oncogene in this disease.

Show MeSH
Related in: MedlinePlus