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Retinoic acid induces HL-60 cell differentiation via the upregulation of miR-663.

Jian P, Li ZW, Fang TY, Jian W, Zhuan Z, Mei LX, Yan WS, Jian N - J Hematol Oncol (2011)

Bottom Line: We used a lentivirus (LV) backbone incorporating the spleen focus forming virus (SFFV-F) promoter to drive miR-663 expression, as the CMV (Cytomegalovirus) promoter is ineffective in some lymphocyte cells.Transfection of LV-miR-663 induced significant HL-60 cell differentiation in vitro.Our results show miR-663 may play an important role in ATRA induced HL-60 cell differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, China.

ABSTRACT

Background: Differentiation of the acute myeloid leukemia (AML) cell line HL-60 can be induced by all trans-retinoic acid (ATRA); however, the mechanism regulating this process has not been fully characterized.

Methods: Using bioinformatics and in vitro experiments, we identified the microRNA gene expression profile of HL-60 cells during ATRA induced granulocytic differentiation.

Results: Six microRNAs were upregulated by ATRA treatment, miR-663, miR-494, miR-145, miR-22, miR-363* and miR-223; and three microRNAs were downregulated, miR-10a, miR-181 and miR-612. Additionally, miR-663 expression was regulated by ATRA. We used a lentivirus (LV) backbone incorporating the spleen focus forming virus (SFFV-F) promoter to drive miR-663 expression, as the CMV (Cytomegalovirus) promoter is ineffective in some lymphocyte cells. Transfection of LV-miR-663 induced significant HL-60 cell differentiation in vitro.

Conclusions: Our results show miR-663 may play an important role in ATRA induced HL-60 cell differentiation. Lentivirus delivery of miR-663 could potentially be used directly as an anticancer treatment in hematological malignancies.

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Related in: MedlinePlus

ATRA treatment significantly upregulates miR-663 in HL-60 cells. (A) TaqMan qRT-PCR miRNA assays were used to quantify the time course of mature miR-663 expression in HL-60 cells treated with ATRA. Expression was normalized to endogenous U6 expression. (B) Summary of TaqMan qRT-PCR miRNA assay results showing miR-663 was significantly upregulated in ATRA treated cells. At 72 h, mir-633 expression in ATRA treated cells (6.93 ± 1.31) was significantly increased compared to cells treated with 1% ethanol (1.17 ± 0.24, p < 0.01).
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Figure 3: ATRA treatment significantly upregulates miR-663 in HL-60 cells. (A) TaqMan qRT-PCR miRNA assays were used to quantify the time course of mature miR-663 expression in HL-60 cells treated with ATRA. Expression was normalized to endogenous U6 expression. (B) Summary of TaqMan qRT-PCR miRNA assay results showing miR-663 was significantly upregulated in ATRA treated cells. At 72 h, mir-633 expression in ATRA treated cells (6.93 ± 1.31) was significantly increased compared to cells treated with 1% ethanol (1.17 ± 0.24, p < 0.01).

Mentions: We confirmed miR-663 was significantly upregulated by ATRA treatment using TaqMan mircoRNA qRT-PCR assays. MiR-663 is a challenging molecule to amplify using PCR as the microRNA precursor consists of a highly stable hairpin due to GC base paring; however, novel technologies have been developed to successfully amplify and quantify the mature miR-663. Real-time PCR has become the gold standard of nucleic acid quantification due the high specificity and sensitivity and technological advancements have enabled quantification of microRNAs in a comparable manner to mRNAs. The time course of mature miR-663 expression determined by qRT-RCR (Figure 3A) indicated miR-663 was significantly upregulated in ATRA treated HL-60 cells. After 72 h, expression of miR-633 in the ATRA treated group was 6.93 ± 1.31 compared with the control group 1.17 ± 0.24, Figure 3B, p < 0.01.


Retinoic acid induces HL-60 cell differentiation via the upregulation of miR-663.

Jian P, Li ZW, Fang TY, Jian W, Zhuan Z, Mei LX, Yan WS, Jian N - J Hematol Oncol (2011)

ATRA treatment significantly upregulates miR-663 in HL-60 cells. (A) TaqMan qRT-PCR miRNA assays were used to quantify the time course of mature miR-663 expression in HL-60 cells treated with ATRA. Expression was normalized to endogenous U6 expression. (B) Summary of TaqMan qRT-PCR miRNA assay results showing miR-663 was significantly upregulated in ATRA treated cells. At 72 h, mir-633 expression in ATRA treated cells (6.93 ± 1.31) was significantly increased compared to cells treated with 1% ethanol (1.17 ± 0.24, p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3094392&req=5

Figure 3: ATRA treatment significantly upregulates miR-663 in HL-60 cells. (A) TaqMan qRT-PCR miRNA assays were used to quantify the time course of mature miR-663 expression in HL-60 cells treated with ATRA. Expression was normalized to endogenous U6 expression. (B) Summary of TaqMan qRT-PCR miRNA assay results showing miR-663 was significantly upregulated in ATRA treated cells. At 72 h, mir-633 expression in ATRA treated cells (6.93 ± 1.31) was significantly increased compared to cells treated with 1% ethanol (1.17 ± 0.24, p < 0.01).
Mentions: We confirmed miR-663 was significantly upregulated by ATRA treatment using TaqMan mircoRNA qRT-PCR assays. MiR-663 is a challenging molecule to amplify using PCR as the microRNA precursor consists of a highly stable hairpin due to GC base paring; however, novel technologies have been developed to successfully amplify and quantify the mature miR-663. Real-time PCR has become the gold standard of nucleic acid quantification due the high specificity and sensitivity and technological advancements have enabled quantification of microRNAs in a comparable manner to mRNAs. The time course of mature miR-663 expression determined by qRT-RCR (Figure 3A) indicated miR-663 was significantly upregulated in ATRA treated HL-60 cells. After 72 h, expression of miR-633 in the ATRA treated group was 6.93 ± 1.31 compared with the control group 1.17 ± 0.24, Figure 3B, p < 0.01.

Bottom Line: We used a lentivirus (LV) backbone incorporating the spleen focus forming virus (SFFV-F) promoter to drive miR-663 expression, as the CMV (Cytomegalovirus) promoter is ineffective in some lymphocyte cells.Transfection of LV-miR-663 induced significant HL-60 cell differentiation in vitro.Our results show miR-663 may play an important role in ATRA induced HL-60 cell differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou, China.

ABSTRACT

Background: Differentiation of the acute myeloid leukemia (AML) cell line HL-60 can be induced by all trans-retinoic acid (ATRA); however, the mechanism regulating this process has not been fully characterized.

Methods: Using bioinformatics and in vitro experiments, we identified the microRNA gene expression profile of HL-60 cells during ATRA induced granulocytic differentiation.

Results: Six microRNAs were upregulated by ATRA treatment, miR-663, miR-494, miR-145, miR-22, miR-363* and miR-223; and three microRNAs were downregulated, miR-10a, miR-181 and miR-612. Additionally, miR-663 expression was regulated by ATRA. We used a lentivirus (LV) backbone incorporating the spleen focus forming virus (SFFV-F) promoter to drive miR-663 expression, as the CMV (Cytomegalovirus) promoter is ineffective in some lymphocyte cells. Transfection of LV-miR-663 induced significant HL-60 cell differentiation in vitro.

Conclusions: Our results show miR-663 may play an important role in ATRA induced HL-60 cell differentiation. Lentivirus delivery of miR-663 could potentially be used directly as an anticancer treatment in hematological malignancies.

Show MeSH
Related in: MedlinePlus