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This study was performed under a protocol approved by the Institutional Animal Care and Use Committee of Yale University. All mice were maintained at the Yale Animal Resource Center. Ctsg-PML-RARA (PR) mice on a C57BL/6 background were gifted by Dr. Timothy Ley’s group, as published[6]. C57BL/6 mice and the Rosa26-rtTA-M2 mice2 were from Jackson Laboratory[15]. The doxycycline-inducible miR-125b allele (i125b) was recently generated by our group. Briefly, mouse A2Lox.cre embryonic stem cells were used with an amiR-125b-containing targeting vector to generate the knock-in line 818-7, which generated knock-in mice through blastocyst injection performed by Yale Animal Genomics Services. Germline transmitted mice were crossed with Rosa26-rtTAm2 mice and backcrossed for six generations onto the C57BL/6 background (National Cancer Institute strain no. 01B96) to generate Ri125b mice[12,14]. For simplicity, the resultant mice are referred to as Ri125b mice, with male Ri125b mice having a genotype of rtTA/rtTA, i125b/i125y and female Ri125b mice having a genotype rtTA/rtTA, i125b/i125b. Genotyping was performed as published[6,12,14].
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A total of 366 patients with AML data who were admitted between June 2000 and May 2020 were recruited following a protocol approved by the Chinese PLA General Hospital. A total of 264 males and 102 females were included, aged 18 to 64 years. AML was diagnosed by morphology, immunology, cytogenetics, and molecular biology according to the French-American-British (FAB) cooperative group subtype and World Health Organization classification system at the first visit. Fifty cases of normal data were from healthy volunteers aged above 18 years. WBC counts and bone marrow blast percentage were obtained from routine clinical tests. The patient characteristics are summarized in Table 1.
Table 1. Clinical characteristics of 366 clinical patients with AML
FAB subtype Patients (n) Gender (M/F) Median age at
diagnosis (Years)WBC
(1 x 103/μL)BM Blast% Ratio of
WBC/Blast%M1 30 23/7 41 75.89 ± 4.06 88.9 ± 4.56 0.83 ± 1.12 M2 57 38/19 41 28.43 ± 44.85 51.48 ± 20.93 0.56 ± 0.84 M3 (APL) 109 66/43 37 9.24 ± 15.60 79.03 ± 14.56 0.11 ± 0.18 M4 61 37/24 50 44.57 ± 60.24 52.79 ± 20.42 0.80 ± 1.06 M5 59 33/26 41 50.47 ± 79.37 68.80 ± 19.57 0.66 ± 0.88 Normal 50 35/15 55 6.15 ± 1.46 N/A N/A Note. FAB: French-American-Britiish1cooperative1group; WBC: White blood cells; BM: Bone marrow; AML, acute myeloid leukemia; APL, Acute promyelocytic leukemia. M1: Acute myeloid leukemia of FAB subtype M1; M2: Acute myeloid leukemia of FAB subtype M2; M3: Acute promyelocytic leukemia; M4: Acute myelomonocytic leukemia; M5: Acute monocytic leukemia. The study design and protocols were approved by the Ethics Committee of the Chinese PLA General Hospital. Written informed consent was obtained from each enrolled subject.
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PR mice were crossed with Rosa26-rtTA-M2 (rtTA allele) and i125b alleles to generate the PR+rtTA, Ri125b, and PR+Ri125b genotypes. Mice were administered with 2 mg/mL Dox water supplemented with 1% sucrose (+Dox) or with regular water (−Dox) from 6 weeks of age. The survival of the mice was monitored with moribund events documented for the Kaplan–Meier curve.
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Recipient wild-type C57/Bl6 mice were sublethally irradiated at 6 Gy on the day or the day before the transplantation. Leukemia cells for secondary transplantation were harvested from the bone marrow of primary moribund mice, which contained –60% to > 90% leukemia blasts. Cells from primary PR+Ri125b+Dox leukemia mice were used, with 125,000 cells transplanted into each recipient through tail vein injection. On the same day of transplantation, recipient mice were administered with 2 mg/mL Dox water supplemented with 1% sucrose or with regular water.
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Peripheral blood (PB) was collected from the tail vein of mice. Complete blood count (CBC) count was performed with the PB samples on a Hemavet machine (Drew Scientific). Red blood cells were lysed using ACK lysis buffer (Thermo Fisher) before the PB samples were analyzed by performing flow cytometry[16]. Bone marrow cells from moribund mice were collected from the femur and tibia. Bone marrow cells were brushed with fetal bovine serum onto slides for bone marrow smears. Slides were stained with May–Grünwald–Giemsa (Sigma MG500) following the manufacturer’s protocol.
To isolate Mac1+ cells, cells were stained with PE-CD11b antibody (Biolegend) and sorted on a FACS Aria machine (BD).
For flow cytometry, all data were acquired on LSRII (BD) and analyzed with FlowJo 8.6.
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Total RNA was extracted from cells using TRIzol (Invitrogen) according to the manufacturer’s instructions. For qRT-PCR of miR-125a and miR-125b, cDNA synthesis was performed with 500 ng of total RNA with miRScript II RT KIT (Qiagen) and subjected to SYBR green (Applied Biosystems) qPCR following the manufacturer’s protocol. Real-time PCR was performed using CFX96 Real-Time System (Bio-Rad). Primers for miR-125a and miR-125b were purchased from Qiagen. U6 small RNA was used as a control in qPCR. Relative expressions were calculated by 2−ΔCT.
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Continuous variables underlying normal distribution were presented as mean ± standard deviations, and the between-group comparisons were tested by using two independent samples t-tests. If a variable was skewed, then it was described using the median (interquartile range) and compared using a nonparametric approach. Survival analyses were identified with bivariate analyses using Kaplan–Meier curves and log-rank testing for comparison. All the tests were two-tailed at a significance level of 0.05 unless otherwise stated. All the analyses were performed by using SPSS 22.0 (IBM Corp., Armonk, NY, USA).
doi: 10.3967/bes2022.067
MicroRNA-125b Accelerates and Promotes PML-RARa-driven Murine Acute Promyelocytic Leukemia
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Abstract:
Objective Most acute promyelocytic leukemia cases are characterized by the PML-RARa fusion oncogene and low white cell counts in peripheral blood. Methods Based on the frequent overexpression of miR-125-family miRNAs in acute promyelocytic leukemia, we examined the consequence of this phenomenon by using an inducible mouse model overexpressing human miR-125b. Results MiR-125b expression significantly accelerates PML-RARa-induced leukemogenesis, with the resultant induced leukemia being partially dependent on continued miR-125b overexpression. Interestingly, miR-125b expression led to low peripheral white cell counts to bone marrow blast percentage ratio, confirming the clinical observation in acute promyelocytic leukemia patients. Conclusion This study suggests that dysregulated miR-125b expression is actively involved in disease progression and pathophysiology of acute promyelocytic leukemia, indicating that targeting miR-125b may represent a new therapeutic option for acute promyelocytic leukemia. -
Key words:
- miR-125b /
- PML-RARA /
- White blood cell /
- Bone marrow blast
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Figure 1. miR-125b accelerates the leukemogenesis induced by PR. (A) Expression levels of miR125b and miR125a in the TCGA dataset plotted across multiple AML subtypes, in reads per million. (B) Schematics of the experiment of PML-RARA (PR) and miR-125b-induced leukemogenesis, including control groups. (C) Kaplan–Meier survival curves of the indicated cohorts of mice. P values are indicated. (D) Representative Giemsa staining of bone marrow cells from moribund PR+Ri125b+Dox or PR mice. (E) Schematics of the experiment to analyze leukemia dependence on miR-125b overexpression. Primary AML cells from PR+Ri125b+Dox mice were transplanted into lethally irradiated wild-type mice. Secondary recipients were randomized into cohorts for continued Dox or withdrawal of Dox. (F) Representative flow cytometry plots of PB four weeks after Dox withdrawal in secondary PR+Ri125b recipients. (G) Relative miR-125a and miR-125b expression levels in bone marrow cells of PR mice, PR+Ri125b mice with continued Dox or Dox withdrawal. U6 was used as a control. *P < 0.05. (H) Kaplan–Meier survival curves of PR+Ri125b mice with continued Dox (n = 7) or Dox withdrawal (n = 7), P < 0.001. AML, acute myeloid leukemia; GFP, green fluorescent protein.
Figure 2. Features associated with peripheral WBC counts in human and mouse APL. (A) Relative miR-125a-5p and miR-125b-5p expression in bone marrow cells in wild-type mice, PR mice before APL onset, and PR mice with APL was determined by qRT-PCR, n = 3. (B) Mac1+ myeloid cells sorted from the bone marrow of wild-type and PR mice before APL onset. Relative miR-125a-5p and miR-125b-5p expression was determined by qRT-PCR. (C) WBC counts from a cohort of human AMLs at disease presentation plotted according to their FAB subtypes. For the M3 subtype, only PML-RARA+ (PR+) cases are shown. WBC data from individuals without hematological disorders were used as normal (n = 50). The horizontal lines represent the medians of the corresponding groups. FAB-M1 (n = 30); FAB-M2 (n = 57); FAB-M3 (PR+) (n = 109); FAB-M4 (n = 61); FAB-M5 (n = 59). (D) Bone marrow blast percentage data from the same patient cohort as in (C). (E) Ratios of WBC (103/μL) to bone marrow blast percentage for data in (C, D). (F) WBC from secondary PR+Ri125b AML mice treated with Dox (continued, n = 5) or without Dox (withdrawal, n = 9). WBC was evaluated only in moribund mice with APL. (G) Bone marrow blast percentage data from the same mice described in (F) are shown. *P < 0.05. (H) Ratios of WBC (103/uL) to bone marrow blast percentage. *P < 0.05. Error bars represent standard deviations. WBC, white blood cell. M1: Acute myeloid leukemia of FAB subtype M1; M2: Acute myeloid leukemia of FAB subtype M2; M3: Acute promyelocytic leukemia; M4: Acute myelomonocytic leukemia; M5: Acute monocytic leukemia
Table 1. Clinical characteristics of 366 clinical patients with AML
FAB subtype Patients (n) Gender (M/F) Median age at
diagnosis (Years)WBC
(1 x 103/μL)BM Blast% Ratio of
WBC/Blast%M1 30 23/7 41 75.89 ± 4.06 88.9 ± 4.56 0.83 ± 1.12 M2 57 38/19 41 28.43 ± 44.85 51.48 ± 20.93 0.56 ± 0.84 M3 (APL) 109 66/43 37 9.24 ± 15.60 79.03 ± 14.56 0.11 ± 0.18 M4 61 37/24 50 44.57 ± 60.24 52.79 ± 20.42 0.80 ± 1.06 M5 59 33/26 41 50.47 ± 79.37 68.80 ± 19.57 0.66 ± 0.88 Normal 50 35/15 55 6.15 ± 1.46 N/A N/A Note. FAB: French-American-Britiish1cooperative1group; WBC: White blood cells; BM: Bone marrow; AML, acute myeloid leukemia; APL, Acute promyelocytic leukemia. M1: Acute myeloid leukemia of FAB subtype M1; M2: Acute myeloid leukemia of FAB subtype M2; M3: Acute promyelocytic leukemia; M4: Acute myelomonocytic leukemia; M5: Acute monocytic leukemia. -
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