MiRNA182 regulates percentage of myeloid and erythroid cells in chronic myeloid leukemia

Arya D, Sachithanandan SP, Ross C, Palakodeti D, Li S, Krishna S.
Source: Cell Death Dis.
Publication Date: (2017)
Issue: 2017;8(1): e2547
Research Area:
Basic Research
Cells used in publication:
Species: human
Tissue Origin: blood
4D-Nucleofector® X-Unit

Hes1 knockdown and overexpression.

In brief, K562 cells were nucleofected with Hes1 overexpression and knockdown plasmids on Amaxa 4d nucleofector from Lonza and incubated for 3 days. Hes1 was overexpressed by miGR1-Hes1 plasmid. Hes1 knockdown was performed by shRNA against Hes1 open-reading frame. ShRNA-Hes1 plasmids were a kind gift from Dr Adolo Ferrando, Columbia University. MigR1 control and MigR1-Hes1 plasmids were a kind gift from Avinash Bhandoola lab (NIH, USA).

Generation of CRISPR-based knockout system for MIR182 locus.

CRISPR/Cas9 system has been used to modify genomic loci of clinical importance. A stepwise process to delete the MIR182 locus was undertaken starting with HEK293 to standardise knockout method (Supplementary Figure 5a). To reduce the incidence of offtarget effects, three CRISPR plasmids (targeting 129409678, 129410425 and 129410482 according to GRCh37.p13 of human genome assembly) were nucleofected in K562 cell line (Figure 3c).


The deregulation of lineage control programs is often associated with the progression of haematological malignancies. The molecular regulators of lineage choices in the context of tyrosine kinase inhibitor (TKI) resistance remain poorly understood in chronic myeloid leukemia (CML). To find a potential molecular regulator contributing to lineage distribution and TKI resistance, we undertook an RNA-sequencing approach for identifying microRNAs (miRNAs). Following an unbiased screen, elevated miRNA182-5p levels were detected in Bcr-Abl-inhibited K562 cells (CML blast crisis cell line) and in a panel of CML patients. Earlier, miRNA182-5p upregulation was reported in several solid tumours and haematological malignancies. We undertook a strategy involving transient modulation and CRISPR/Cas9 (clustered regularly interspersed short palindromic repeats)-mediated knockout of the MIR182 locus in CML cells. The lineage contribution was assessed by methylcellulose colony formation assay. The transient modulation of miRNA182-5p revealed a biased phenotype. Strikingly, ?182 cells (homozygous deletion of MIR182 locus) produced a marked shift in lineage distribution. The phenotype was rescued by ectopic expression of miRNA182-5p in ?182 cells. A bioinformatic analysis and Hes1 modulation data suggested that Hes1 could be a putative target of miRNA182-5p. A reciprocal relationship between miRNA182-5p and Hes1 was seen in the context of TK inhibition. In conclusion, we reveal a key role for miRNA182-5p in restricting the myeloid development of leukemic cells. We propose that the ?182 cell line will be valuable in designing experiments for next-generation pharmacological interventions