Let-7b inhibits the development of AML through inducing repolarization of leukemia-associated macrophages

doi:10.11958/20252591

Abstract

Abstract:

Objective To study the mechanism of let-7b regulating acute myeloid leukemia (AML) by leukemia-associated macrophages (LAMs). Methods Bone marrow cells were collected from AML patients prior to any treatment and cryopreserved. Patients subsequently received induction chemotherapy with the "DA 3+7" regimen, and therapeutic responses were evaluated post-treatment. According to the efficacy assessment results, patients were stratified into two groups: the complete remission (CR) group and the non-complete remission (refractory) group. AML blasts of pre-cryopreserved patients were then respectively transplanted into NOD/SCID mice to establish AML xenograft mouse model corresponding to the CR group and the refractory group. After miRNA sequencing and polymerase chain reaction (PCR) verification, let-7b was selected as the target gene to investigate its role in MLL-AF9-induced AML. Transfection of LAMs in MLL-AF9-induced AML mice with let-7b shRNA or scramble control was validated by quantitative real-time PCR (RT-qPCR), Western blot assay and enzyme-linked immunosorbent assay (ELISA) in vitro and in vivo experiments. Results Compared with the AML CR group, the apoptosis rate of LAM cells was lower in the AML refractory group. Furthermore, let‐7b was a potentially aberrant gene in LAMs contributing to M2‐subtype characteristics. Knockdown of let‐7b in LAMs could inhibit the development of AML by repolarizing LAMs toward M1‐subtype characteristics through the activation of Toll‐like receptor and NF‐κB pathway. The results of in vivo experiments showed that knockdown of let-7b in LAMs inhibited the proliferation of leukemia cells and decreased infiltration into spleen, thereby inhibiting the progression of AML. Conclusion Let-7b interference in LAMs can activate Toll-like receptors (TLRs) and NF-κB pathway, promote the repolarization of LAMs to M1 macrophages, thereby inhibiting the development of AML.

Key words: leukemia, myeloid, acute, tumor-associated macrophages, NF-kappa B, Toll-like receptors, let-7b, leukemia-associated macrophages

CLC Number:

R733.71

Figures/Tables 4

References 20

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基因名称	引物序列（5'→3'）	产物大小/bp
let-7b	上游：CTGAGTGAGGTAGTAGGTTGTAT	127
let-7b	下游：CCCTCTACACTCTTTCTCTCCAT	127
let-7e	上游：CGGGCTGAGGTAGTAGGTTGT	89
let-7e	下游：GTGCAGGGTCCGAGGTATTC	89
let-7i	上游：GCTGAGGTAGTAGTTTGTGCTG	95
let-7i	下游：TGCGCTGTGGAATGTAAGCTA	95
miR146	上游：TGCCTTCAGTGCGTTACCCAGA	120
miR146	下游：GTGCAGGGTCCGAGGTATTC	120
miR105	上游：GCGCTGTGTCCTGGAGTTTGA	98
miR105	下游：GTGCAGGGTCCGAGGTATT	98
miR223	上游：GCCCTGTGTTGGAGCTGG	85
miR223	下游：TCCAGTTTTTCAACATATCCAC	85
miR147	上游：CTGTGCCCTGGTGCTGTCT	121
miR147	下游：GGTCCGAGGTATTCGCACTG	121
TLR1	上游：TCTGCCGGACGGTTGTCTG	100
TLR1	下游：AGCTGTGCATCACGGAACCTC	100
TLR2	上游：GGTGCGGACTGTTTCCTTCT	102
TLR2	下游：CCGGTGATGCAATTCGGATG	102
TLR4	上游：ACTGTTCTTCTCCTGCCTGAC	103
TLR4	下游：AGGGACTTTGCTGAGTTTCTGAT	103
TLR5	上游：CCTGGAGCCGAGTGAGAAAT	90
TLR5	下游：TGACGATCCTGGGGTGTTTG	90
TLR6	上游：TCGGAGAAGGAAGTCTTGAGC	145
TLR6	下游：TATTAAGGCCAGGGCGCAAA	145
TLR10	上游：GCCAATTCTGACCGTGTCAAC	83
TLR10	下游：ATGAGCTCAAAACCCCCACG	83
TLR11	上游：CCAGGCTACGTGTGCTAGAG	146
TLR11	下游：GCTGGACACAGAGAGGTTCC	146
TLR12	上游：GCTCCGGCAGTATTGTTCCT	131
TLR12	下游：CCGCCGCTTATAGTCAAGGT	131
LR13	上游：GCGTTTGTCTCTTTCTCGGC	124
LR13	下游：TGCCTGGTTCAAAATCCCGT	124
IKBKG	上游：CAAGTCACCCAAGGGAGGTG	83
IKBKG	下游：GTGGAGACCAGACTTGGACC	83
CARD9	上游：CGAGAACTACCGCAGGAAGC	82
CARD9	下游：TGTCGTATTCCCGCGATCC	82
CARD11	上游：CAAGGGGCACGAAGAGGATT	96
CARD11	下游：CTCCGATGGGGTTGCATCTT	96
CARD14	上游：CTGGTGTAGGAGTAAGCCCG	81
	下游：CTCAAAGTCCAGCGCCAAAG	81
IKBKA	上游：GTTCTGCCCGCTCTCTTGTA	111
IKBKA	下游：TCACACATGTCAGAGGATGTTCA	111
IKBKB	上游：CTGTGCACGTCATTTGTGGG	81
IKBKB	下游：GTCAACGGTCACGGTGTACT	81
caspase8	上游：TCTCACACGGAGAGAAGGGC	83
caspase8	下游：TCCGGCAAAAGGCAAAGTGA	83
RELA	上游：CTTTACTTTAGCGCGCCGTG	147
RELA	下游：TGCTTCGGCTGTTCGATGAT	147
RELB	上游：CGGTCAACGTGTTCTTGCAG	112
RELB	下游：CGCTTTCGCTTCTTGTCCAC	112
ICAM3	上游：CCAGAACCCTGTGCTCTCTG	144
ICAM3	下游：CTGAGATTGAAGGCTGCCCA	144
EGLN3	上游：AAGGAGCGGTCCAAGGCAAT	95
EGLN3	下游：ATACAGCGGCCATCACCATT	95
INHBA	上游：AAATCAGAACGCCTCCGCTA	92
INHBA	下游：TCCCGAGTGTAGAGTTCGGT	92
MMP12	上游：AGGCACAGGACACCTATAAACT	96
MMP12	下游：GTCATTCATGGGAGCAGCCC	96
CLEC5A	上游：GGCACATGATCATCTCGGGG	114
CLEC5A	下游：TGGGGACGAAGCCATCATTAC	114
TNF-α	上游：GATCGGTCCCCAAAGGGATG	92
TNF-α	下游：CCACTTGGTGGTTTGTGAGTG	92
IL6	上游：TGGTCTTCTGGAGTACCATAGC	140
IL6	下游：TGTGACTCCAGTTATCTCTTGG	140
NOS2	上游：CAACAGGGAGAAAGCGCAAA	125
NOS2	下游：ATTCTGTGCTGTCCCAGTGAG	125
CCR7	上游：ATTCTGTGCTGTCCCAGTGAG	135
CCR7	下游：GGCAGGAACCAGGCCTTAAA	135
ALDH2	上游：TGGCACTGTGTGGTAAGAGC	90
ALDH2	下游：TCTCCGGCATAGGAGAACCA	90
FRB	上游：CCGCTACTGTGTCTTGGCAT	118
FRB	下游：CAGCTCGCGGATCTCAAAGA	118
STAB1	上游：ACGTCATCGTGTGGGACATC	131
STAB1	下游：AACCCCCAAGCTTAATGCCA	131
CCL2	上游：TGACCCCAAGAAGGAATGGG	104
CCL2	下游：ACCTTAGGGCAGATGCAGTT	104
Arg1	上游：ACATTGGCTTGCGAGACGTA	109
Arg1	下游：ATCACCTTGCCAATCCCCAG	109
Mgl1	上游：ATCAGAGAGGATTTGGCCGC	122
	下游：AGGAAGATCGGTGTCGCTTG	122

基因名称	引物序列（5'→3'）	产物大小/bp
let-7b	上游：CTGAGTGAGGTAGTAGGTTGTAT	127
let-7b	下游：CCCTCTACACTCTTTCTCTCCAT	127
let-7e	上游：CGGGCTGAGGTAGTAGGTTGT	89
let-7e	下游：GTGCAGGGTCCGAGGTATTC	89
let-7i	上游：GCTGAGGTAGTAGTTTGTGCTG	95
let-7i	下游：TGCGCTGTGGAATGTAAGCTA	95
miR146	上游：TGCCTTCAGTGCGTTACCCAGA	120
miR146	下游：GTGCAGGGTCCGAGGTATTC	120
miR105	上游：GCGCTGTGTCCTGGAGTTTGA	98
miR105	下游：GTGCAGGGTCCGAGGTATT	98
miR223	上游：GCCCTGTGTTGGAGCTGG	85
miR223	下游：TCCAGTTTTTCAACATATCCAC	85
miR147	上游：CTGTGCCCTGGTGCTGTCT	121
miR147	下游：GGTCCGAGGTATTCGCACTG	121
TLR1	上游：TCTGCCGGACGGTTGTCTG	100
TLR1	下游：AGCTGTGCATCACGGAACCTC	100
TLR2	上游：GGTGCGGACTGTTTCCTTCT	102
TLR2	下游：CCGGTGATGCAATTCGGATG	102
TLR4	上游：ACTGTTCTTCTCCTGCCTGAC	103
TLR4	下游：AGGGACTTTGCTGAGTTTCTGAT	103
TLR5	上游：CCTGGAGCCGAGTGAGAAAT	90
TLR5	下游：TGACGATCCTGGGGTGTTTG	90
TLR6	上游：TCGGAGAAGGAAGTCTTGAGC	145
TLR6	下游：TATTAAGGCCAGGGCGCAAA	145
TLR10	上游：GCCAATTCTGACCGTGTCAAC	83
TLR10	下游：ATGAGCTCAAAACCCCCACG	83
TLR11	上游：CCAGGCTACGTGTGCTAGAG	146
TLR11	下游：GCTGGACACAGAGAGGTTCC	146
TLR12	上游：GCTCCGGCAGTATTGTTCCT	131
TLR12	下游：CCGCCGCTTATAGTCAAGGT	131
LR13	上游：GCGTTTGTCTCTTTCTCGGC	124
LR13	下游：TGCCTGGTTCAAAATCCCGT	124
IKBKG	上游：CAAGTCACCCAAGGGAGGTG	83
IKBKG	下游：GTGGAGACCAGACTTGGACC	83
CARD9	上游：CGAGAACTACCGCAGGAAGC	82
CARD9	下游：TGTCGTATTCCCGCGATCC	82
CARD11	上游：CAAGGGGCACGAAGAGGATT	96
CARD11	下游：CTCCGATGGGGTTGCATCTT	96
CARD14	上游：CTGGTGTAGGAGTAAGCCCG	81
	下游：CTCAAAGTCCAGCGCCAAAG	81
IKBKA	上游：GTTCTGCCCGCTCTCTTGTA	111
IKBKA	下游：TCACACATGTCAGAGGATGTTCA	111
IKBKB	上游：CTGTGCACGTCATTTGTGGG	81
IKBKB	下游：GTCAACGGTCACGGTGTACT	81
caspase8	上游：TCTCACACGGAGAGAAGGGC	83
caspase8	下游：TCCGGCAAAAGGCAAAGTGA	83
RELA	上游：CTTTACTTTAGCGCGCCGTG	147
RELA	下游：TGCTTCGGCTGTTCGATGAT	147
RELB	上游：CGGTCAACGTGTTCTTGCAG	112
RELB	下游：CGCTTTCGCTTCTTGTCCAC	112
ICAM3	上游：CCAGAACCCTGTGCTCTCTG	144
ICAM3	下游：CTGAGATTGAAGGCTGCCCA	144
EGLN3	上游：AAGGAGCGGTCCAAGGCAAT	95
EGLN3	下游：ATACAGCGGCCATCACCATT	95
INHBA	上游：AAATCAGAACGCCTCCGCTA	92
INHBA	下游：TCCCGAGTGTAGAGTTCGGT	92
MMP12	上游：AGGCACAGGACACCTATAAACT	96
MMP12	下游：GTCATTCATGGGAGCAGCCC	96
CLEC5A	上游：GGCACATGATCATCTCGGGG	114
CLEC5A	下游：TGGGGACGAAGCCATCATTAC	114
TNF-α	上游：GATCGGTCCCCAAAGGGATG	92
TNF-α	下游：CCACTTGGTGGTTTGTGAGTG	92
IL6	上游：TGGTCTTCTGGAGTACCATAGC	140
IL6	下游：TGTGACTCCAGTTATCTCTTGG	140
NOS2	上游：CAACAGGGAGAAAGCGCAAA	125
NOS2	下游：ATTCTGTGCTGTCCCAGTGAG	125
CCR7	上游：ATTCTGTGCTGTCCCAGTGAG	135
CCR7	下游：GGCAGGAACCAGGCCTTAAA	135
ALDH2	上游：TGGCACTGTGTGGTAAGAGC	90
ALDH2	下游：TCTCCGGCATAGGAGAACCA	90
FRB	上游：CCGCTACTGTGTCTTGGCAT	118
FRB	下游：CAGCTCGCGGATCTCAAAGA	118
STAB1	上游：ACGTCATCGTGTGGGACATC	131
STAB1	下游：AACCCCCAAGCTTAATGCCA	131
CCL2	上游：TGACCCCAAGAAGGAATGGG	104
CCL2	下游：ACCTTAGGGCAGATGCAGTT	104
Arg1	上游：ACATTGGCTTGCGAGACGTA	109
Arg1	下游：ATCACCTTGCCAATCCCCAG	109
Mgl1	上游：ATCAGAGAGGATTTGGCCGC	122
	下游：AGGAAGATCGGTGTCGCTTG	122