Contributed equally
In preliminary experiments, it was found that the expression of early growth response-1 (Egr-1) was upregulated during the committed differentiation of leukemia cells into monocytes/macrophages. The cross-analysis of gene chip detection and database prediction indicated that Egr-1 was associated with upstream microRNA (miR)-let-7c-3p, thus the present study focused on the role of the miR-let-7c-3p/Egr-1 signaling axis in the committed differentiation of leukemia cells into monocytes/macrophages. Phorbol 12-myristate 13-acetate (PMA) was used to induce the directed differentiation of human K562 leukemia cells into monocytes/macrophages and the differentiation of K562 leukemia cells was determined by cell morphology observation and expression of differentiation antigens CD11b and CD14 by flow cytometry. The expression levels of Egr-1 and miR-let-7c-3p were detected by reverse transcription-quantitative PCR and the protein expression of Egr-1 was detected by western blotting. The effect of Egr-1 on the differentiation of K562 cells was detected by short interfering (si)RNA interference assay. A dual-luciferase reporter assay was used to detect target binding of miR-let-7c-3p on the 3′UTR of Egr-1. Cell transfection of miR-let-7c-3p mimics and inhibitors was used to modulate the expression of miR-let-7c-3p, as indicated by RT-qPCR assays. Western blotting was also used to examine the effect of miR-let-7c-3p on Egr-1 expression. The PMA-induced differentiation of K562 cells was transfected with miR-let-7c-3p and the expression of differentiation antigen was detected by flow cytometry. A differentiation model of K562 leukemia cells into monocytes/macrophages was induced by PMA, which was indicated by morphological observations and upregulation of CD11b and CD14 antigens. The gene or protein expression of Egr-1 was significantly higher compared with that of the control group, while the expression of miR-let-7c-3p was significantly lower compared with that of the control group. siRNA interference experiments showed that the expression of cell differentiation antigen CD14 in the 100 µg/ml PMA + si-Egr-1 group was significantly lower compared with that in the 100 µg/ml PMA + si-ctrl group. The dual luciferase reporter gene results showed that the luciferase activity of the co-transfected mimic and Egr-1 WT groups was significantly lower than that of the NC control group, while the luciferase activity of the co-transfected mimic and Egr-1 MUT groups was comparable to that of the NC control group. Therefore, the dual-luciferase reporter gene assay confirmed that miR-let-7c-3p can target Egr-1. Western blotting showed that the expression of Egr-1 following transfection with miR-let-7c-3p inhibitor was significantly higher compared with that of the negative control and the expression of Egr-1 after transfection with miR-let-7c-3p mimic was significantly lower than that of the negative control. Following exposure to PMA, the expressions of CD11b and CD14 in the miR-let-7c-3p inhibitor group were significantly higher than those in the miR-let-7c-3p NC group, as indicated by CD11b and CD14 respectively. In conclusion, miR-let-7c-3p could bind to the 3′UTR of Egr-1 and negatively regulated Egr-1 expression. The miR-let-7c-3p/Egr-1 signaling axis was closely associated with the committed differentiation of K562 cells from leukemia cells to monocytes/macrophages.
Myeloid leukemia is a type of hematopoietic stem cell malignant tumor, with differentiation disorder, uncontrolled proliferation, or the inability of terminal differentiation of primitive cells to retain malignant proliferation ability and accounting for ~15% of new cases of adult leukemia (
Expression of early growth response-1 (Egr-1) is a member of the early growth response protein family, which has been considered to be of great significance in a variety of physiological processes and has been extensively studied (
micro (mi)RNAs are non-coding small RNAs with post-transcriptional regulation. They are endogenous small RNAs with a length of 18–24 nucleotides. Usually, they can base pair with the 3′ untranslated region (UTR) of target mRNAs and silence genes at the post-transcriptional level by inhibiting mRNA translation or directly causing mRNA degradation and abnormal expression often appears in the occurrence and development of tumors (
In the authors' previous work (data not published), distinct changes in miR-let-7c-3p and Egr-1 expression were detected in a PMA-induced differentiation model of K562 cells. The present study focused on the role of the miR-let-7c-3p/Egr-1 signaling axis in the committed differentiation of K562 leukemia cells into more mature monocytes/macrophages. The results demonstrated that the miR-let-7c-3p/Egr-1 axis was closely associated with the differentiation of K562 cells from leukemia cells into more matured monocytes/macrophages induced by PMA.
Human chronic myeloid leukemia cell line K562 was purchased from Shanghai Cell Bank, Chinese Academy of Sciences. PMA was purchased from American Sigma Company (cat. no. P1585-1MG). Fetal bovine serum (FBS), BCA protein assay kit and SDS-PAGE gel rapid preparation kit were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. RPMI 1640 medium was purchased from HyClone (Cytiva). Swiss-Giemsa staining solution, double antibody, RIPA protein lysis solution and 5X protein loading buffer were purchased from Beijing Solarbio Science & Technology Co., Ltd. Standard protein marker and Lipofectamine® 2000 transfection kit were purchased from Thermo Fisher Scientific, Inc. The ECL luminescence kit was purchased from Shandong Sparkjade Scientific Instruments Co., Ltd. Egr-1 (cat. no. 22008-1-AP), GAPDH (cat. no. 10494-1-AP) and β-actin (cat. no. 20536-1-AP) primary antibodies were purchased from ProteinTech Group, Inc. HRP-labeled rabbit secondary antibody (cat. no. ZB-2301) was purchased from OriGene Technologies, Inc. Primer design was provided by Sangon Biotech Co., Ltd. Reverse transcription kit PrimeScript RT reagent kit with gDNA Eraser (Perfect Real Time), chimeric fluorescence detection kit and TB Green Premix Ex Taq™ (Tli RNaseH Plus) kit was purchased from Takara Biotechnology Co., Ltd. The cycle kit was purchased from Jiangsu KGI Biotechnology Co., Ltd. PE-CD11b (cat. no. 301306) and FITC-CD14 (cat. no. 301804) fluorescent conjugated antibodies were purchased from BioLegend, Inc. TRIzol® reagent was purchased from Thermo Fisher Scientific. Inc.
K562 cells were grown in culture flasks containing 10% FBS in RPMI-1640 complete medium, cultured at 37°C, 5% CO2. In the logarithmic growth phase, an appropriate amount of K562 cell suspension and PMA solution were added to 96-well plates, so that the cell concentration in each well was 1×105/ml and the corresponding dose of PMA solution was added. A total of three duplicate wells were set up in each group and one zero-adjusting well was set up in each plate with only an equal volume of RPMI1640 culture medium (100 µl) added and then cultured at 37°C, 5% CO2 with saturated humidity. At 24, 48 and 72 h of culture 10 µl of CCK8 solution was added to each well, except the blank well and incubated at 37°C for 2 h and detected at 450 nm. According to the IC50 experimental results of 48 h of culture, the control group (PMA 0 ng/ml) and the experimental group (the final concentration of PMA 100 ng/ml) were selected.
The control group with 0 ng/ml PMA and the experiment group with 100 ng/ml PMA of K562 cells induced for 48 h without staining were observed directly under an inverted optical microscope. Cells of the above-mentioned control and experiment group were collected at 48 h, washed twice with cold PBS, resuspended with 100 µl PBS and mixed by gently pipetting to make a cell suspension. After centrifugation at 200 × g for 3 min at 4°C, the centrifuged smears were dried and stained with Wright-Giemsa Stain Solution for 5 min at room temperature. The changes of cell morphology were observed under different magnifications of the optical microscope and the resulting images were captured.
K562 cells were collected into flow tubes, resuspended in PBS, washed twice by centrifugation at 200 × g for 3 min at 4°C, and adjusted to a cell concentration of 1×106 cells/ml. PBS (100 µl) was added to each tube, followed by 5 µl PE-labeled mouse anti-human CD11b fluorescent antibody and FITC-labeled mouse anti-human CD14 fluorescent antibody respectively and incubated at 4°C for 30 min in the dark. The cells were centrifuged at 200 × g for 4 min at 4°C and washed twice with PBS to remove excess monoclonal antibody. The cells were resuspended in 200 µl PBS and then fixed in 1% paraformaldehyde. The expressions of CD11b and CD14 in different treatment groups were analyzed on FACSVerse (BD Biosciences Pharmingen) Flow cytometer. Isotypic rat IgG was also used to check for nonspecific binding. The experiment was repeated three times.
The cells of the control group and the experimental group were collected, washed twice with pre-cooled PBS, cells were lysed with Protein Extraction reagent (Beijing Solarbio Science & Technology Co., Ltd.), the total cell protein was extracted, the protein concentration was determined by BCA method and 5× protein loading buffer was added and boiled for denaturation at 95°C for 10 min. Protein (20 µg) was loaded for SDS-PAGE (10%) electrophoresis, the separated protein was transferred to PVDF membrane, blocked with 5% skimmed milk for 90 min at room temperature and then incubated with the corresponding primary antibody; Egr-1 (1:800), GAPDH (1:8,000) and β-actin (1:2,000) at 4°C overnight, then washed with 1X TBST for 30 min, then incubated with goat anti-rabbit IgG secondary antibody (1:20,000) at room temperature for 90 min and finally washed for 30 min, and proteins were detected using an ECL kit (Sparkjade ECL super, ED0015-B, Shandong Sparkjade Scientific Instruments Co., Ltd.). ImageJ v1.51j8 was used for densitometry (National Institutes of Health). The experiment was repeated three times.
TargetScan, PITA and microRNAorg databases were used to predict target genes of possible upstream miRNAs of Egr-1 and intersected the predicted target miRNAs by crosstalk of the three databases. The common target miRNAs in the three databases were obtained, and the top 10 miRNAs were selected according to the P-value and literature research. In addition, GeneChip miRNA 4.0 (Affymetrix Co., Ltd.) was used to detect the different miRNA profiles between control and PMA-induced K562 cells, and 10 miRNAs that were reduced after induced-differentiation were screened according to the P-value.
When the K562 cells were cultured to the logarithmic growth phase, the cells (1×106) were collected and the total RNA was extracted by TRIzol® (Thermo Fisher Scientific, Inc.) method and the total RNA concentration was measured by an ultra-trace nucleic acid and protein analyzer. cDNA was synthesized according to the instructions of the PrimeScript RT reagent kit with gDNA Eraser (Perfect Real Time) reverse transcription kit. For reverse transcription, samples were incubated in an Eppendorf PCR system at 42°C for 30 min, then at 90°C for 5 min and at 5°C for 5 min. cDNA was used as a template for PCR amplification. The sense primer of miR-let-7c-3p was 5′-GCGCGCTGTACAACCTTCTAG-3′, the antisense primer was 5′-AGTGCAGGGTCCGAGGTATT-3′; the U6 sense primer was 5′-AGAGAAGATTAGCATGGCCCCTG-3′, Antisense is 5′-AGTGCAGGGTCCGAGGTATT-3′; Egr-1 sense primer was 5′-AGCAGCAGCAGCACCTTCAAC-3′, antisense was 5′-CCACCAGCACCTTCTCGTTGTTC-3′; GAPDH sense primer is 5′-CAACTTTGGTATCGTGGAAGG-3′, antisense was 5′-GCCATCACGCCAGAGTTTC-3′. The real-time fluorescence quantitative amplification reaction was performed according to the instructions of the TB Green® Premix Ex Taq (Tli RNaseH Plus) kit, PCR was conducted with the following conditions: 10 sec at 95°C; 40 cycles of 5 sec at 60°C and 10 sec at 72°C; 34 sec at 60°C, and the relative quantitative analysis was performed using the 2-ΔΔCq method (
Using the bioinformatics prediction website (
An appropriate amount of short interfering RNA (siRNA) and its corresponding negative control were mixed with the transfection reagent to form a transfection complex, which was added to the 6-well plate that had been seeded with cells and cultured at 37°C for 48 h for subsequent experiments. Egr-1 siRNA-1: 5′-CCAUGGACAACUACCCUAATT-3′, Egr-1 siRNA-2: 5′-GCCUAGUGAGCAUGACCAATT-3′ and Egr-1 siRNA-3: 5′-GCUGUCACCAACUCCUUCATT-3′ synthesized by Shanghai BioSune Co., Ltd. were selected. According to the preliminary experimental results, the Egr-1 siRNA-1 with the most obvious interference effect (5′-CCAUGGACAACUACCCUAATT-3′) was selected as the target siRNA (hereinafter referred to as siEgr-1). As to the si-RNA interference experiment, there were four groups. In addition to the above 100 µg/l PMA experimental group and 0 µg/l PMA control group, the 100 µg/l PMA experimental group was transfected with si-ctrl at the same time as the PMA + si-Ctrl group. The 100 µg/l PMA experimental group was transfected with siEgr-1 at the same time as PMA + si-Egr-1 group.
miR-let-7c-3p mimic and inhibitor were synthesized by Shanghai GenePharma Co., Ltd. and their sequences were 5′-CUGUACAACCUUCUAGCUUUCC-3′, 5′-GGAAAGCUAGAAGGUUGUACAG-3′, corresponding NCs were: 5′-UUCUCCGAACGUGUCACGUTT-3′ and 5′-GGAAAGCUAGAAGGUUGUACAG-3′, respectively. The conventional transfection method of Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.) was used. Following transfection, RT-qPCR and western blotting were used to detect the expression changes of miR-let-7c-3p and downstream Egr-1 protein expression, respectively. iii) The effect of miR-let-7c-3p on the expression of differentiation antigens in K562 cells: In the above experiments of transfection of miR-let-7c-3p inhibitor, the expression changes of K562 cell differentiation antigens CD11b and CD14 were detected at the same time and let-7c-3p inhibitor NC was used as a control. The expression levels of differentiation antigens in the 100 µg/l PMA experimental group and the 0 µg/l PMA control group were also observed.
GraphPad Prism 8 software (GraphPad Software, Inc.) was used for data processing and Shapiro-Wilk (S-W) normal distribution was used for quantitative data. Each experiment was repeated three times and the measurement data were expressed as mean ± standard deviation. Comparisons between two groups were performed using independent samples t-test and ANOVA was used for multiple-group comparisons. The Bonferroni test was used as the post-hoc test for the one-way ANOVA test. Pearson analysis was used for the correlation between miR-let-7c-3p and Egr-1. All data were analyzed by two-tailed test. P<0.05 was considered to indicate a statistically significant difference.
The
To further validate the committed differentiation of leukemia cells into monocytes/macrophages, the present study examined the expression of monocyte/macrophage-specific surface markers CD11b and CD14 in K562 cells treated with PMA for 48 h. The results of flow cytometry showed that the expression of CD11b in the PMA-induced group was significantly higher compared with that in the control group (49.47±3.48 vs. 3.54±0.54; t=24.070; P=0.002) and CD14 in the PMA-induced group was significantly higher compared with that in the control group (59.84±5.26 vs. 6.79±0.66; t=16.670; P=0.004;
The expression level of Egr-1 in normal human peripheral blood mononuclear cells and K562 cells was detected by western blotting and the results showed that the expression of Egr-1 protein in K562 cells was significantly lower compared with that in normal controls (
The expression changes of Egr-1 in K562 cells before and after PMA-induced differentiation were detected. The results confirmed that compared with the control group, the expression of Egr-1 was significantly increased after PMA induction (0.19±0.02 vs. 0.85±0.03; t=24.800; P<0.001;
In the preliminary experiments, TargetScan, PITA and microRNAorg databases were used to predict target genes of possible upstream miRNAs of Egr-1 and intersected the predicted target miRNAs by crosstalk of the three databases. According to the results of target gene prediction, the common target miRNAs in the three databases were obtained. After sorting according to the P-value and literature research, the top 10 miRNAs were selected (
The regulatory effect of miR-let-7c-3p mimic and inhibitor on the expression of miR-let-7c-3p were first verified and the results confirmed that the expression level of miR-let-7c-3p in the miR-let-7c-3p inhibitor group was significantly lower compared with that in its negative control group (0.44±0.42 vs. 0.96±0.05; t=12.870; P=0.006). The expression level of miR-let-7c-3p in the miR-let-7c-3p mimic group was significantly higher compared with that in the negative control group (418.80±17.33 vs. 1.01±0.02; t=41.760; P<0.001;
Further analysis of the StarBase database revealed a binding site between miR-let-7c-3p and Egr-1, while the TargetScan database predicted a pairing site between miR-let-7c-3p and Egr-1 (
To explore the effect of miR-let-7c-3p on PMA-induced differentiation of K562 cells, K562 cells were transfected with miR-let-7c-3p inhibitor and its negative control, treated with PMA (100 ng/ml) for 48 h to induce differentiation and the expression of cell surface markers CD11b and CD14 in each group was detected by flow cytometry. The results, as shown in
PMA also significantly increased the expression of CD14 in K562 cells. The expression levels of CD14 in the control group, PMA group, PMA + NC group and PMA + inhibitor group were 4.91±0.42%, 70.54±1.44%, 71.91±0.74% and 85.98±0.52%, respectively (
Myeloid leukemia is a type of hematopoietic stem cell malignant tumor with the characteristics of differentiation disorder and uncontrolled proliferation (
The Egr-1 gene, as a member of the zinc finger structure transcription factor family, is located on human chromosome 5q31 and encodes a DNA-binding transcription factor of ~80 kDa (
It has been demonstrated that some important transcriptional regulatory proteins are often regulated by epigenetic factors, such as non-coding RNAs, at the same time that some important transcriptional regulatory proteins are involved in transcriptional regulation. Usually, one important pathway for microRNAs (miRNAs) to result in gene silencing or translational repression was mainly contributed to binding to 3′UTR of target mRNAs (
miR-let-7, as the earliest discovered human miRNA, is one of the most widely studied miRNAs. Its family members are abnormally expressed in various malignant tumors and become a new target for tumor prevention and treatment (
In the preliminary experiments of the present study, it was found that some miRNAs changed significantly during the process of differentiation of leukemia cells into mature monocytes/macrophages induced by PMA. Further analysis of the StarBase database revealed a binding site between miR-let-7c-3p and Egr-1, while the TargetScan database predicted a pairing site between miR-let-7c-3p and Egr-1. In addition, following Agilent miRNA Chip assay, miR-let-7c-3p also reduced after induced-differentiation by PMA with the upregulation of Egr-1 mRNA. Furthermore, it was found that the expressions of miR-let-7c-3p and Egr-1 showed an inverse relationship by Pearson analysis in the differentiation process induced by PMA and that they had a nucleic acid sequence basis for targeted binding. In addition, Egr-1 is widely associated with miRNAs and can be regulated by miRNAs in a variety of tumors (
Some other studies (
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
GSJ, XDW, FQ, XPW and CZW made substantial contributions to the conception and design and also critically reviewed the study. FQ, XPW, CZW, RJS and HW performed the experiments. FQ, SZZ, PCD and JW analyzed the data and wrote the manuscript. GSJ and XDW confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Changes in morphology and proliferation of K562 cells after 48 h of PMA treatment. (A) Cell growth density before induction (magnification, ×200). (B) Cell growth density at 48 h after induction (magnification, ×200). (C) CCK8 detection of cell proliferation prior to and following induction. (D) Swiss-Giemsa staining prior to induction (magnification, ×400). (E) Swiss-Giemsa staining of cells following induction (magnification, ×400). (F) Changes in percentage of mature cells prior to and following induction (****P<0.0001; NS, no significance). PMA, phorbol 12-myristate 13-acetate; Ctrl, control.
Effect of PMA on the expression of CD11b and CD14 on the surface molecule of K562 cells. (A) CD11b expression group prior to and following induction (B) CD14 expression group prior to and following induction. (C) Average expression levels of CD11b and CD14 (**P<0.01). PMA, phorbol 12-myristate 13-acetate.
Differences in expression of Egr-1 between K562 cell control group and induced group. (A and B) Egr-1 expression levels in normal human PBMC and K562 cells. (C) Egr-1 mRNA relative expression statistics prior to and following induction. (D and E) Egr-1 expression prior to and following K562 cell differentiation variety (*P<0.05, **P<0.01, ***P<0.001). Egr-1, early growth response-1; PBMC, peripheral blood mononuclear cells.
The effect of siRNA-Egr-1 on differentiation of K562 cells induced by PMA. (A and B) Changes of Egr-1 expression following PMA induction. (C and D) Changes of expression levels of differentiation antigen CD14 following PMA induction. (E and F) Egr-1 protein expression in PMA + siEgr-1 co-action group. (G and H) Changes in the expression of CD14 differentiating antigen in the PMA + siEgr-1 group (**P<0.01, ***P<0.001). si, short interfering; Egr-1, early growth response-1; PMA, phorbol 12-myristate 13-acetate.
Relationship between miR-let-7c-3p and Egr-1. (A) TargetScan, PITA and microRNAorg databases predicted the top ten miRNAs. (B) Agilent miRNA Chip detected 10 reduced miRNAs. (C) Reverse transcription-quantitative PCR detected the expression level of miR-let-7c-3p following PMA induction. (D) Correlation of changes in miR-let-7c-3p and Egr-1 (**P<0.01). miR, microRNA; Egr-1, early growth response-1; PMA, phorbol 12-myristate 13-acetate.
Effect of upregulation or downregulation of miR-let-7c-3p on the expression of Egr-1 in K562 cells. (A) Verification of the regulation of miR-let-7c-3p inhibitor on miR-let-7c-3p. (B) Verification of the regulation of miR-let-7c-3p mimic on miR-let-7c-3p. (C) The effect of miR-let-7c-3p inhibitor on Egr-1 protein expression. (D) The effect of miR-let-7c-3p inhibitor on the average level of Egr-1 protein expression. (E) The effect of miR-let-7c-3p mimic on Egr-1 protein expression. (F) The effect of miR-let-7c-3p mimic on the average level of Egr-1 protein expression (*P<0.05, **P<0.01, ***P<0.001). miR, microRNA; Egr-1, early growth response-1; NC, negative control.
Expression of miR-let-7c-3p and targeting on Egr1. (A) 3′ UTR binding region of miR-let-7c-3p and Egr-1. (B) Luciferase activity detection (ns, no significance; ***P<0.001). miR, microRNA; Egr-1, early growth response-1; WT, wild-type; MUT, mutant.
Effect of miR-let-7c-3p on PMA-induced differentiation of K562 cells. (A) CD11b expression in control group. (B) CD11b expression in K562 cells induced by PMA. (C) CD11b expression in PMA + NC group. (D) CD11b expression in PMA + miR-let-7c-3p inhibitor group. (E) Average level of CD11b in each group. (F) CD14 expression in control group. (G) CD14 expression in K562 cells induced by PMA. (H) CD14 expression in PMA + NC group. (I) CD14 expression in PMA + miR-let-7c-3p inhibitor group. (J) Average level of CD14 in each group (**P<0.01, ***P<0.001). miR, microRNA; PMA, phorbol 12-myristate 13-acetate; NC, negative control.