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Circ_0009910 promotes imatinib resistance through ULK1-induced autophagy by sponging miR-34a-5p in chronic myeloid leukemia
Life Sciences 243 (2020) 117255
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Circ_0009910 promotes imatinib resistance through ULK1-induced autophagy by sponging miR-34a-5p in chronic myeloid leukemia
T
Hai-xia Caoa, Chao-feng Miaob, Li-na Sanga, Yu-min Huanga, Ran Zhanga, Ling Suna, ⁎ Zhong-xing Jianga, a b
Department of Hematology, The First Affiliated Hospital of Zhengzhou University, 450052 Zhengzhou, Henan, China Department of Vascular surgery, The First Affiliated Hospital of Zhengzhou University, 450052 Zhengzhou, Henan, China
A R T I C LE I N FO
A B S T R A C T
Keywords: CML Circ_0009910 MiR-34a-5p Autophagy ULK1
Background: The occurrence in drug resistance of chronic myeloid leukemia (CML) was accompanied by autophagy activation. Abnormal circular RNAs (circRNAs) participated in this progression. This study attempted to investigate the potential role of circ_0009910 in imatinib resistance of CML cells. Methods: The expression of circ_0009910 and miR-34a-5p was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The characterization of circ_0009910 was investigated using oligo (dT)18 primers, Actinomycin D and RNase R. Cell viability (IC50 value) and apoptosis were assessed by Cell Counting Kit-8 (CCK8) assay and flow cytometry assay, respectively. The relative protein expression was quantified by western blot. The relationship among miR-34a-5p, circ_0009910 and ULK1 was predicted by online bioinformatics tool, and verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP). Results: The expression of circ_0009910 was up-regulated in the serum of imatinib-resistance CML patients and K562/R cells, and associated with unfavorable clinicopathologic features. Circ_0009910 in K562 and K562/R cells was mainly localized in the cytoplasm. Circ_0009910 knockdown inhibited cell proliferation and autophagy, but induced apoptosis in K562/R cells. Circ_0009910 targeted miR-34a-5p to regulate ULK1. MiR-34a-5p depression rescued the effects of circ_0009910 knockdown on apoptosis and autophagy in K562/R cells. Conclusion: Circ_0009910 accelerated imatinib-resistance in CML cells by modulating ULK1-induced autophagy via targeting miR-34a-5p, providing a potential target in imatinib resistance of CML.
1. Introduction Chronic myeloid leukemia (CML), prevalent malignant tumor, is one of the leading causes of hematological tumor-related mortality with a long-duration remedy for chemotherapy [1,2]. The clinical results of CML still remained unsatisfied due to the occurrence of metastasis and drug resistance [3–5]. Although plenty of research has illuminated the pathogenesis of CML, whereas, the underlying molecular mechanisms and effective treatment way during CML therapy were mostly deficient. Therefore, it was essential to understand the pathogenesis and found a novel treatment strategy for CML. Circular RNAs (circRNAs) are a series of endogenous noncoding RNAs with covalently closed loop structure [6]. A previous study clarified that circRNAs were more stable than linear mRNA, due to the lack of 5′ cap and 3′ polyadenylated tail [7]. CircRNAs were specifically expressed in hematological malignancies, indicating that circRNAs
might serve as post-transcriptional regulators [8,9]. For instance, the high expression of circ_0009910 predicted poor prognosis in patients with CML and was associated with imatinib resistance [10]. Overexpression of circ_0009910 promoted tumor cell proliferation, metastasis, and inhibited cell apoptosis [11–13]. The role of autophagy in drug-resistance has been widely reported in various cancers, including CML. MiR-199a/b-5p inhibited autophagy via repressing WNT2 signaling and might provide novel therapeutic strategy for imatinib-resistant CML therapy [14]. Recent studies have shown that the combination of TKIs with autophagy inhibitors was an effective method for the treatment of CML [15,16]. We aimed to explore the underlying mechanism of circ_0009910 in autophagy-mediated CML cell resistance. Furthermore, it is widely reported that miRNA could negatively regulate gene expression at the post-transcriptional level by degrading or repressing translation of mRNA [17,18]. MiR-34a-5p, located on
⁎ Corresponding author at: Department of Hematology, The First Affiliated Hospital of Zhengzhou University, No. 1 Jianshedong Road, Zhengzhou 450052, Henan, China. E-mail address: [email protected] (Z.-x. Jiang).
https://doi.org/10.1016/j.lfs.2020.117255 Received 16 October 2019; Received in revised form 27 December 2019; Accepted 31 December 2019 Available online 07 January 2020 0024-3205/ © 2020 Elsevier Inc. All rights reserved.
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CTTAG-3′), miR-34a-5p reverse (5′-CAGTGCGTGTCGTGGAGT-3′), ULK1 forward (5′-CAGCAAAGGCATCATCCAC-3′), ULK1 reverse ( 5′-GGTTGCGTTGCAGTAGGG-3′), GAPDH forward (5′-ACCACAGTCCA TGCCATCAC-3′), GAPDH reverse (5′-TCCACCACCCTGTTGCTGTA-3′), U6 forward (5′-GCUUCGGCAGCACAUAUACUAAAAU-3′), U6 reverse ( 5′-CGCUUCACGAAUUUGCGUGUCAU-3′).
chromosome 1p36 [19], was decreased in breast cancer [20], bladder cancer [21], non-small cell lung cancer [22] and metastatic colorectal cancer [23]. Besides, the expression level of miR-34a-5p was associated with resistance in several diseases, including osteosarcoma [24], ovarian cancer [25], prostate cancer [26], and non-small cell lung cancer [27]. Autophagy may take part in mediating resistance of cancer cells to anticancer therapy [28–30]. Interestingly, miR-34a-5p was found to be involved in autophagy in the pathological and physiological processes, including hypoxia-reoxygenation injury, proliferation, and fibrosis [31,32]. However, it has not been reported that the potential role of miR-34a-5p in CML resistance. Thus, we explore whether miR34a-5p was involved in the regulation of autophagy in CML. UNC-51-like kinase (ULK1), as a homologous gene of yeast Atg1, is a serine-threonine kinase and a mammalian autophagy promoter [33]. The autophagy signal was mediated by activation of the ULK complex consisting of ULK1 or ULK2, FIP200, and mATG13, which was prior to autophagic lysosomal assembly [34]. The ULK1 complex was a bridge between the upstream nutrient or energy receptors mTOR and AMPK and downstream autophagosomes in the body [35]. Meanwhile, the ULK1-induced autophagy activation signals can be regulated by miRNA [36]. This study was designed to elucidate the molecular mechanisms of CML resistance and provided the novel perspectives for the treatment of CML.
2.4. Cell Counting Kit-8 (CCK8) assay The CCK-8 reagent (DOJINDO, Kumamoto, Japan) was used to detect cell viability. Two cell lines with or without transfection were treated with imatinib (0.02 μM, 0.1 μM, 0.5 μM, 2.5 μM, 12.5 μM, 62.5 μM, 312.5 μM) for 48 h, then 10 μL CCK-8 solution was added into each well for another 4 h. The proliferation of K562 and K562/R cells was detected at wavelength of 450 nm each well on a microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA). IC50 values were calculated as previously described [37]. 2.5. Circularization detection of circ_0009910 Random primers (Invitrogen, Carlsbad, CA, USA) and oligo (dT)18 primers (Invitrogen) were used for qRT-PCR. 2.6. Actinomycin D treatment
2. Materials and methods
To detect the stability of circ_0009910, 2 mg/mL Actinomycin D (Sigma-Aldrich, St. Louis, MO, USA) was added before RNA isolation. Then, qRT-PCR analysis was carried out.
2.1. Serum samples collection This study acquired the authorization of the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. A total of 65 serum specimens from CML patients (imatinib-sensitive n = 30, imatinib-resistance n = 35) were collected from the First Affiliated Hospital of Zhengzhou University. Written informed consent was obtained from each subject before imatinib chemotherapy. All blood samples were collected by anticoagulant tubes containing heparin and centrifuged to obtain supernatants and stored at −80 °C condition.
2.7. Rnase R treatment
2.2. Cell lines, cell culture and drug treatment
RNAs in cytoplasm and nucleus were extracted using the Cytoplasmic and Nuclear RNA Purification Kit (Norgen Biotek, Thorold, Canada). Total RNA in each fraction was used for qRT-RCR analysis. GAPDH and U6 were acted as cytoplasm and nucleus internal references, respectively.
To confirm the existence of circ_0009910, the RNA extraction was incubated with or without Rnase R (Applied Biological Materials Inc., Vancouver, Canada) at 37 °C for 10 min. Then, qRT-PCR analysis was conducted. 2.8. Nuclear and cytoplasmic localization analysis
Parental and resistant cell lines of CML (K562 and K562/R, individually) were purchased from Shanghai Innovation Biotechnology Co., Ltd. (Shanghai, China). All cells were cultured in 90% Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) with 10% fetal Bovine Serum (FBS; Gibco) at 37 °C condition with 5% CO2. For drug treatment, K562 and K562/R cells were treated with different concentrations of imatinib (Sigma, St. Louis, MO, USA). According to IC50 value, the concentration of 0.5 μM for K562 or 12.5 μM for K562/R cells was used in subsequent experiments. 3-Methyladenine (5 mM, 3MA, Sigma) or rapamycin (100 nmol, Rap, Sigma) was added to overnight-cultured cells for the indicated time in K562 or K562R cells.
2.9. Cell transfection For circ_0009910 downregulation, short hairpin RNA (shRNA) against circ_0009910 (sh-circ_0009910) and its negative control (shNC) were constructed by Genepharma (Shanghai, China). For circ_0009910 upregulation, circ_0009910 sequences were amplified by PCR and inserted into pcDNA3.1 vector (Invitrogen) (circ_0009910), and the corresponding control was pcDNA3.1 empty vector (vector). MiR-34a-5p mimics (miR-34a-5p), miR-34a-5p inhibitor (anti-miR-34a5p) and their own negative control (miR-NC and anti-NC) were purchased from Ribobio (Guangzhou, China). All sequences were transfected into K562 and K562/R cells using Lipofectamine 2000 (Invitrogen). The used sequences were listed as below: sh-circ_0009910 sequence (3′-TGGCCGCGCAATGTCCCTGCT-5′), sh-NC sequence ( 3′-UCACCCAGAUGCCGCUAU-5′). Rapamycin (200 nM) and 3-MA (10 mM) (Sigma Aldrich, St Louis, MO, USA) were added into cells after transfection.
2.3. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was separated from CML serums and cells using Total RNA Extractor (Sangon Biotech, Shanghai, China). Then the HiScript III First Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) or Mir-X™ miRNA First Strand Synthesis Kit (Clontech, Mountain View, CA, USA) was used to assemble complementary DNA (cDNA). Next, qRT-PCR was performed using AceQ Universal SYBR qPCR Master Mix (Vazyme) on CFX Connect system (Bio-Rad, Hercules, CA, USA). The fold-change of expression was calculated using the 2−ΔΔCt method and normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or small nuclear RNA U6. The used primers were listed as below: circ_0009910 forward ( 5′-TGAGAGGCATCAGTGAGGTG-3′), circ_0009910 reverse (5′-AAGTG CTTAAGTGGGGATGC-3′), miR-34a-5p forward (5′-GCCCTGGCAGTGT
2.10. Flow cytometry assay Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme, Nanjing, China) was used for flow cytometry assay. In brief, K562 and K562/R 2
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circ_0009910 in serum, and the ROC analysis demonstrated an area under the curve (AUC) of 0.831, with a diagnostic sensitivity and specificity reaching 65.71% and 95.33%, respectively (95% CI = 0.7317–0.9331) (Fig. 1B). Based on the cut-offs (1.729) established by ROC, the proportion of patients not responding to gefitinib therapy was significantly higher in the high circ_0009910 expressing group than in the low expressing group (Fig. 1C). We further analyzed the relationship between circ_0009910 level and survival time in CML patients. The Kaplan-Meier survival curve showed that the patients with high circ_0009910 expression had shorter overall survival rate than those with low circ_0009910 expression (Fig. 1D).
cells were seeded into 6-well plates, and then treated with 0.25% trypsin and washed using pre-cooled phosphate buffer saline (PBS). Then, cells (2 × 105) were resuspended by 100 μL 1× binding buffer and then incubated with 5 μL Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining solution for 10 min at room temperature without light. Finally, the apoptotic cells were analyzed using flow cytometer (BD Biosciences, San Jose, CA, USA). 2.11. Western blot Western blot analysis was performed as previously described [14]. The primary antibodies against Cleaved Caspase-3 (ab2302, 1:1000, Abcam, Cambridge, UK), Bax (Bcl-2-associated X protein, ab53154, 1:1000, Abcam), Bcl-2 (B-cell lymphoma-2, ab196495, 1:1000, Abcam), LC3-I/LC3-II (ab62721, 1:1000, Abcam), Beclin1 (ab62557, 1:1000, Abcam), P62 (ab155686, 1:1000, Abcam), ULK1 (ab167139, 1:1000, Abcam), p-ULK1 (ab133747, 1:1000, Abcam), GAPDH (ab9485, 1:2500, Abcam) and goat anti-rabbit secondary antibodies (ab205718, 1:5000, Abcam) were used in this study.
3.2. Confirmation of the circular structure and subcellular localization of circ_0009910 As shown in Fig. 2A, compared with parental cells, the established resistant cells showed imatinib-resistance, as shown by increased IC50 value and elevated cell viability. Next, the circ_0009910 was highly expressed in K562/R cells (K562 resistant cells) compared with that of K562 cells (Fig. 2B). Circ_0009910 was derived from MFN2 gene exon 3–4, with a spliced mature sequence length of 315 base pairs (bp) (Fig. 2C). Next, we investigated the stability and localization of circ_0009910 in K562 and K562/R cells. Total RNAs from K562 and K562/R cells were isolated at the indicated time points after treatment with Actinomycin D, an inhibitor of transcription. Then qRT-PCR was performed to measure the level of circ_0009910 and MFN2 mRNA. The results showed that the half-life of circ_0009910 exceeded 24 h, whereas that of MFN2 mRNA was about 3.5 h in both K562 and K562/R cells (Fig. 2D–E). Furthermore, we found that circ_0009910 was resistant to RNase R digestion (Fig. 2F–G). These data confirmed that circ_0009910 was a circular RNA. We then investigated the localization of circ_0009910. qRT-PCR of RNAs from nuclear and cytoplasmic fractions indicated that circ_0009910 was predominantly localized in the cytoplasm of K562 and K562/R cells (Fig. 2H–I). Collectively, the above data suggested that circ_0009910 harbored a loop structure and was predominantly localized in the cytoplasm.
2.12. Bioinformatics analysis and dual-luciferase reporter assay The online bioinformatics tool starBase was used to predict the potential target genes and the specific binding sites. The relationship among miR-34a-5p, circ_0009910 and ULK1 was verified by dual-reporter assay. In brief, the sequence of wild type circ_0009910 (WTcirc_0009910) containing the binding sites of miR-34a-5p, and the corresponding mutant circ_0009910 (MUT-circ_0009910) sequence was amplified and cloned into the pRL-CMV vector (Promega, Madison, WI, USA). Then, WT-circ_0009910 or MUT-circ_0009910 and miR-34a-5p or miR-NC were co-transfected into K562 and K562/R cells. After transfection for 48 h, the luciferase activity was detected using the Dual-Luciferase Reporter Assay Kit (Promega). Similarly, WT-ULK1 and MUT-ULK1 were also constructed and used for luciferase activity analysis. 2.13. RNA immunoprecipitation (RIP) assay Magna RIP Kit (Millipore, Billerica, MA, USA) was used for RIP assay. In brief, K562 and K562/R cells were collected and resuspended in RNA immunoprecipitation lysis buffer containing magnetic beads, and then incubated with anti‑argonaute 2 (anti-Ago2) or IgG antibodies. Next, the protein was digested using proteinase K buffer, followed by RNA purification. Subsequently, the enrichment of circ_0009910, miR-34a-5p, or ULK1 was determined by qRT-PCR.
3.3. Circ_0009910 enhanced imatinib resistance but reduced the imatinibinduced apoptosis in K562 and K562/R cells In order to further explore the role of circ_0009910 in imatinib-resistance cells, we performed overexpression or silencing of circ_0009910 experiments in K562 or K562/R cells, respectively. As shown in Fig. 3A–B, the expression of circ_0009910 was decreased in K562 cells, or increased in K562/R cells, individually, compared to its negative controls. Meanwhile, overexpression of circ_0009910 led to an increase of IC50 in K562 cells, while knockdown of circ_0009910 resulted in a significant decreased IC50 in K562/R cells and repressed anchorage-independent growth upon imatinib treatment (Fig. 3C–D). After that, we measured the ability of apoptosis in K562 and K562/R cells, the rate of apoptosis was inhibited in K562 cells treated with circ_0009910 vector, and apoptosis was enhanced in K562/R cells administrated with sh-circ_0009910 (Fig. 3E–F). Western blot assay showed that upregulation of circ_0009910 triggered the expression of Bcl-2, but impeded that of Cleaved caspase-3 and Bax in K562 cells. Opposite results could be seen in K562/R cells with suppression of circ_0009910 (Fig. 3G–H).
2.14. Statistical analysis Data were collected from at least 3 independent experiments, processed by GraphPad Prism 5.0 (GraphPad software Inc., San Die, CA, USA), and exhibited as mean ± standard deviation (SD). The difference analyses between groups were performed using one-way analysis of variance (ANOVA) or Student's t-test. P value < 0.05 was regarded as statistically significant. 3. Results 3.1. Serum expression of circ_0009910 is associated with imatinib resistance in CML patients
3.4. Circ_0009910 enhanced imatinib resistance by triggering autophagy in K562 and K562/R cells
First, we extracted the serum samples from patients with or without imatinib-resistance and tested the expression of circ_0009910 in serum components by qRT-PCR. Results showed that compared with imatinibsensitive corresponding controls, a significant promotion of circ_0009910 was observed in the imatinib-resistance group (Fig. 1A). After that, we used the ROC curve to evaluate the diagnostic value of
In order to further explore the regulatory mechanism of circ_0009910 in drug-resistant cells, according to previous researches reported that autophagy might be involved in the occurrence and development of tumor resistance [10], we hypothesized that circ_0009910 3
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Fig. 1. Serum expression of circ_0009910 was associated with imatinib resistance in CML patients. (A) QRT-PCR assay was used for differential expression of circ_0009910 in imatinib-sensitive (n = 30) or imatinib-resistance (n = 35) samples from CML patients. (B) ROC curve analysis showed that the diagnostic value of circ_0009910 in CML patients. (C) qRT-PCR for the proportion of patients showed that high expression of circ_0009910 was observed in imatinib-resistant group. (D) The Kaplan-Meier survival cure showed the survival rate in patients with high circ_0009910 expression (n = 40) and with low circ_0009910 expression (n = 25). ⁎⁎⁎P < 0.001.
starBase predicted that miR-34-5p contained the binding sites of circ_0009910 and ULK1 (Fig. 5E). Then, dual-luciferase reporter assay clarified that the luciferase activity was markedly decreased in K562/R cells co-transfected with WT-circ_0009910 and miR-34a-5p compared with miR-NC, while the luciferase activity had no noticeable change in K562/R cells co-transfected with MUT-circ_0009910 and miR-34a-5p compared with miR-NC (Fig. 5F). Meanwhile, several binding sites existed between miR-34a-5p and ULK1 3′ UTR, and the transfection of miR-34a-5p significantly reduced the luciferase activity in K562/R cells transfected with WT-ULK1-3′ UTR but did not alter the luciferase activity with the transfection of MUT-ULK1-3′ UTR (Fig. 5G). The similar results were discovered in K562 cells (Supplement Fig. 2B–C). The enrichment of circ_0009910, miR-34a-5p, and ULK1 could be combined to Ago2 protein in both K562 (Fig. 5H) and K562/R cells (Supplement Fig. 2D). Moreover, miR-34a-5p showed the successful overexpression efficiency in K562/R cells (Fig. 5I), while anti-miR-34a-5p showed the successful knockdown efficiency in K562 cells (Supplement Fig. 2E). Moreover, the protein expression of ULK1 was inhibited in K562/R cells transfected with miR-34a-5p, while circ_0009910 reversed the inhibitory effect of miR-34a-5p overexpression on ULK1 expression (Fig. 5J). In addition, miR-34a-5p deletion promoted the protein expression of ULK1 in K562 cells, which was reversed by circ_0009910 knockdown (Supplement Fig. 2F). These data suggested that miR-34a5p was a target of circ_0009910 and targeted ULK1, and the expression of ULK1 was modulated by circ_0009910 through miR-34a-5p.
might mediate the activation of autophagy, which subsequently lead to the development of resistance. Western blot assessment examined the expression of autophagy-related proteins, and K562/R cells exhibited an aberrant expression of LC3B-I/II and Beclin1, but limited pattern of P62 compared to the parental cells (Fig. 4A). The above results indicated that circ_0009910 might promote the activation process of autophagy in K562/R cells. Then, we performed loss- or gain-functional assays, and results suggested that autophagy response was reactivated after K562/R cells transfected with sh-circ_0009910 (Fig. 4B), while deactivated response could be seen in K562 cells with circ_0009910 overexpression (Supplement Fig. 1A). Besides, inhibitor (3-MA) or agonist (Rap, rapamycin) of autophagy was used for rescue experiments in K562 or K562/R cells, respectively. Rap impaired cell apoptosis induced by downregulating circ_0009910 in K562/R cells (Fig. 4C), while 3-MA reserved the inhibition effect of circ_0009910 upregulation on cell apoptosis in K562 cells (Supplement Fig. 1B). 3-MA administration inverted the effects of circ_0009910 upregulation on apoptosis-related protein expression in K562 cells (Supplement Fig. 1C), while Rap reversed the effects of circ_0009910 knockdown on Cleaved caspase-3, Bax and Bcl-2 expression in K562 cells (Fig. 4D). 3.5. MiR-34a-5p was a target of circ_0009910and directly targeted ULK1 ULK1, a kind of serine-threonine kinase, is regarded as a mammalian autophagy promoter. In previous reports, He and Yu et al. showed that ULK1 was highly expressed in imatinib-resistance CML cells [38,39]. Thus, we tested the protein level of ULK1 and found that it was upregulated in imatinib-resistance K562 cells (Fig. 5A). We found that the expression of ULK1 was inhibited by circ_0009910 downregulation in K562/R cells (Fig. 5B), and ULK1 expression was upregulated by circ0009910 overexpression in K562/R cells (Supplement Fig. 2A). The binding sites between miR-34a-5p and circ_0009910 or ULK1 that were elaborated by Venn diagram showed that mniR-34a-5p was one of the target genes of circ_0009910 and ULK1, which was associated with drug-resistance (Fig. 5C). And we observed that the expression of miR34a-5p was lowly expressed in K562/R cells (Fig. 5D). Moreover,
3.6. Circ_0009910 could regulate ULK1-induced autophagy via sponging miR-34a-5p Next, we investigated whether circ_0009910 could regulate ULK1induced autophagy through targeting miR-34a-5p. The levels of ULK1, p-ULK1, LC3B-I/II and Beclin1 were inhibited and p62 expression was facilitated by circ_0009910 deletion in K562/R cells, while these effects of circ_0009910 deletion were blocked by miR-34a-5p deletion (Fig. 6A). The apoptosis rate was promoted in K562/R cells transfected with sh-circ_0009910, while miR-34a-5p downregulation harbored the promotion effect (Fig. 6B). In addition, the effects of circ_0009910 4
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Fig. 2. Characteristics of circ_0009910 in K562 and K562/R cells. (A) The IC50 value of imatinib at different concentrations (0.02 μM, 0.1 μM, 0.5 μM, 2.5 μM, 12.5 μM, 62.5 μM, 312.5 μM) was detected in both sensitive and resistant cells by cell viability assay. (B) The expression of circ_0009910 was verified by qRT-PCR in K562 and K562/R cells. (C) Schematic diagram of circ_0009910 that derived from exon3 and exon4 of MFN2. (D–E) qRT-PCR assay was used to detect the expression of circ_0009910 and MFN2 mRNA in K562 and K562/R cells treated with imatinib at the indicated time points. The results suggested that circ_0009910 was more stable compared with its linear counterpart. (F–G) qRT-PCR used to detect the expression of circ_0009910 and MFN2 mRNA in K562 and K562/R cells treated with or without RNase R. The results indicated that circ_0009910 was resistant to RNase R digestion. (H–I) qRT-PCR was carried out to detect the expression of circ_0009910 in the nuclear and cytoplasmic fractions. U6 snRNA and 18S rRNA were employed as positive control for nuclear and cytoplasmic fractions, respectively. The results showed that circ_0009910 was predominantly localized in the cytoplasm. ⁎P < 0.05, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001.
that circ_0009910 could regulate ULK1-induced autophagy by miR-34a5p.
downregulation on apoptosis-related protein expression were reversed by miR-34a-5p deletion in K562/R cells (Fig. 6C). K562/R cells were transfected with Vector, circ_0009910, circ_0009910 + miR-NC or circ_0009910 + miR-34a-5p. we found that miR-34a-5p blocked the effects of circ_0009910 overexpression on ULK1, p-ULK1, LC3B-I/II, Beclin1 and p62 expression in K562 cells (Supplement Fig. 3A). Moreover, the effects of circ-0009910 overexpression on cell apoptosis and apoptosis-related proteins expression were reversed by miR-34a-5p upregulation in K562 cells (Supplement Fig. 3B–C). These data implied
4. Discussion CML is a myeloproliferative disease originating from granulocytic cell proliferation and expansion that affected older adults typically. And imatinib is a common chemotherapy agent used in CML. Drug resistance was confirmed to induce the low survival rate of CML patients. 5
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Fig. 3. Circ_0009910 enhanced imatinib resistance but reduced the imatinib-induced apoptosis in K562 and K562/R cells. (A–B) QRT-PCR was conducted to measure the expression of circ_0009910 in K562 cells transfected with circ_0009910 or in K562/R cells transfected with sh-circ_0009910. (C–D) The IC50 value of imatinib was detected by CCK8 assay for the sensitivity and resistance of K562 and K562/R cells. (E–F) Flow cytometry analysis was used to detect the cell apoptosis. (G–H) The expression of apoptosis-related proteins including Cleaved caspase-3, Bax and Bcl-2 was measured by western blot assay in K562 and K562/R cells. ⁎P < 0.05, ⁎⁎ P < 0.01, ⁎⁎⁎P < 0.001.
Fig. 4. Circ_0009910 enhanced imatinib resistance by triggering autophagy in K562/R cells. (A) Expression levels of autophagy-associated proteins (LC3B-I/II, Beclin1, and P62) were analyzed by western blotting in K562 and K562/R cells. (B) The autophagy-associated proteins were analyzed after downregulation in K562/ R cells transfected with sh-NC or sh-circ_0009910. (C) K562/R cells were pre-treated with/without Rap (rapamycin, 100 nM, 24 h) and subsequently transfected with sh-circ_0009910, then cell apoptosis was detected. (D) Western blot assay was carried out to explore the apoptosis-related proteins levels after treating with Rap in K562/R cells. ⁎P < 0.05, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001. 6
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Fig. 5. Circ_0009910 could combine with miR-34a-5p and regulate downstream gene ULK1. (A) Western blot assay measured the protein level of ULK1 in K562 and K562/R cells. (B) The expression pattern of ULK1 was assessed by western blot. (C) Venn diagram revealed that the miR-34a-5p was targeted-intersection in circ_0009910 group and ULK1 group. (D) The expression of miR-34a-5p was detected in K562 and K562/R cells. (E) It was exhibited that putative sequences of miR34a-5p and circ_0009910 with 7 paired nucleotides, and predicted binding sequences of miR-34a-5p in the 3′-UTR of ULK1. (F) The luciferase activity was determined after transfection of WT-circ_0009910 (or MUT) and miR-34a-5p (or miR-NC). (G) MiR-34a-5p decreased the luciferase activity of wild-type 3′-UTR of ULK1. (H) The enrichment of circ_0009910, miR-34a-5p and ULK1 were measured by RIP and qRT-PCR. (I) Upregulation of miR-34a-5p level was conducted by gainfunction assay in K562/R cells. (J) Western blot was conducted to determine the protein level of ULK1 in K562/R cells transfected with miR-NC, miR-34a-5p, Mir34A-5P + Vector or miR-34a-5p + circ_0009910. ⁎P < 0.05, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001.
promotion in CML. Our data demonstrated that miR-34a-5p was a direct target of circ_0009910. Interestingly, miR-34a-5p was confirmed to regulate the expression of the autophagy-related gene ULK1. ULK1 was found to be highly expressed in imatinib-resistance CML cells [44] The ULK1 complex bridged between the upstream nutrient or energy receptors (mTOR and AMPK) and downstream autophagosomes in vivo [45]. Phosphorylated ULK1 has been recognized as a key regulator of autophagy [46]. Chung SJ and his colleagues reported that AMPK-ULK1 axis was involved in the cytotoxic autophagy in breast cancer [47]. Considering that ULK1 participated in drug phenotype of resistance changes as an autophagy regulator, we subsequently explored the expression level of ULK1 in drug-resistant CML cells. Consistent with the results in previous researches, ULK1 is highly expressed in imatinibresistance CML cells. And our results suggested that circ_0009910 could regulate ULK1 expression via sponging miR-34a-5p. We additionally tested the phosphorylated protein pattern of ULK1 in order to verify the autophagy induced by ULK1. Moreover, our data confirmed that circ_0009910 facilitated imatinib-resistance in CML by mediating autophagy through miR-34a-5p/ULK1 axis. However, our study also has some limitations. The commercial cells
Consistently, our data revealed that circ_0009910 was up-regulated in imatinib-resistance CML serum samples and positively associated with the overall survival rate, suggesting that circ_0009910 played a promotion role in imatinib-resistance of CML. CircRNAs and their effects in pathogenesis of various diseases have been widely studies. Accumulating evidence suggested that circRNAs acted vital roles in the development of myeloid and leukemogenesis. For instance, Liu and his colleagues indicated that has_circ_0080145 regulated cell growth in CML via targeting miR-29b [40]. Circ_0004136 was dramatically increased in acute myeloid leukemia and exerted promotion effect on cell proliferation [41]. Moreover, circ-Foxo3 was demonstrated to be associated with the prognosis of patients that diagnosed as acute myeloid leukemia [42]. Besides, increased circ_100053 expression was observed in CML and contributed to imatinib resistance [43]. In our study, the data revealed that circ_0009910 knockdown decreased the imatinibresistance in CML cells, as shown by inhibited cell growth, promoted cell apoptosis, as well as increased phenotypic changes of the autophagic activation. Overall, our research revealed a novel functional role of circ_0009910 in CML. Next, we explored the underlying mechanism of circ_0009910 7
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Fig. 6. Circ_0009910 could regulate ULK1-induced autophagy via sponging miR-34a-5p. (A) The protein expression of ULK1, p-ULK1, LC3B-I/II, Beclin1, and P62 in K562/R cells was assayed by western blot. (B) The apoptosis rate of K562/R cells was assessed using flow cytometry. (C) The apoptosis-related proteins expression in K562/R cells was detected using western blot. ⁎P < 0.05, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001.
Author contributions
cannot completely simulate the situation in vivo and the targeting relationship between miR-34a-5p and circ_0009910 or ULK1 would be explored in further research. In conclusion, circ_0009910 and ULK1 were enhanced, while miR34a-5p was decreased in imatinib-resistance of CML. Furthermore, circ_0009910 promoted the autophagy-mediated resistance by promoting the expression of ULK1 via targeting miR-34a-5p. Also, it was the first report of the promotion role and its mechanism in the progression of tumor resistance. Our findings demonstrated that circ_0009910 involved in the imatinib-resistance of CML via miR-34a5p/ULK1 axis (Fig. 7), providing a potential target for the treatment of CML. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lfs.2020.117255.
Hai-xia Cao, Chao-feng Miao, Li-na Sang and Zhong-xing Jiang: conceived, designed and did statistical analysis & writing of the manuscript; Yu-min Huang and Ran Zhang: review and final approval of the manuscript. Ling Sun: participated in revising the manuscript.
Declaration of competing interest The authors have no conflict of interest to declare.
Acknowledgment None.
Fig. 7. A Schematic model of circ_0009910 promoting imatinib resistance through ULK1-induced autophagy via sponging miR-34a-5p in CML. Pointed arrows and blunted arrows indicate activation and repression, respectively. 8
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Funding
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Circ_0009910 promotes imatinib resistance through ULK1-induced autophagy by sponging miR-34a-5p in chronic myeloid leukemia
T
Hai-xia Caoa, Chao-feng Miaob, Li-na Sanga, Yu-min Huanga, Ran Zhanga, Ling Suna, ⁎ Zhong-xing Jianga, a b
Department of Hematology, The First Affiliated Hospital of Zhengzhou University, 450052 Zhengzhou, Henan, China Department of Vascular surgery, The First Affiliated Hospital of Zhengzhou University, 450052 Zhengzhou, Henan, China
A R T I C LE I N FO
A B S T R A C T
Keywords: CML Circ_0009910 MiR-34a-5p Autophagy ULK1
Background: The occurrence in drug resistance of chronic myeloid leukemia (CML) was accompanied by autophagy activation. Abnormal circular RNAs (circRNAs) participated in this progression. This study attempted to investigate the potential role of circ_0009910 in imatinib resistance of CML cells. Methods: The expression of circ_0009910 and miR-34a-5p was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The characterization of circ_0009910 was investigated using oligo (dT)18 primers, Actinomycin D and RNase R. Cell viability (IC50 value) and apoptosis were assessed by Cell Counting Kit-8 (CCK8) assay and flow cytometry assay, respectively. The relative protein expression was quantified by western blot. The relationship among miR-34a-5p, circ_0009910 and ULK1 was predicted by online bioinformatics tool, and verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP). Results: The expression of circ_0009910 was up-regulated in the serum of imatinib-resistance CML patients and K562/R cells, and associated with unfavorable clinicopathologic features. Circ_0009910 in K562 and K562/R cells was mainly localized in the cytoplasm. Circ_0009910 knockdown inhibited cell proliferation and autophagy, but induced apoptosis in K562/R cells. Circ_0009910 targeted miR-34a-5p to regulate ULK1. MiR-34a-5p depression rescued the effects of circ_0009910 knockdown on apoptosis and autophagy in K562/R cells. Conclusion: Circ_0009910 accelerated imatinib-resistance in CML cells by modulating ULK1-induced autophagy via targeting miR-34a-5p, providing a potential target in imatinib resistance of CML.
1. Introduction Chronic myeloid leukemia (CML), prevalent malignant tumor, is one of the leading causes of hematological tumor-related mortality with a long-duration remedy for chemotherapy [1,2]. The clinical results of CML still remained unsatisfied due to the occurrence of metastasis and drug resistance [3–5]. Although plenty of research has illuminated the pathogenesis of CML, whereas, the underlying molecular mechanisms and effective treatment way during CML therapy were mostly deficient. Therefore, it was essential to understand the pathogenesis and found a novel treatment strategy for CML. Circular RNAs (circRNAs) are a series of endogenous noncoding RNAs with covalently closed loop structure [6]. A previous study clarified that circRNAs were more stable than linear mRNA, due to the lack of 5′ cap and 3′ polyadenylated tail [7]. CircRNAs were specifically expressed in hematological malignancies, indicating that circRNAs
might serve as post-transcriptional regulators [8,9]. For instance, the high expression of circ_0009910 predicted poor prognosis in patients with CML and was associated with imatinib resistance [10]. Overexpression of circ_0009910 promoted tumor cell proliferation, metastasis, and inhibited cell apoptosis [11–13]. The role of autophagy in drug-resistance has been widely reported in various cancers, including CML. MiR-199a/b-5p inhibited autophagy via repressing WNT2 signaling and might provide novel therapeutic strategy for imatinib-resistant CML therapy [14]. Recent studies have shown that the combination of TKIs with autophagy inhibitors was an effective method for the treatment of CML [15,16]. We aimed to explore the underlying mechanism of circ_0009910 in autophagy-mediated CML cell resistance. Furthermore, it is widely reported that miRNA could negatively regulate gene expression at the post-transcriptional level by degrading or repressing translation of mRNA [17,18]. MiR-34a-5p, located on
⁎ Corresponding author at: Department of Hematology, The First Affiliated Hospital of Zhengzhou University, No. 1 Jianshedong Road, Zhengzhou 450052, Henan, China. E-mail address: [email protected] (Z.-x. Jiang).
https://doi.org/10.1016/j.lfs.2020.117255 Received 16 October 2019; Received in revised form 27 December 2019; Accepted 31 December 2019 Available online 07 January 2020 0024-3205/ © 2020 Elsevier Inc. All rights reserved.
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CTTAG-3′), miR-34a-5p reverse (5′-CAGTGCGTGTCGTGGAGT-3′), ULK1 forward (5′-CAGCAAAGGCATCATCCAC-3′), ULK1 reverse ( 5′-GGTTGCGTTGCAGTAGGG-3′), GAPDH forward (5′-ACCACAGTCCA TGCCATCAC-3′), GAPDH reverse (5′-TCCACCACCCTGTTGCTGTA-3′), U6 forward (5′-GCUUCGGCAGCACAUAUACUAAAAU-3′), U6 reverse ( 5′-CGCUUCACGAAUUUGCGUGUCAU-3′).
chromosome 1p36 [19], was decreased in breast cancer [20], bladder cancer [21], non-small cell lung cancer [22] and metastatic colorectal cancer [23]. Besides, the expression level of miR-34a-5p was associated with resistance in several diseases, including osteosarcoma [24], ovarian cancer [25], prostate cancer [26], and non-small cell lung cancer [27]. Autophagy may take part in mediating resistance of cancer cells to anticancer therapy [28–30]. Interestingly, miR-34a-5p was found to be involved in autophagy in the pathological and physiological processes, including hypoxia-reoxygenation injury, proliferation, and fibrosis [31,32]. However, it has not been reported that the potential role of miR-34a-5p in CML resistance. Thus, we explore whether miR34a-5p was involved in the regulation of autophagy in CML. UNC-51-like kinase (ULK1), as a homologous gene of yeast Atg1, is a serine-threonine kinase and a mammalian autophagy promoter [33]. The autophagy signal was mediated by activation of the ULK complex consisting of ULK1 or ULK2, FIP200, and mATG13, which was prior to autophagic lysosomal assembly [34]. The ULK1 complex was a bridge between the upstream nutrient or energy receptors mTOR and AMPK and downstream autophagosomes in the body [35]. Meanwhile, the ULK1-induced autophagy activation signals can be regulated by miRNA [36]. This study was designed to elucidate the molecular mechanisms of CML resistance and provided the novel perspectives for the treatment of CML.
2.4. Cell Counting Kit-8 (CCK8) assay The CCK-8 reagent (DOJINDO, Kumamoto, Japan) was used to detect cell viability. Two cell lines with or without transfection were treated with imatinib (0.02 μM, 0.1 μM, 0.5 μM, 2.5 μM, 12.5 μM, 62.5 μM, 312.5 μM) for 48 h, then 10 μL CCK-8 solution was added into each well for another 4 h. The proliferation of K562 and K562/R cells was detected at wavelength of 450 nm each well on a microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA). IC50 values were calculated as previously described [37]. 2.5. Circularization detection of circ_0009910 Random primers (Invitrogen, Carlsbad, CA, USA) and oligo (dT)18 primers (Invitrogen) were used for qRT-PCR. 2.6. Actinomycin D treatment
2. Materials and methods
To detect the stability of circ_0009910, 2 mg/mL Actinomycin D (Sigma-Aldrich, St. Louis, MO, USA) was added before RNA isolation. Then, qRT-PCR analysis was carried out.
2.1. Serum samples collection This study acquired the authorization of the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. A total of 65 serum specimens from CML patients (imatinib-sensitive n = 30, imatinib-resistance n = 35) were collected from the First Affiliated Hospital of Zhengzhou University. Written informed consent was obtained from each subject before imatinib chemotherapy. All blood samples were collected by anticoagulant tubes containing heparin and centrifuged to obtain supernatants and stored at −80 °C condition.
2.7. Rnase R treatment
2.2. Cell lines, cell culture and drug treatment
RNAs in cytoplasm and nucleus were extracted using the Cytoplasmic and Nuclear RNA Purification Kit (Norgen Biotek, Thorold, Canada). Total RNA in each fraction was used for qRT-RCR analysis. GAPDH and U6 were acted as cytoplasm and nucleus internal references, respectively.
To confirm the existence of circ_0009910, the RNA extraction was incubated with or without Rnase R (Applied Biological Materials Inc., Vancouver, Canada) at 37 °C for 10 min. Then, qRT-PCR analysis was conducted. 2.8. Nuclear and cytoplasmic localization analysis
Parental and resistant cell lines of CML (K562 and K562/R, individually) were purchased from Shanghai Innovation Biotechnology Co., Ltd. (Shanghai, China). All cells were cultured in 90% Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) with 10% fetal Bovine Serum (FBS; Gibco) at 37 °C condition with 5% CO2. For drug treatment, K562 and K562/R cells were treated with different concentrations of imatinib (Sigma, St. Louis, MO, USA). According to IC50 value, the concentration of 0.5 μM for K562 or 12.5 μM for K562/R cells was used in subsequent experiments. 3-Methyladenine (5 mM, 3MA, Sigma) or rapamycin (100 nmol, Rap, Sigma) was added to overnight-cultured cells for the indicated time in K562 or K562R cells.
2.9. Cell transfection For circ_0009910 downregulation, short hairpin RNA (shRNA) against circ_0009910 (sh-circ_0009910) and its negative control (shNC) were constructed by Genepharma (Shanghai, China). For circ_0009910 upregulation, circ_0009910 sequences were amplified by PCR and inserted into pcDNA3.1 vector (Invitrogen) (circ_0009910), and the corresponding control was pcDNA3.1 empty vector (vector). MiR-34a-5p mimics (miR-34a-5p), miR-34a-5p inhibitor (anti-miR-34a5p) and their own negative control (miR-NC and anti-NC) were purchased from Ribobio (Guangzhou, China). All sequences were transfected into K562 and K562/R cells using Lipofectamine 2000 (Invitrogen). The used sequences were listed as below: sh-circ_0009910 sequence (3′-TGGCCGCGCAATGTCCCTGCT-5′), sh-NC sequence ( 3′-UCACCCAGAUGCCGCUAU-5′). Rapamycin (200 nM) and 3-MA (10 mM) (Sigma Aldrich, St Louis, MO, USA) were added into cells after transfection.
2.3. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was separated from CML serums and cells using Total RNA Extractor (Sangon Biotech, Shanghai, China). Then the HiScript III First Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) or Mir-X™ miRNA First Strand Synthesis Kit (Clontech, Mountain View, CA, USA) was used to assemble complementary DNA (cDNA). Next, qRT-PCR was performed using AceQ Universal SYBR qPCR Master Mix (Vazyme) on CFX Connect system (Bio-Rad, Hercules, CA, USA). The fold-change of expression was calculated using the 2−ΔΔCt method and normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or small nuclear RNA U6. The used primers were listed as below: circ_0009910 forward ( 5′-TGAGAGGCATCAGTGAGGTG-3′), circ_0009910 reverse (5′-AAGTG CTTAAGTGGGGATGC-3′), miR-34a-5p forward (5′-GCCCTGGCAGTGT
2.10. Flow cytometry assay Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme, Nanjing, China) was used for flow cytometry assay. In brief, K562 and K562/R 2
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circ_0009910 in serum, and the ROC analysis demonstrated an area under the curve (AUC) of 0.831, with a diagnostic sensitivity and specificity reaching 65.71% and 95.33%, respectively (95% CI = 0.7317–0.9331) (Fig. 1B). Based on the cut-offs (1.729) established by ROC, the proportion of patients not responding to gefitinib therapy was significantly higher in the high circ_0009910 expressing group than in the low expressing group (Fig. 1C). We further analyzed the relationship between circ_0009910 level and survival time in CML patients. The Kaplan-Meier survival curve showed that the patients with high circ_0009910 expression had shorter overall survival rate than those with low circ_0009910 expression (Fig. 1D).
cells were seeded into 6-well plates, and then treated with 0.25% trypsin and washed using pre-cooled phosphate buffer saline (PBS). Then, cells (2 × 105) were resuspended by 100 μL 1× binding buffer and then incubated with 5 μL Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining solution for 10 min at room temperature without light. Finally, the apoptotic cells were analyzed using flow cytometer (BD Biosciences, San Jose, CA, USA). 2.11. Western blot Western blot analysis was performed as previously described [14]. The primary antibodies against Cleaved Caspase-3 (ab2302, 1:1000, Abcam, Cambridge, UK), Bax (Bcl-2-associated X protein, ab53154, 1:1000, Abcam), Bcl-2 (B-cell lymphoma-2, ab196495, 1:1000, Abcam), LC3-I/LC3-II (ab62721, 1:1000, Abcam), Beclin1 (ab62557, 1:1000, Abcam), P62 (ab155686, 1:1000, Abcam), ULK1 (ab167139, 1:1000, Abcam), p-ULK1 (ab133747, 1:1000, Abcam), GAPDH (ab9485, 1:2500, Abcam) and goat anti-rabbit secondary antibodies (ab205718, 1:5000, Abcam) were used in this study.
3.2. Confirmation of the circular structure and subcellular localization of circ_0009910 As shown in Fig. 2A, compared with parental cells, the established resistant cells showed imatinib-resistance, as shown by increased IC50 value and elevated cell viability. Next, the circ_0009910 was highly expressed in K562/R cells (K562 resistant cells) compared with that of K562 cells (Fig. 2B). Circ_0009910 was derived from MFN2 gene exon 3–4, with a spliced mature sequence length of 315 base pairs (bp) (Fig. 2C). Next, we investigated the stability and localization of circ_0009910 in K562 and K562/R cells. Total RNAs from K562 and K562/R cells were isolated at the indicated time points after treatment with Actinomycin D, an inhibitor of transcription. Then qRT-PCR was performed to measure the level of circ_0009910 and MFN2 mRNA. The results showed that the half-life of circ_0009910 exceeded 24 h, whereas that of MFN2 mRNA was about 3.5 h in both K562 and K562/R cells (Fig. 2D–E). Furthermore, we found that circ_0009910 was resistant to RNase R digestion (Fig. 2F–G). These data confirmed that circ_0009910 was a circular RNA. We then investigated the localization of circ_0009910. qRT-PCR of RNAs from nuclear and cytoplasmic fractions indicated that circ_0009910 was predominantly localized in the cytoplasm of K562 and K562/R cells (Fig. 2H–I). Collectively, the above data suggested that circ_0009910 harbored a loop structure and was predominantly localized in the cytoplasm.
2.12. Bioinformatics analysis and dual-luciferase reporter assay The online bioinformatics tool starBase was used to predict the potential target genes and the specific binding sites. The relationship among miR-34a-5p, circ_0009910 and ULK1 was verified by dual-reporter assay. In brief, the sequence of wild type circ_0009910 (WTcirc_0009910) containing the binding sites of miR-34a-5p, and the corresponding mutant circ_0009910 (MUT-circ_0009910) sequence was amplified and cloned into the pRL-CMV vector (Promega, Madison, WI, USA). Then, WT-circ_0009910 or MUT-circ_0009910 and miR-34a-5p or miR-NC were co-transfected into K562 and K562/R cells. After transfection for 48 h, the luciferase activity was detected using the Dual-Luciferase Reporter Assay Kit (Promega). Similarly, WT-ULK1 and MUT-ULK1 were also constructed and used for luciferase activity analysis. 2.13. RNA immunoprecipitation (RIP) assay Magna RIP Kit (Millipore, Billerica, MA, USA) was used for RIP assay. In brief, K562 and K562/R cells were collected and resuspended in RNA immunoprecipitation lysis buffer containing magnetic beads, and then incubated with anti‑argonaute 2 (anti-Ago2) or IgG antibodies. Next, the protein was digested using proteinase K buffer, followed by RNA purification. Subsequently, the enrichment of circ_0009910, miR-34a-5p, or ULK1 was determined by qRT-PCR.
3.3. Circ_0009910 enhanced imatinib resistance but reduced the imatinibinduced apoptosis in K562 and K562/R cells In order to further explore the role of circ_0009910 in imatinib-resistance cells, we performed overexpression or silencing of circ_0009910 experiments in K562 or K562/R cells, respectively. As shown in Fig. 3A–B, the expression of circ_0009910 was decreased in K562 cells, or increased in K562/R cells, individually, compared to its negative controls. Meanwhile, overexpression of circ_0009910 led to an increase of IC50 in K562 cells, while knockdown of circ_0009910 resulted in a significant decreased IC50 in K562/R cells and repressed anchorage-independent growth upon imatinib treatment (Fig. 3C–D). After that, we measured the ability of apoptosis in K562 and K562/R cells, the rate of apoptosis was inhibited in K562 cells treated with circ_0009910 vector, and apoptosis was enhanced in K562/R cells administrated with sh-circ_0009910 (Fig. 3E–F). Western blot assay showed that upregulation of circ_0009910 triggered the expression of Bcl-2, but impeded that of Cleaved caspase-3 and Bax in K562 cells. Opposite results could be seen in K562/R cells with suppression of circ_0009910 (Fig. 3G–H).
2.14. Statistical analysis Data were collected from at least 3 independent experiments, processed by GraphPad Prism 5.0 (GraphPad software Inc., San Die, CA, USA), and exhibited as mean ± standard deviation (SD). The difference analyses between groups were performed using one-way analysis of variance (ANOVA) or Student's t-test. P value < 0.05 was regarded as statistically significant. 3. Results 3.1. Serum expression of circ_0009910 is associated with imatinib resistance in CML patients
3.4. Circ_0009910 enhanced imatinib resistance by triggering autophagy in K562 and K562/R cells
First, we extracted the serum samples from patients with or without imatinib-resistance and tested the expression of circ_0009910 in serum components by qRT-PCR. Results showed that compared with imatinibsensitive corresponding controls, a significant promotion of circ_0009910 was observed in the imatinib-resistance group (Fig. 1A). After that, we used the ROC curve to evaluate the diagnostic value of
In order to further explore the regulatory mechanism of circ_0009910 in drug-resistant cells, according to previous researches reported that autophagy might be involved in the occurrence and development of tumor resistance [10], we hypothesized that circ_0009910 3
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Fig. 1. Serum expression of circ_0009910 was associated with imatinib resistance in CML patients. (A) QRT-PCR assay was used for differential expression of circ_0009910 in imatinib-sensitive (n = 30) or imatinib-resistance (n = 35) samples from CML patients. (B) ROC curve analysis showed that the diagnostic value of circ_0009910 in CML patients. (C) qRT-PCR for the proportion of patients showed that high expression of circ_0009910 was observed in imatinib-resistant group. (D) The Kaplan-Meier survival cure showed the survival rate in patients with high circ_0009910 expression (n = 40) and with low circ_0009910 expression (n = 25). ⁎⁎⁎P < 0.001.
starBase predicted that miR-34-5p contained the binding sites of circ_0009910 and ULK1 (Fig. 5E). Then, dual-luciferase reporter assay clarified that the luciferase activity was markedly decreased in K562/R cells co-transfected with WT-circ_0009910 and miR-34a-5p compared with miR-NC, while the luciferase activity had no noticeable change in K562/R cells co-transfected with MUT-circ_0009910 and miR-34a-5p compared with miR-NC (Fig. 5F). Meanwhile, several binding sites existed between miR-34a-5p and ULK1 3′ UTR, and the transfection of miR-34a-5p significantly reduced the luciferase activity in K562/R cells transfected with WT-ULK1-3′ UTR but did not alter the luciferase activity with the transfection of MUT-ULK1-3′ UTR (Fig. 5G). The similar results were discovered in K562 cells (Supplement Fig. 2B–C). The enrichment of circ_0009910, miR-34a-5p, and ULK1 could be combined to Ago2 protein in both K562 (Fig. 5H) and K562/R cells (Supplement Fig. 2D). Moreover, miR-34a-5p showed the successful overexpression efficiency in K562/R cells (Fig. 5I), while anti-miR-34a-5p showed the successful knockdown efficiency in K562 cells (Supplement Fig. 2E). Moreover, the protein expression of ULK1 was inhibited in K562/R cells transfected with miR-34a-5p, while circ_0009910 reversed the inhibitory effect of miR-34a-5p overexpression on ULK1 expression (Fig. 5J). In addition, miR-34a-5p deletion promoted the protein expression of ULK1 in K562 cells, which was reversed by circ_0009910 knockdown (Supplement Fig. 2F). These data suggested that miR-34a5p was a target of circ_0009910 and targeted ULK1, and the expression of ULK1 was modulated by circ_0009910 through miR-34a-5p.
might mediate the activation of autophagy, which subsequently lead to the development of resistance. Western blot assessment examined the expression of autophagy-related proteins, and K562/R cells exhibited an aberrant expression of LC3B-I/II and Beclin1, but limited pattern of P62 compared to the parental cells (Fig. 4A). The above results indicated that circ_0009910 might promote the activation process of autophagy in K562/R cells. Then, we performed loss- or gain-functional assays, and results suggested that autophagy response was reactivated after K562/R cells transfected with sh-circ_0009910 (Fig. 4B), while deactivated response could be seen in K562 cells with circ_0009910 overexpression (Supplement Fig. 1A). Besides, inhibitor (3-MA) or agonist (Rap, rapamycin) of autophagy was used for rescue experiments in K562 or K562/R cells, respectively. Rap impaired cell apoptosis induced by downregulating circ_0009910 in K562/R cells (Fig. 4C), while 3-MA reserved the inhibition effect of circ_0009910 upregulation on cell apoptosis in K562 cells (Supplement Fig. 1B). 3-MA administration inverted the effects of circ_0009910 upregulation on apoptosis-related protein expression in K562 cells (Supplement Fig. 1C), while Rap reversed the effects of circ_0009910 knockdown on Cleaved caspase-3, Bax and Bcl-2 expression in K562 cells (Fig. 4D). 3.5. MiR-34a-5p was a target of circ_0009910and directly targeted ULK1 ULK1, a kind of serine-threonine kinase, is regarded as a mammalian autophagy promoter. In previous reports, He and Yu et al. showed that ULK1 was highly expressed in imatinib-resistance CML cells [38,39]. Thus, we tested the protein level of ULK1 and found that it was upregulated in imatinib-resistance K562 cells (Fig. 5A). We found that the expression of ULK1 was inhibited by circ_0009910 downregulation in K562/R cells (Fig. 5B), and ULK1 expression was upregulated by circ0009910 overexpression in K562/R cells (Supplement Fig. 2A). The binding sites between miR-34a-5p and circ_0009910 or ULK1 that were elaborated by Venn diagram showed that mniR-34a-5p was one of the target genes of circ_0009910 and ULK1, which was associated with drug-resistance (Fig. 5C). And we observed that the expression of miR34a-5p was lowly expressed in K562/R cells (Fig. 5D). Moreover,
3.6. Circ_0009910 could regulate ULK1-induced autophagy via sponging miR-34a-5p Next, we investigated whether circ_0009910 could regulate ULK1induced autophagy through targeting miR-34a-5p. The levels of ULK1, p-ULK1, LC3B-I/II and Beclin1 were inhibited and p62 expression was facilitated by circ_0009910 deletion in K562/R cells, while these effects of circ_0009910 deletion were blocked by miR-34a-5p deletion (Fig. 6A). The apoptosis rate was promoted in K562/R cells transfected with sh-circ_0009910, while miR-34a-5p downregulation harbored the promotion effect (Fig. 6B). In addition, the effects of circ_0009910 4
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Fig. 2. Characteristics of circ_0009910 in K562 and K562/R cells. (A) The IC50 value of imatinib at different concentrations (0.02 μM, 0.1 μM, 0.5 μM, 2.5 μM, 12.5 μM, 62.5 μM, 312.5 μM) was detected in both sensitive and resistant cells by cell viability assay. (B) The expression of circ_0009910 was verified by qRT-PCR in K562 and K562/R cells. (C) Schematic diagram of circ_0009910 that derived from exon3 and exon4 of MFN2. (D–E) qRT-PCR assay was used to detect the expression of circ_0009910 and MFN2 mRNA in K562 and K562/R cells treated with imatinib at the indicated time points. The results suggested that circ_0009910 was more stable compared with its linear counterpart. (F–G) qRT-PCR used to detect the expression of circ_0009910 and MFN2 mRNA in K562 and K562/R cells treated with or without RNase R. The results indicated that circ_0009910 was resistant to RNase R digestion. (H–I) qRT-PCR was carried out to detect the expression of circ_0009910 in the nuclear and cytoplasmic fractions. U6 snRNA and 18S rRNA were employed as positive control for nuclear and cytoplasmic fractions, respectively. The results showed that circ_0009910 was predominantly localized in the cytoplasm. ⁎P < 0.05, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001.
that circ_0009910 could regulate ULK1-induced autophagy by miR-34a5p.
downregulation on apoptosis-related protein expression were reversed by miR-34a-5p deletion in K562/R cells (Fig. 6C). K562/R cells were transfected with Vector, circ_0009910, circ_0009910 + miR-NC or circ_0009910 + miR-34a-5p. we found that miR-34a-5p blocked the effects of circ_0009910 overexpression on ULK1, p-ULK1, LC3B-I/II, Beclin1 and p62 expression in K562 cells (Supplement Fig. 3A). Moreover, the effects of circ-0009910 overexpression on cell apoptosis and apoptosis-related proteins expression were reversed by miR-34a-5p upregulation in K562 cells (Supplement Fig. 3B–C). These data implied
4. Discussion CML is a myeloproliferative disease originating from granulocytic cell proliferation and expansion that affected older adults typically. And imatinib is a common chemotherapy agent used in CML. Drug resistance was confirmed to induce the low survival rate of CML patients. 5
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Fig. 3. Circ_0009910 enhanced imatinib resistance but reduced the imatinib-induced apoptosis in K562 and K562/R cells. (A–B) QRT-PCR was conducted to measure the expression of circ_0009910 in K562 cells transfected with circ_0009910 or in K562/R cells transfected with sh-circ_0009910. (C–D) The IC50 value of imatinib was detected by CCK8 assay for the sensitivity and resistance of K562 and K562/R cells. (E–F) Flow cytometry analysis was used to detect the cell apoptosis. (G–H) The expression of apoptosis-related proteins including Cleaved caspase-3, Bax and Bcl-2 was measured by western blot assay in K562 and K562/R cells. ⁎P < 0.05, ⁎⁎ P < 0.01, ⁎⁎⁎P < 0.001.
Fig. 4. Circ_0009910 enhanced imatinib resistance by triggering autophagy in K562/R cells. (A) Expression levels of autophagy-associated proteins (LC3B-I/II, Beclin1, and P62) were analyzed by western blotting in K562 and K562/R cells. (B) The autophagy-associated proteins were analyzed after downregulation in K562/ R cells transfected with sh-NC or sh-circ_0009910. (C) K562/R cells were pre-treated with/without Rap (rapamycin, 100 nM, 24 h) and subsequently transfected with sh-circ_0009910, then cell apoptosis was detected. (D) Western blot assay was carried out to explore the apoptosis-related proteins levels after treating with Rap in K562/R cells. ⁎P < 0.05, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001. 6
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Fig. 5. Circ_0009910 could combine with miR-34a-5p and regulate downstream gene ULK1. (A) Western blot assay measured the protein level of ULK1 in K562 and K562/R cells. (B) The expression pattern of ULK1 was assessed by western blot. (C) Venn diagram revealed that the miR-34a-5p was targeted-intersection in circ_0009910 group and ULK1 group. (D) The expression of miR-34a-5p was detected in K562 and K562/R cells. (E) It was exhibited that putative sequences of miR34a-5p and circ_0009910 with 7 paired nucleotides, and predicted binding sequences of miR-34a-5p in the 3′-UTR of ULK1. (F) The luciferase activity was determined after transfection of WT-circ_0009910 (or MUT) and miR-34a-5p (or miR-NC). (G) MiR-34a-5p decreased the luciferase activity of wild-type 3′-UTR of ULK1. (H) The enrichment of circ_0009910, miR-34a-5p and ULK1 were measured by RIP and qRT-PCR. (I) Upregulation of miR-34a-5p level was conducted by gainfunction assay in K562/R cells. (J) Western blot was conducted to determine the protein level of ULK1 in K562/R cells transfected with miR-NC, miR-34a-5p, Mir34A-5P + Vector or miR-34a-5p + circ_0009910. ⁎P < 0.05, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001.
promotion in CML. Our data demonstrated that miR-34a-5p was a direct target of circ_0009910. Interestingly, miR-34a-5p was confirmed to regulate the expression of the autophagy-related gene ULK1. ULK1 was found to be highly expressed in imatinib-resistance CML cells [44] The ULK1 complex bridged between the upstream nutrient or energy receptors (mTOR and AMPK) and downstream autophagosomes in vivo [45]. Phosphorylated ULK1 has been recognized as a key regulator of autophagy [46]. Chung SJ and his colleagues reported that AMPK-ULK1 axis was involved in the cytotoxic autophagy in breast cancer [47]. Considering that ULK1 participated in drug phenotype of resistance changes as an autophagy regulator, we subsequently explored the expression level of ULK1 in drug-resistant CML cells. Consistent with the results in previous researches, ULK1 is highly expressed in imatinibresistance CML cells. And our results suggested that circ_0009910 could regulate ULK1 expression via sponging miR-34a-5p. We additionally tested the phosphorylated protein pattern of ULK1 in order to verify the autophagy induced by ULK1. Moreover, our data confirmed that circ_0009910 facilitated imatinib-resistance in CML by mediating autophagy through miR-34a-5p/ULK1 axis. However, our study also has some limitations. The commercial cells
Consistently, our data revealed that circ_0009910 was up-regulated in imatinib-resistance CML serum samples and positively associated with the overall survival rate, suggesting that circ_0009910 played a promotion role in imatinib-resistance of CML. CircRNAs and their effects in pathogenesis of various diseases have been widely studies. Accumulating evidence suggested that circRNAs acted vital roles in the development of myeloid and leukemogenesis. For instance, Liu and his colleagues indicated that has_circ_0080145 regulated cell growth in CML via targeting miR-29b [40]. Circ_0004136 was dramatically increased in acute myeloid leukemia and exerted promotion effect on cell proliferation [41]. Moreover, circ-Foxo3 was demonstrated to be associated with the prognosis of patients that diagnosed as acute myeloid leukemia [42]. Besides, increased circ_100053 expression was observed in CML and contributed to imatinib resistance [43]. In our study, the data revealed that circ_0009910 knockdown decreased the imatinibresistance in CML cells, as shown by inhibited cell growth, promoted cell apoptosis, as well as increased phenotypic changes of the autophagic activation. Overall, our research revealed a novel functional role of circ_0009910 in CML. Next, we explored the underlying mechanism of circ_0009910 7
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Fig. 6. Circ_0009910 could regulate ULK1-induced autophagy via sponging miR-34a-5p. (A) The protein expression of ULK1, p-ULK1, LC3B-I/II, Beclin1, and P62 in K562/R cells was assayed by western blot. (B) The apoptosis rate of K562/R cells was assessed using flow cytometry. (C) The apoptosis-related proteins expression in K562/R cells was detected using western blot. ⁎P < 0.05, ⁎⁎P < 0.01, ⁎⁎⁎P < 0.001.
Author contributions
cannot completely simulate the situation in vivo and the targeting relationship between miR-34a-5p and circ_0009910 or ULK1 would be explored in further research. In conclusion, circ_0009910 and ULK1 were enhanced, while miR34a-5p was decreased in imatinib-resistance of CML. Furthermore, circ_0009910 promoted the autophagy-mediated resistance by promoting the expression of ULK1 via targeting miR-34a-5p. Also, it was the first report of the promotion role and its mechanism in the progression of tumor resistance. Our findings demonstrated that circ_0009910 involved in the imatinib-resistance of CML via miR-34a5p/ULK1 axis (Fig. 7), providing a potential target for the treatment of CML. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lfs.2020.117255.
Hai-xia Cao, Chao-feng Miao, Li-na Sang and Zhong-xing Jiang: conceived, designed and did statistical analysis & writing of the manuscript; Yu-min Huang and Ran Zhang: review and final approval of the manuscript. Ling Sun: participated in revising the manuscript.
Declaration of competing interest The authors have no conflict of interest to declare.
Acknowledgment None.
Fig. 7. A Schematic model of circ_0009910 promoting imatinib resistance through ULK1-induced autophagy via sponging miR-34a-5p in CML. Pointed arrows and blunted arrows indicate activation and repression, respectively. 8
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Funding
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