EVI-1-Mediated Up-Regulation of lncRNA TUG1 Interacts with miR-186-5p to Promote Proliferation and Drug Resistance in Pediatric Acute Myeloid Leukemia

Background: Deregulated lncRNAs have been well-documented to be closely associated with resistance to chemotherapeutic agents in malignancies including acute myeloid leukemia (AML). Herein, we intended to explore the roles and underlying mechanism of lncRNA taurine-upregulated gene 1 (TUG1) in pediatric AML cell adriamycin (ADR) resistance. Methods: TUG1 and ecotropic viral integration site-1 (EVI-1) expressions in pediatric AML patients and cells were detected by using qRT-PCR and western blot, respectively. Western blot analysis, ow cytometry and CCK-8 assays were conducted for evaluating the drug sensitivity, apoptosis, and cell viability. Luciferase reporter assay and qRT-PCR were implemented to determine the interaction between miR-186-5p and TUG1. The western blot was employed to analyze the changes of proteins. on exploring the impacts of TUG1 on resistance to ADR in AML cells and the functional We observed upregulated expression of TUG1 in AML cells and TUG1 level was higher in ADR-resistant AML cells than their parental cells. TUG1 overexpression signicantly promoted cell proliferation and ADR resistance, and enhanced P-gp level in HL60 cells while TUG1 knockdown exhibited the opposite effects on HL60/ADR cells, suggesting the oncogenic role of TUG1 in AML.


Background
Acute leukemia, a heterogeneous group of aggressive hematopoietic system malignancies, is believed to be one of the most frequently diagnosed childhood cancers in China, accompanied by variable clinical outcomes [1]. Acute myeloid leukemia (AML), a common pediatric acute leukemia, originates from abnormal differentiation and rapid proliferation of immature myeloid progenitors, accounting for ~ 30% of leukemia-associated pediatric mortalities [2]. Despite considerable improvements in AML remedy including targeted drugs, the overall prognosis for AML is still hard to be estimated, and the ve-year survival rate is only 20-40% [3]. During the recent decades, traditional cytotoxic chemotherapy remains one of the major therapies for AML [4]. Currently, adriamycin (ADR) was a widely used clinical chemotherapeutic agent for AML [5]. However, the therapeutic ine ciency and frequent recurrence in AML attributed to drug resistance signi cantly limits the e cacy of chemotherapy [6]. Consequently, an indepth understanding of the potential mechanism for the chemoresistance in pediatric AML is helpful for developing e cient treatment interventions for overcoming chemoresistance.
Researches on human transcriptome discover that merely less than 2% of the human genome is capable of encoding proteins, while non-coding RNAs (ncRNAs), including recently discovered long non-coding RNA (lncRNAs) and extensively investigated microRNAs (miRNAs), accounts for the vast majority of transcripts [7]. LncRNAs is a group of endogenous single-stranded ncRNAs longer than 200 nt and devoid of the potential of protein encoding [8]. Growing literatures indicate that abnormally expressed lncRNAs closely related to the tumorigenesis of human malignancies, such as AML by modulating a multitude of biological process, including cell development, metastasis, and death [9,10]. Moreover, increasing experimental data have proposed that deregulated lncRNAs are closely associated with resistance to chemotherapeutic agents in miscellaneous cancers [11]. Originally, Taurine-upregulated gene 1 (TUG1) was acknowledged as an upregulated lncRNA in the developing mouse retinal cells under taurine treatment [12]. Widespread evidence has shown that dysregulation of TUG1 is frequent in diverse carcinomas and is closely implicated in cancer carcinogenesis by exerting either cancer-promoting or tumor-suppressing effects in different cancers [13]. Interestingly, recent studies demonstrated that TUG1 is overly expressed in AML cells and closely correlated with worse outcome, which induces cell proliferation as well as ADR resistance, and represses apoptosis in AML cells [14,15]. Nevertheless, the detailed regulation of TUG1 on AML resistance to ADR still remains to be more deeply investigated.
Ecotropic viral integration site-1 (EVI-1), situated at chromosome 3q26, was rstly recognized in an integration locus of retrovirus in an AML mouse model [16]. EVI-1 is believed as an oncogenic dual domain zinc nger transcription regulator involved in myeloid leukemia [17]. Moreover, it is widely recognized that EVI-1 is implicated in the maintenance and proliferation of hemopoietic stem cells and myeloid progenitor cell differentiation [18].
We identi ed that TUG1 expression was increased in pediatric AML patients, which is transcriptionally activated by EVI-1. Moreover, it was demonstrated that depletion of TUG1 hindered cell proliferation and ADR resistance in AML cells by suppressingmiR-186-5p through inhibiting the phosphatidylinositol 3kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signals, contributing to the development of effective treatments to overcome chemoresistance in AML cells.

Clinical samples
Prior to any interventional therapies at the First A liated Hospital of Zhengzhou University from March 2018 to April 2019, bone marrow (BM) specimens were harvested during the biopsy from 21 pediatric patients who were newly diagnosed with AML and 10 healthy children as normal controls. The French-American-British (FAB) classi cation was used as the standard criteria for the diagnosis of AML patients [19,20]. Signed consent for clinical studies was signed by their guardians prior to the study and our study was approved by Institutional Ethics Board of the First A liated Hospital of Zhengzhou University.

Cell culture and transfection
The AML cell lines (HL60 and K562), as well as human bone marrow stromal cell line (HS-5 ) were get from the American Type Culture Collection (ATCC) (Manassas, VA, USA). These cells were cultivated in RPMI-1640 medium (BOSTER, Wuhan, China) complemented with 10% heat-inactivated fetal calf serum (ExCell Bio, Shanghai, China), together with 1% penicillin/streptomycin in a water-saturated culture incubator ushed with 5% CO 2

Cell viability assay
The cell counting kit-8 (CCK-8) assay was used for cell viability evaluation. 96-well plates were used to carry the logarithmically growing HL60/ ADR and HL60 cells with a density of 5 × 10 3 cells per well and all the cells were exposed to 8 μM ADR or infected with si-TUG1, TUG1, miR-186-5p, anti-miR-186-5p, or matched controls in the absence or presence of 8 μM ADR. After incubated for the indicated time, the cells were fostered for another 3 h after adding 10 μL CCK-8 solutions. A MultiSkan3 ELISA Reader (Thermo Fisher Scienti c, Waltham, MA, USA) was adopted to record the cell viability.
Quantitative real-time polymerase chain reaction (qRT-PCR) RNAiso Plus (Takara, Dalian, China) was taken to extract total RNA. Using SuperScript III reverse transcriptase (Invitrogen), the synthesis of rst strand cDNA was carried out. To examine TUG1 and EVI-1 mRNA expressions, qRT-PCR was implemented on a Chromo4 instrument (Bio-Rad) with THUNDERBIRD SYBR qPCR mix (Toyobo, Osaka, Japan), with GAPDH as the normalization. Additionally, miR-186-5p expression was examined using miScript SYBR Green PCR Kit (Qiagen) on a Chromo4 instrument (Bio-Rad) and normalized to U6 small nuclear RNA (snRNA). The fold changes were detected according to the 2 -ΔΔCt method.
Western blot analysis RIPA lysis buffer (KeyGEN, Nanjing, China) containing phenylmethylsulphonyi uoride (PMSF) was used to lyse collected samples and the protein content was quanti ed by using the BCA™ Protein Assay Kit (Beyotime, Shanghai, China) . The 10% SDS-JPAGE gel was used to separate collected cell lysates, and separated brands were then electro-transferred onto nitrocellulose membranes. Next, the membranes were exposed to 5% skimmed milk for 1 hour. Then, the blocked membranes were probed with primary antibodies for 12 hours at 4°C and secondary antibody marked by horseradish peroxidase (Abcam, Cambridge, MA, USA) at room temperature. An ECL detection reagent (Solarbio, Shanghai, China) was implemented to measure the signals. The primary antibodies in this study include proliferating cell nuclear antigen (PCNA), Bcl-2, EVI-1 (Abcam); P-glycoprotein (P-gp), PI3K, phosphorylated PI3K (p-PI3K), phosphorylated mTOR (p-mTOR), phosphorylated Akt (p-Akt) and β-actin (Abcam).

Luciferase reporter assay
The fragments from TUG1 cDNA encompassing two potential miR-186-5p-targeting sites and its two mutated (MUT) counterparts were synthesized and subcloned into p-MIR-reporter plasmid (Thermo Fisher Scienti c) to produce TUG1 wild type, TUG1 mutant type 1 and TUG1 mutant type 2. Subsequently, HL60/ADR and HL60 cells were cotransfected with TUG1 wild type, TUG1 mutant type 1, or TUG1 mutant type 2 and anti-miR-186-5p, miR-186-5p, or respective controls by means of Lipofectamine 2000 (Invitrogen). A luminometer was used to measure the luciferase activity under the Luciferase Reporter System (Promega, Madison, WI, USA).

Statistics
GraphPad software 6.0 (GraphPad Inc., San Diego, CA, USA) was taken for statistical analysis. All results were displayed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and two-tailed Student's t-test and were conducted to determine the signi cance of differences. P values < 0.05 between differences were regarded statistically signi cant.

Results
TUG1 was upregulated with a high positive correlation with EVI-1 in BM samples of pediatric AML patients Firstly, we detected the TUG1 and EVI-1 expression in collected clinical pediatric AML samples by qRT-PCR. Upregulated expressions of TUG1 and EVI-1 in 21 BM samples of pediatric AML patients were demonstrated when compared with that in 10 BM samples from healthy controls (Fig. 1A and 1B). A positive correlation between TUG1 and EVI-1 expressions in the BM samples of pediatric AML patients was shown in Fig. 1C. These results suggested that TUG1 was upregulated with a signi cant positive correlation with EVI-1 in pediatric AML patients.

EVI-1 enhanced TUG1 expression in AML cells
The TUG1 and EVI-1 expression in ADR-resistant AML cells and their parental cells were further evaluated. As shown in Fig. 2A and 2B, TUG1 and EVI-1 expressions were both signi cantly upregulated in HL60 and K562 cells relative to HS-5 cells. Particularly, higher expressions of TUG1 and EVI-1 were observed in HL60/ADR and K562/ADR cells versus those in their parental cells ( Fig. 2A and 2B). It is widely recognized that transcription factors could contribute to the activation of lncRNAs in human diseases [21]. Bioinformatics analysis using UCSC (http://genome.ucsc.edu/) showed that EVI-1 was a potential transcriptional regulator for TUG1. Determination of EVI-1 effect on TUG1 transcription was accomplished via evaluating the changes of TUG1 expression after delivering 1 μg or 2 μg si-EVI-1 to HL60/ADR and HL60 cells. As a result, EVI-1 expression was dramatically inhibited in HL60/ADR and HL60 cells after si-EVI-1 transfection (Fig. 2C). Of note, introduction with 1 μg or 2 μg si-EVI-1 led to a remarkable reduction of TUG1 expression in these cells (Fig. 2D). Meanwhile, the regulatory effect of TUG1 on EVI-1 expression was analyzed by introducing TUG1, si-TUG1, or respective controls into HL60/ ADR and HL60 cells. The expression of TUG1 was observed being strikingly increased followed by delivery with TUG1 and distinctly declined in si-TUG1-treated HL60/ADR and HL60 cells (Fig. 2E). However, no signi cant change of EVI-1 expression was observed in TUG1-or si-TUG1-transfected cells ( Fig. 2F). Thus, we concluded that EVI-1 promoted TUG1 expression by serving as a transcriptional activator.

TUG1 promoted cell proliferation and the resistance to ADR in AML cells
For characterizing the TUG1 regulation on the resistance to ADR in AML cells, TUG1 or Vector were delivered to HL60 cells, and si-TUG1 or si-NC was transfected to HL60/ADR cells. CCK-8 assay proved that ectopic expression of TUG1 dramatically enhanced cell proliferation in HL60 cells compared to control group but TUG1 knockdown by si-TUG1 presented a reverse phenomenon in HL60/ADR cells (Fig.  3A). Consistently, the expression of PCNA, a marker of cell proliferation, was pronouncedly augmented in response to TUG1 overexpression in HL60 cells, but was drastically blocked after TUG1 was silenced in HL60/ADR cells (Fig. 3B). In addition, CCK-8 assay demonstrated that exposure of HL60/ADR and HL60 cells to 8 μM ADR resulted in an evident decrease of cell viability. However, increased expression of TUG1 effectively restored ADR-induced reduction of cell viability in HL60 cells while TUG1 downregulation markedly intensi ed ADR-induced decrease of cell viability in HL60/ADR cells (Fig. 3C). Also ow cytometry analyses proved that TUG1 upregulation reversed the apoptosis induced by ADR in HL60 cells, whereas TUG1 downregulation accelerated the apoptosis induced by ADR in HL60/ADR cells (Fig 3D). Furthermore, it was demonstrated that ADR treatment prominently restrained the Bcl-2 expression, Bcl-2 protein, in cells, which was partially reversed following promotion of TUG1 in HL60 cells (Fig. 3E) and further promoted by delivery with si-TUG1 in HL60/ADR cells (Fig. 3F). The above ndings demonstrated that TUG1 facilitated cell proliferation and ADR resistance in AML cells.
TUG1 acted as a miR-186-5p sponge in AML cells Widespread evidence has well-documented the close association between lncRNAs and miRNAs in regulating cellular processes in various types of human malignancies [22]. To clarify the regulatory basis of TUG1 on AML cells, the bioinformatic database starBase v2.0. was applied to predict the potential target miRNAs binding to TUG1. TUG1 was proposed harboring two binding sequences complementary to miR-186-5p (Fig. 4A), a well-characterized tumor suppressor in different types of tumors. qRT-PCR analysis showed a remarkable reduction of miR-186-5p in HL60 and K562 cells relative to that in HS-5 cells, particularly in HL60/ADR and K562/ADR cells (Fig. 4B). Intriguingly, TUG1 was reversely correlated with the expression of miR-186-5p in 21 BM specimens from pediatric AML patients (Fig. 4C). To doublecon rm the TUG1 interaction with miR-186-5p, luciferase reporter assay was conducted. miR-186-5p overexpression greatly reduced but miR-186-5p inhibition greatly boosted the luciferase activity of wildtype reporter vector (TUG1 wild type) in HL60/ADR and HL60 cells (Fig. 4D). However, amti-miR-186-5p and miR-186-5p mimics failed to generate any alternation of mutant reporters (TUG1 mutant type 1 and TUG1 mutant type 2) in these cells (Fig. 4D). Next, HL60/ADR and HL60 cells were introduced with TUG1, si-TUG1, or corresponding controls to investigate whether TUG1 could modulate miR-186-5p expression. As a result, miR-186-5p was apparently repressed in TUG1-overexpressing cells and successfully enhanced in TUG1-silencing cells (Fig. 4E). We deduced that TUG1 sponged miR-186-5p in AML cells.

miR-186-5p upregulation constrained cell proliferation and ADR resistance in AML cells
To determine the functional basis of miR-186-5p in regulating AML progression, anti-miR-186-5p or anti-miR-NC was delivered into HL60 cells and miR-186-5p or miR-NC was introduced into HL60/ADR cells. miR-186-5p silencing notably contributed to cell proliferation in HL60 cells relative to anti-miR-NC group (Fig. 5A). On the contrary, cell proliferation was dramatically suppressed in response to overexpression of miR-186-5p (Fig. 5A). Moreover, miR-186-5p downregulation signi cantly increased PCNA expression in HL60 cells versus that in anti-miR-NC group while miR-186-5p overexpression showed the opposite effect in HL60/ADR cells (Fig. 5B). The CCK-8 assay was performed to assess the miR-186-5p impact on ADR cytotoxicity. Anti-miR-186-5p effectively restored ADR-induced viability inhibition of HL60 cells while increased expression of miR-186-5p distinctly promoted ADR-induced viability inhibition of HL60/ADR cells (Fig. 5C). In line with the results of CCK-8 assay, ADR-induced decrease of Bcl-2 level in HL60 cells was remarkably rescued following the suppression of miR-186-5p.
Conversely, we observed an enhancement of ADR-induced reduction of Bcl-2 expression by promotion of miR-186-5p in HL60/ADR cells (Fig. 5D). Moreover, inhibition of miR-186-5p hindered apoptosis induced by ADR in HL60 cells and robust expression of miR-186-5p elevated ADR-induced apoptosis in HL60/ADR cells (Fig 5E). Altogether, the inhibition of miR-186-5p overexpression on cell proliferation as well as ADR resistance in AML cells was con rmed. TUG1 increased P-gp expression and activated the PI3K/Akt/mTOR signaling by targeting miR-186-5p in AML cells Next, western blot analysis manifested that promotion of TUG1 signi cantly enhanced P-gp level in HL60 cells while TUG1 downregulation elicited the opposite effects in HL60/ADR cells (Fig. 6A and 6B). Additionally, we found that overexpression of TUG1 resulted in a signi cant increase of the phosphorylation of PI3K, Akt and mTOR in HL60 cells (Fig. 6C) while depletion of TUG1 resulted in a substantial decrease of p-PI3K, p-mTOR, and p-Akt expression in HL60/ADR cells (Fig. 6D). Moreover, enforced expression of miR-186-5p evidently dampened the protein levels of p-PI3K, p-Akt and p-mTOR in HL60/ADR cells, which was strikingly ameliorated after reintroduction with TUG1 (Fig. 6E). Collectively, these results demonstrated that TUG1 increased P-gp expression and activated the PI3K/Akt/mTOR signals via sponging miR-186-5p in AML cells.

Discussion
It has been proposed that lncRNAs inactivate tumor suppressors and activate oncogenes in tumorigenesis. Considerable evidence shows an upregulation of TUG1, as an tumor-promoting lncRNA, in miscellaneous malignancies, such as osteosarcoma [23], prostate cancer [24], and AML [15]. On the contrary, TUG1 is well-documented to retard tumorigenesis in non-small-cell lung carcinoma [25] and glioma [13]. Accordingly, TUG1 presents great protentional in prognosis and therapeutic strategies for various cancers including AML [9]. Lately, numerous studies have implicated the association between aberrantly expressed lncRNAs and the resistance to chemotherapy of diverse carcinomas [26]. For example, in colorectal cancer cells, TUG1 knockdown re-sensitizes methotrexate (MTX) resistance via miR-186/CPEB2 axis [27]. TUG1 is overexpressed in osteosarcoma cells resistant to cisplatin and underexpression of TUG1 suppresses cisplatin resistance and facilitates apoptosis in cisplatin-treated osteosarcoma cells through the MET/Akt signaling [28]. Intriguingly, it has been previously proved that TUG1 is upregulated in AML cells and tissues resistant to ADR, and underexpression of TUG1 facilitates sensitivity to ADR in ADR-resistant AML cells in vivo and in vitro by upregulating miR-34a [14]. Our study further focused on exploring the impacts of TUG1 on resistance to ADR in AML cells and the functional basis. We observed upregulated expression of TUG1 in AML cells and pediatric AML patients. Moreover, TUG1 level was higher in ADR-resistant AML cells than their parental cells. Functionally, TUG1 overexpression signi cantly promoted cell proliferation and ADR resistance, and enhanced P-gp level in HL60 cells while TUG1 knockdown exhibited the opposite effects on HL60/ADR cells, suggesting the oncogenic role of TUG1 in AML.
Mechanistically, increasing experimental data have shown that transcriptional regulation contributes to lncRNA upregulation in human cancers by activating lncRNA transcriptions [29]. Herein, we explored whether overexpression of TUG1 was induced by transcription activation. Bioinformatics prediction presented that EVI-1 was a potential transcriptional activator of TUG1. The present thesis demonstrated that EVI-1 expression was boosted in pediatric AML patients and cells, particularly in the ones resistant to ADR, and positively correlated with the expression of TUG1 in pediatric AML patients. Robust expression of EVI-1 frequently occurs in up to one fth of pediatric AML and leads to unfavorable prognosis and low survival rate with currently used chemotherapy regimens [30]. The subsequent analyses demonstrated that EVI-1 knockdown suppressed TUG1 expression while TUG1 knockdown or silencing did not affect EVI-1 expression in HL60/ADR and HL60 cells. Therefore, EVI-1 activated the transcription of TUG1 to upregulate the expression of TUG1. miRNAs are de ned as a group of short, endogenous single-stranded ncRNAs (typically 19-25 nucleotides) long with limited or no capacity in encoding proteins [31]. The roles of miRNAs in cancer carcinogenesis and drug resistance have been extensively studied [32]. Substantive studies have suggested that lncRNAs may block the expression levels and activities of miRNAs by competitively binding to miRNAs [33]. According to our bioinformatics analysis, we found that TUG1 contained two potential binding sites pairing with the seed region of miR-186-5p. We further manifested that TUG1 repressed the expression of miR-186-5p via targeting miR-186-5p. As a tumor-speci c miRNA located at chromosomal 1p31.1, miR-186-5p is documented to be upregulated and exert cancer-promoting effects in several tumors such as colorectal cancer [34] and lung adenocarcinoma [35]. Conversely, miR-186-5p is down-regulated and suppresses tumorigenesis in human malignancies including non-small cell lung cancer [36], ovarian cancer [37] and osteosarcoma [38]. These results suggest the cancer type-dependent role of miR-186-5p in different tumors. Interestingly, miR-186-5p is reported to be downregulated in AML patients and predict poor prognosis [39]. Herein, a low miR-186-5p expression was observed in AML cells, especially in ADR-resistant AML cells. Moreover, functional experiments revealed that anti-miR-186-5p facilitated cell proliferation and ADR resistance in HL60 cells while miR-186-5p overexpression presented a reverse effect in HL60/ADR cells, indicating that miR-186-5p suppressed oncogenesis in AML.
As we all know, the PI3K/Akt/mTOR signaling cascade is regarded as a key prototypic survival signaling network that participates in modulating diverse physiological processes in hematological malignancies, including cellular apoptosis, metabolism, and cell proliferation [40]. Constitutively activated PI3K/AKT/mTOR pathway is observed in various tumors including AML [41]. Notably, extensive researches within the past decades have demonstrated that the PI3K/Akt/mTOR signals is closely related to drug resistance and its overactivation contributes to resistance to diverse chemotherapeutic agents in human malignancies [42]. Thus, inhibiting the PI3K/Akt/mTOR cascade may be a potential treatment candidate to reverse drug resistance. We proved that the PI3K/Akt/mTOR cascade was activated by TUG1 overexpression in HL60 cells and inhibited after TUG1 downregulation in HL60/ADR cells. Moreover, we further proved that robust expression of miR-186-5p blocked the PI3K/Akt/mTOR signals in HL60/ADR cells while ectopically expressing TUG1 rescued miR-186-5p-induced repression of the PI3K/Akt/mTOR signals in HL60/ADR cells. Altogether, these ndings suggested that TUG1 activated the PI3K/Akt/mTOR cascade by suppressing the expression of miR-186-5p in AML cells.
Collectively, we con rmed the upregulation of TUG1 in pediatric AML cells and patients, particularly in AML cells resistant to ADR. Moreover, we rstly claimed that upregulation of TUG1, which was induced by EVI-1, promoted cell proliferation and ADR resistance in pediatric AML via interacting with miR-186-5p through activating the PI3K/Akt/mTOR cascade. Our study provided a novel regulatory mechanism by which TUG1 exerted the oncogenic role in AML progression and TUG1 has the potential to be a potential treatment candidate for conquering drug resistance in AML.

Declarations
Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions E. L. and Y. L. contributed equally to this study. Y. L. designed the study and revised and edited the manuscript. Y. L. and Y. W. analyzed the data, interpreted the results, and wrote the manuscript. E. L., Y.
L. and H. Z. designed and guided the whole experiment. L. Z. and Y. T. helped with the sample preparation and performed the experiments. The author(s) read and approved of the nal manuscript.

Ethics approval and consent to participate
Signed consent for clinical studies was signed by their guardians prior to the study and our study was approved by Institutional Ethics Board of the First A liated Hospital of Zhengzhou University.

Competing interest
All the authors state that they have no con icts of interest.
Author details   human bone marrow stromal cell line HS-5, respectively. Western blot and qRT-PCR were implemented to analyze EVI-1 (C) and TUG1 (D) expressions in HL60 and HL60/ADR cells received with si-NC, 1 μg or 2 μg si-EVI-1 delivery. qRT-PCR and western blot were taken to evaluate TUG1 (E) and EVI-1(F) expressions in HL60 and HL60/ADR cells after transfection with TUG1, si-TUG1 or matched controls. *P < 0.05.   In uences of miR-186-5p on cell proliferation and ADR resistance in AML cells. (A and B) Anti-miR-186-5p or anti-miR-NC was delivered to HL60 cells and miR-186-5p or miR-NC was introduced into HL60/ADR cells, followed by assessment of cell proliferation and PCNA expression by CCK-8 assay and western blot analysis. (C-E) HL60 cells were received with anti-miR-186-5p or anti-miR-NC transfection and HL60/ADR cells were introduced with miR-186-5p or miR-NC in the presence of 8 μM ADR. At 48 h post-transfection, cell viability, Bcl-2 expression and apoptosis were detected by CCK-8, western blot, and ow cytometry analyses, respectively. *P < 0.05. Figure 6