LncRNA UCA1 elevates the resistance of human leukemia cells to daunorubicin by the PI3K/AKT pathway via sponging miR-613

Abstract Acute leukemia is a hematological malignant tumor. Long non-coding RNA urothelial cancer-associated 1 (UCA1) is involved in the chemo-resistance of diverse cancers, but it is unclear whether UCA1 is associated with the sensitivity of acute leukemia cells to daunorubicin (DNR). DNR (100 nM) was selected for functional analysis. The viability, cell cycle progression, apoptosis, and invasion of treated acute leukemia cells (HL-60 and U-937) were evaluated by cell counting kit-8 (CCK-8) assay, flow cytometry assay, or transwell assay. Protein levels were detected with Western blot analysis. Expression patterns of UCA1 and miR-613 were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between UCA1 and microRNA-613 (miR-613) was verified by dual-luciferase reporter assay. We observed that UCA1 expression was elevated in HL-60 and U-937cells. DNR constrained viability, cell cycle progression, invasion, and facilitated apoptosis of HL-60 and U-937 cells in a dose-dependent manner, but these impacts mediated by DNR were reverted after UCA1 overexpression. MiR-613 was down-regulated in HL-60 and U-937 cells, and UCA1 was verified as a miR-613 sponge. MiR-613 inhibitor reversed DNR treatment-mediated effects on viability, cell cycle progression, apoptosis, and invasion of HL-60 and U-937 cells, but these impacts mediated by miR-613 inhibitor were counteracted after UCA1 inhibition. Notably, the inactivation of the PI3K/AKT pathway caused by DNR treatment was reversed after miR-613 inhibitor introduction, but this influence mediated by miR-613 inhibitor was offset after UCA1 knockdown. In conclusion, UCA1 up-regulation facilitated the resistance of acute leukemia cells to DNR via the PI3K/AKT pathway by sponging miR-613.


Introduction
Acute leukemia is a malignant clonal disease of hematopoietic stem/progenitor cells, which includes acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML) [1]. The treatment of acute leukemia includes chemotherapy, immunotherapy, radiotherapy, targeted therapy, or stem cell therapy [2]. Although these treatments improve the prognosis of patients with acute leukemia, chemotherapy resistance remains a major problem [3,4].
Long non-coding RNAs (lncRNAs) are a kind of non-protein encoding RNAs that exert crucial regulatory roles in gene regulatory networks [5]. Studies have revealed that lncRNAs are involved in many pathological and physiological processes, such as tumorigenesis, organogenesis, cell lineage choice, and tissue homeostasis [6][7][8][9]. Increased researches have revealed that lncRNAs are associated with tumor chemo-resistance [10]. Long non-coding RNA urothelial cancer-associated 1 (UCA1) has been reported to be involved in the occurrence and progression of multiple tumors [11]. Mounting researches have pointed out that UCA1 participates in the chemo-resistance in a series of tumors. For instance, UCA1 exerts a promoting influence on cisplatin resistance in oral squamous cell cancer [12] and bladder cancer [13]. Also, UCA1 up-regulation elevates the resistance of ovarian cancer cells to paclitaxel [14]. Furthermore, UCA1 also facilitates the progression of chronic myeloid leukemia and AML [15,16]. Additionally, UCA1 is associated with Adriamycin resistance in pediatric AML [17]. Nevertheless, the mechanism by which UCA1 regulates daunorubicin (DNR) resistance in acute leukemia remains unclear.
MicroRNAs (miRNAs) are a large class of non-coding RNAs that generally bind to mRNAs to cause translation inhibition or degradation [18]. Researchers have reported that miRNAs play vital roles in intracellular transformation, benign and malignant states, and the progression of cancer [11,19]. MicroRNA-613 (miR-613) has been revealed to play a repressive role in a range of tumors, such as osteosarcoma [20], colorectal cancer [21], and glioma [22]. Moreover, miR-613 increase cisplatin sensitivity in gastric cancer [23]. However, the role of miR-613 in DNR resistance in acute leukemia is unclear.
Consequently, we aimed to survey the function and regulatory mechanism of UCA1 in DNR resistance in acute leukemia.

Flow cytometry assay
The apoptosis of HL-60 and U-937 cells with or without DNR treatment was analyzed with the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (BD Biosciences, San Jose, CA, U.S.A.). In brief, HL-60 and U-937 cells were cultured in RPMI-1640 medium supplemented with DNR for 48 h. After washing, HL-60 and U-937 cells (3 × 10 5 ) were re-suspended in binding buffer (100 μl). Then, the cells were stained with Annexin V-FITC (5 μl) and PI (100 μl) and incubated for 30 min in the dark. In the end, the cells were analyzed using the FACScan flow cytometry (BD Biosciences).

Transwell assay
The invasion ability of HL-60 and U-937 cells with or without DNR treatment was assessed by transwell assay. In brief, the cell medium containing HL-60 and U-937 cells (1 × 10 5 ) were added to the top chamber (8 μm, BD Biosciences) coated with matrigel matrix (BD Biosciences). Contemporaneously, RPMI-1640 medium with FBS (10%) were added to the down chamber. After culture for 24 h, the cells in the lower chamber were counted by the CCK-8 assay.

Dual-luciferase reporter assay
The potential binding sites between UCA1 and miR-613 were predicted by the starbase, LncBase and mircode databases. The sequences of wild-type (WT) UCA1 (with predicted miR-613 binding sites) and mutant (MUT) UCA1 were synthesized and inserted into the pGL3-control vector (Promega, Madison, WI, U.S.A.). After that, A luciferase reporter vector were cotransfected into HL-60 and U-937 cells together with miR-NC or miR-613. A dual-luciferase reporter assay kit (Promega) was employed to analyze the luciferase activities.

Statistical analysis
Data derived from at least three independent experiments were presented as mean + − standard deviation. GraphPad Prism 6.0 (GraphPad, San Diego, CA, U.S.A.) and SPSS 18.0 software (SPSS, Chicago, IL, U.S.A.) were utilized for statistical analysis. P<0.05 was deemed to indicate a statistically significant difference. The differences between two or among more groups were analyzed through Student's t test or one-way analysis of variance (ANOVA).

DNR repressed viability, cell cycle progression, invasion, and triggered apoptosis of HL-60 and U-937 cells
To survey the effects of DNR on malignant behaviors of HL-60 and U-937 cells, we first performed CCK-8 assay to assess the viability of HL-60 and U-937 cells treated with different DNR doses (0, 25, 50, and 100 nM). Compared with the control group, DNR treatment suppressed the viability of HL-60 and U-937 cells with the enhancement of DNR dose ( Figure 1A,B). Flow cytometry assay manifested that DNR treatment promoted cell cycle arrest and apoptosis of HL-60 and U-937 cells with the increase of DNR ( Figure 1C,D). Transwell assay was then conducted and the results presented that DNR treatment caused a decrease in the invasion ability of HL-60 and U-937 cells in a dose-dependent manner ( Figure 1E). Afterward, the levels of Cyclin D1 (proliferation-associated protein), Vimentin (migration-associated protein), and Cleaved-casp-3 (apoptosis-rated apoptosis) were detected and the results exhibited that DNR treatment decreased protein levels of Cyclin D1 and Vimentin in HL-60 and U-937 cells in a dose-dependent manner, while elevated the protein level of Cleaved-casp-3 ( Figure 1F,G). Collectively, these results suggested that DNR repressed viability, cell cycle progression, invasion, and induced apoptosis of HL-60 and U-937 cells in a dose-dependent manner.

DNR treatment decreased UCA1 expression in HL-60 and U-937 cells
To verify the expression of UCA1 in HL-60 and U-937 cells, we executed qRT-PCR analysis to detect UCA1 expression in HL-60 and U-937 cells. We discovered that UCA1 expression was markedly elevated in HL-60 and U-937 cells in comparison with the HS-5 cells (Figure 2A). However, DNR treatment resulted in a decrease in UCA1 expression in HL-60 and U-937 cells ( Figure 2B,C). Taken together, these data indicated that DNR treatment reduced UCA1 expression in HL-60 and U-937 cells in a dose-dependent manner.

UCA1 up-regulation decreased the sensitivity of HL-60 and U-937 cells to DNR
Taking into account the above results, we further explored the effects of UCA1 on the viability, apoptosis and invasion of DNR (100 nM)-treated HL-60 and U-937 cells. The overexpression efficiency of pCDNA3.0-UCA1 in HL-60 and U-937 cells was presented in Figure 3A. Moreover, UCA1 overexpression partly reversed the repressive influence of DNR treatment on the viability of HL-60 and U-937 cells ( Figure 3B). Moreover, forced UCA1 expression partially abolished the promoting influence of DNR treatment on cell cycle arrest and apoptosis of HL-60 and U-937 cells ( Figure 3C,D). Also, transwell assay displayed that UCA1 overexpression reversed the decrease of the invasion ability of HL-60 and U-937 cells caused by DNR treatment ( Figure 3E). Additionally, Western blot analysis manifested that UCA1 up-regulation overturned DNR treatment-mediated effects on protein levels of Cyclin D1, Vimentin, and Cleaved-casp-3 in HL-60 and U-937 cells ( Figure 3F,G). Therefore, these data indicated that UCA1 up-regulation reduced the sensitivity of HL-60 and U-937 cells to DNR.

UCA1 served as a sponge of miR-613 in HL-60 and U-937 cells
Further experiments revealed that UCA1 was mostly distributed in the cytoplasm of HL-60 and U-937 cells, indicating that UCA1 might act as a miRNA sponge (Supplementary Figure S1). To explore the regulatory mechanism of UCA1 in HL-60 and U-937 cells, we predicted miRNAs that might interact with UCA1. We discovered that four miRNAs (miR-206, miR-613, miR-4770, and miR-4735-3p) had complementary sites with UCA1 in the overlapping starbase, LncBase and mircode databases (Supplementary Figure S2A). Moreover, miR-613 mimic reduced the luciferase activity of the UCA1-WT reporter in both HL-60 and U-937 cells (Supplementary Figure S2B). The overexpression efficiency of miR-613 mimic was exhibited in Supplementary Figure S3. The binding sties between miR-613 and UCA1 were presented in Figure 4A. Dual-luciferase reporter assay exhibited that miR-613 mimic curbed the luciferase activity of the UCA1-WT reporter in HL-60 and U-937 cells, whereas the luciferase activity of the UCA1-MUT reporter did not change ( Figure 4B). Compared with the HS-5 cells, the expression of miR-613 was markedly down-regulated in HL-60 and U-937 cells ( Figure 4C). QRT-PCR displayed that UCA1 expression was observably reduced in HL-60 and U-937 cells after transfection with si-UCA1 ( Figure 4D). In addition, silenced UCA1 expression distinctly elevated the expression of miR-613 in HL-60 and U-937 cells ( Figure 4E). In sum, these results demonstrated that UCA1 severed as a sponge for miR-613 expression in HL-60 and U-937 cells.

UCA1 decreased the sensitivity of HL-60 and U-937 cells to DNR by interacting with miR-613
Given that UCA1 acted as a sponge for miR-613, we further investigated whether UCA1 affected the sensitivity of HL-60 and U-937 cells to DNR via interacting with miR-613. The knockdown efficiency of miR-613 inhibitor was  Figure S3. We observed that miR-613 was up-regulated in DNR-treated HL-60 and U-937 cells, while this increase was recovered after Anti-miR-613 introduction ( Figure 5A). Moreover, the down-regulation of miR-613 in DNR-treated HL-60 and U-937 cells mediated by miR-613 inhibitor was reversed by UCA1 silencing ( Figure 5A). The viability of HL-60 and U-937 cells was elevated by miR-613 inhibition under DNR treatment, while this elevation was abolished by the knockdown of UCA1 ( Figure 5B). Furthermore, miR-613 inhibitor promoted cell cycle progression and repressed cell apoptosis in DNR-treated HL-60 and U-937 cells, while these effects caused by miR-613 inhibitor were reversed after UCA1 knockdown ( Figure 5C,D). In addition, UCA1 silencing abrogated the promoting impact of miR-613 knockdown on the invasion of DNR-treated HL-60 and U-937 cells ( Figure 5E). Also, silenced miR-613 expression increased protein levels of Cyclin D1 and Vimentin and decreased the protein level of Cleaved-casp-3 in DNR-treated HL-60 and U-937 cells, but these trends were reversed by UCA1 inhibition ( Figure  5F,G). In short, these results manifested that UCA1 affected the sensitivity of HL-60 and U-937 cells to DNR through miR-613.

UCA1 regulated the PI3K/AKT pathway via miR-613
To further survey the regulatory mechanism of UCA1 in HL-60 and U-937 cells, we assessed the levels of p-PI3K and p-AKT in DNR-treated HL-60 and U-937 cells transfected Anti-miR-NC, Anti-miR-613, Anti-miR-613+si-NC, or Anti-miR-613+si-UCA1. The results exhibited that DNR reduced the value of p-PI3K/P13K and p-AKT/AKT in HL-60 and U-937 cells, while this decrease was reversed by miR-613 silencing ( Figure 6A,B). However, the effects of miR-613 knockdown on the value of p-PI3K/PI3K and p-AKT/AKT in DNR-treated HL-60 and U-937 cells were counteracted by UCA1 silencing (Figure 6A,B). Therefore, these data indicated that UCA1 regulated the PI3K/AKT pathway via adsorbing miR-613.

Discussion
DNR is a chemotherapeutic drug widely utilized in the treatment of various malignant tumors. A number of studies have proved that DNR delayed the growth of cancer cells. Also, the synergy of DNR and celecoxib promotes apoptosis and reduces growth of AML cells [24]. At present, the resistance of acute leukemia cells to DNR is one of the main obstacles to its treatment [25].
Accumulated studies have proved that UCA1 is connected with the chemo-resistance of various cancers. Report of Bian et al. claimed that UCA1 reduced the sensitivity of colorectal cancer cells to 5-fluorouracil and promoted the proliferation of colorectal cancer cells [26]. Another report pointed out that UCA1 elevated doxorubicin resistance in gastric cancer [27]. Xiao et al. stated that UCA1 accelerated the resistance of chronic myeloid leukemia cells to imatinib [15]. Additionally, UCA1 silencing could elevate the cytotoxic effect of adriamycin to adriamycin-treated pediatric AML cells [28]. In the present study, DNR induced apoptosis and impeded viability, cell cycle progression, and invasion of HL-60 and U-937 cells in a dose-dependent manner. Also, DNR restrained the expression of UCA1 in HL-60 and U-937 cells, and forced UCA1 reversed DNR treatment-mediated impacts on viability, cell cycle progression, invasion, and apoptosis of HL-60 and U-937 cells. Thus, we concluded that UCA1 upregulation could reduce the sensitivity of HL-60 and U-937 cells to DNR.
Recent researches have pointed out that UCA1 act as a miRNA sponge and participate in the regulation of chemo-resistance in diversified cancers [14,28,29]. MiR-613 had been reported as an anti-tumor gene in bladder cancer [30], non-small cell lung cancer [31], and laryngeal squamous cell cancer [32]. Moreover, miR-613 mimic elevated cell sensitivity to cisplatin by down-regulating SOX9 in gastric cancer [23]. Herein, miR-613 was down-regulated in HL-60 and U-937 cells. Also, UCA1 was identified as a miR-613 sponge. Furthermore, DNR treatment restored the down-regulation of miR-613 in HL-60 and U-937 cells. In addition, the effects of DNR treatment on viability, cell cycle progression, apoptosis, and invasion of HL-60 and U-937 cells were reversed by miR-613 inhibition, but these effects caused by miR-613 silencing were restored after UCA1 silencing. However, miR-613 was revealed as an oncogene in colon cancer [33] and cervical cancer [34], which might be related to tissues specificity. Thus, we inferred that UCA1 mediated the sensitivity of HL-60 and U-937 cells to DNR via miR-613.
It was reported that the Pi3K/AKT pathway was involved in the chemo-resistance of diverse cancers [35,36]. Yang et al. reported that lncRNA linc00239 contributed to malignant behaviors of AML cells and elevated doxorubicin resistance through activating the PI3K/AKT/mTOR pathway in AML [37]. In the present study, DNR treatment blocked the PI3K/AKT pathway, but miR-613 silencing reversed the inactivation of the PI3K/AKT pathway in HL-60 and U-937 cells caused by DNR treatment. Nevertheless, UCA1 suppression offset the influence of miR-613 inhibitor on the PI3K/AKT pathway in DNR-treated HL-60 and U-937 cells. Therefore, we inferred that UCA1 regulated the sensitivity of HL-60 and U-937 cells to DNR through mediating PI3K/AKT pathway via sponging miR-613.
In sum, UCA1 up-regulation decreased the sensitivity of acute leukemia cells to DNR. Moreover, UCA1 overexpression activated the PI3K/AKT pathway via sponging miR-613. The present study provided an evidence to support UCA1 as a promising target for acute leukemia treatment.

Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.