MicroRNA-411-3p motivates methotrexate’s cellular uptake and cytotoxicity via targeting Yin-yang 1 in leukemia cells

This study aimed to figure out how microRNA (miR)- 411-3p’s impacts on methotrexate (MTX)’s cellular uptake and cytotoxicity in acute lymphoblastic leukaemia (ALL) CEM-C1 cells by targeting Yin-yang 1 (YY1). miR-411-3p and YY1 were detected by RT-qPCR or Western blot. Intracellular MTX concentration was measured by enzyme-linked immunosorbent assay. Cell viability and apoptosis were evaluated by CCK-8, clonal formation assay, and flow cytometry. Verification of miR-411-3p and YY1’s targeting link was manifested. It came out that miR-411-3p mimic or si-YY1 elevated intracellular MTX, MTX-induced cytotoxicity and apoptosis rate in CEM-C1. However, the inverse results were noticed in cells introduced with miR-411-3p inhibitor or oe-YY1. Meanwhile, it was found that cell relative luciferase activity was reduced after co-transfection of miR-411-3p mimic with YY1-WT, indicating that miR-411-3p targeted YY1. Elevation of YY1 could turn around elevating miR-411-3p’s impacts on MTX’s cellular uptake and cytotoxicity in CEM-C1 cells. These findings convey that miR-411-3p motivated MTX’s cellular uptake and cytotoxic impacts via targeting YY1 in leukemia cells. This study is helpful for learning about the mechanisms underlying MTX responses in ALL patients.


INTRODUCTION
Acute lymphoblastic leukaemia (ALL) is an extremely common malignant tumor, which frequently shows up in children, taking up about 80% of ALL cases, and occasionally takes place in adults (Liang et al., 2021).ALL is featured by malignant transformation and proliferation of T-or B-cell progenitors which invade blood, bone marrow, and extramedullary sites, thereby preventing normal blood cell production (Ranjbar et al., 2019).Methotrexate (MTX) is a chemotherapeutic drug for ALL and has been applied to cure leukemia and lymphoma patients (Woźniak et al., 2021).Al-though most patients are available to achieve long-term disease-free survival via standardized treatments, including MTX, some ALL treatments for children are still ineffective, and drug resistance is a vital reason for this phenomenon (Jaramillo et al., 2019).Therefore, discovering approaches to suppress chemotherapy resistance in ALL patients is crucial for ALL cure.
MTX is a folate antagonist, whose action is extremely complicated, involving some transporters determining intracellular drug levels and active MTX polyglutamate and some metabolic enzymes (Gervasini et al., 2017).MTX enters cells by passive diffusion via the solute carrier 19A1 transporter (RFC1) (Ando et al., 2013), while the drug is pumped out by some membrane efflux transporters of the ATP-binding cassette family (Aberuyi et al., 2021).MTX is converted into active polyglutamate form (MTX polyglutamate, MTXPG) by folate polyglutamate synthase intracellularly (De Beaumais et al., 2012).MTX and MTXPG inhibit thymidine synthesis by targeting dihydrofolate reductase and thymidine synthase (Rushworth et al., 2015), thereby impairing DNA and RNA synthesis and leading to cell death (Oosterom et al., 2018).In conclusion, the accumulation of MTX and its active metabolite methotrexate polyglutamate (MTXPG) in all cells is an important determinant of its anti-leukemia effect (Panetta et al., 2010).
MicroRNAs (miRNAs), a cluster of endogenous single-stranded non-coding RNAs, modulate gene expression (Lin et al., 2021).Much evidence illustrates that miRNAs participate in almost all biological processes, including drug resistance (Xian et al., 2019;Naghizadeh et al., 2020;Wei et al., 2021).Genetic variation in MTX transporter genes has been reported to be implicated in toxicity, and these transporters are controlled by miRNAs (Iparraguirre et al., 2016).Wang SM (Wang et al., 2017).confirm that two miRNA-binding site polymorphisms (rs3737966 G>A and rs35134728 DEL/TTC) in the 3'-UTR of 5,10-methylenetetrahydrofolate reductase were linked with serum MTX concentrations.Xu Wen (Xu et al., 2018).discovered that miR-29 family restrained MTX resistance but motivated apoptosis by targeting COL3A1 and MCL1 in osteosarcoma.MiR-411-3p, a newly studied miRNA, has been discovered to be aberrantly expressed in some human cancers (Huang et al., 2021;Wang et al., 2019;Wang et al., 2020).However, no studies have been conducted to clarify its role in MTX resistance.In the research, the main focus was on miR-411-3p's function in controlling Yin-yang 1 (YY1) and its impact on ALL CEM-C1 cell uptake and MTX cytotoxicity.

Growth of human ALL CEM-C1 cells was in Roswell
Park Memorial Institute -1640 medium replenished with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 U/ml penicillin, and 100 U/ml streptomycin (Beyotime Biotechnology, Shanghai, China).CEM-C1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

Intracellular MTX Concentration Determination
Cells were dispersed in 96-well plates at 5.0×10 5 cells per well prior to transfection and MTX treatment.Quantification of intracellular MTX was done using human methotrexate, MTX ELISA kit (HB1386-Hu, Shanghai Hengyuan Biotechnology Co., LTD).Absorbance at 450 nm was recorded on a microplate reader (ELX800, Bi-oTek Instruments, Inc.) and MTX concentrations were calculated.

Colony formation assay
Treatment of transfected CEM-C1 cells was done with different concentrations of MTX (10, 20 and 40 ng/ml).The cells were then transferred to a 6-well plate (1×10 3 cells/well) and supplemented with RPMI-1640 medium containing 10% fetal bovine serum at 37°C.After 14 days, the cells were fixed with formaldehyde and stained with 0.1% crystal violet solution (Sigma Aldrich) at 25°C for 15 min and counted under a CKX41 microscope (Olympus).

Apoptosis assay
Annexin V-fluorescein isothiocyanate Apoptosis Detection Kit (BD, USA) was applied to detect apoptosis.In brief, cells were stained with 10 μl Annexin V and 5 μl propidium iodide for 15 min.Then, quantitative analysis was performed using BD FACSCalibur TM (BD, USA).

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA's extraction was conducted using the miRNeasy extraction kit (Qiagen, Valencia, CA, USA).
MiR-411-3p's quantification was conducted using the Hairpin-it miRNA qPCR quantification kit (GenePharma, Shanghai, China) with U6 as a loading control.Detection of YY1 was implemented using Power SYBR Green PCR Master Mix (Applied Biosystems) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control, and all PCR reactions were conducted on a 7500 Fast Real-Time PCR Systems (Applied Biosystems, CA, USA).All primer sequences were manifested in Table 1.Calculation of miR-411-3p or YY1 was implemented using the 2 -ΔΔCT method (Wu et al., 2020).

The luciferase activity assay
Construction of the luciferase reporter gene plasmids (Promega, USA) covering the 3'-UTR of YY1 with forecast miR-411-3p target sites (Wild-type) or the mutated sites of 3'UTR of YY1 (Mutant) was implemented.Transient co-transfection of CEM-C1 cells was then done with reporters and miR-411-3p mimic or mimic NC.Detection of Firefly and renilla luciferase activities was conducted using a dual-luciferase reporter assay system (Promega) (Liu et al., 2020).

Genes
Primer sequence (5'-3') MiR-411-3p Cell lysates were collected.Incubation of the supernatant from cell lysates was implemented with human anti-Ago2 antibody (SCBT, Santa Cruz, CA, USA) or mouse Immunoglobulin G (SCBT, Santa Cruz).Detachment of proteins was done using Proteinase K buffer and the quantitative real time PCR examined co-precipitated RNAs.

2Statistical analysis
Data were presented as mean ± standard deviation (S.D.) unless otherwise noted.Two groups' comparisons were implemented using the unpaired two-tailed Student's t-test.Application of Graphpad Prism software version 4.0 was for all statistical analyses.P<0.05 emphasized obvious statistical meaning.

Elevating miR-411-3p motivates MTX's cellular uptake and cytotoxic influences
CEM/C1 cells were transfected with miR-411-3p mimic or mimic NC. miR-411-3p was elevated after transfection of miR-411-3p mimic (Fig. 1A).CEM/C1 cells were treated with disparate concentrations of MTX (10, 20 and 40 ng/ml).Detection of the intracellular MTX concentration was conducted, and the results displayed that the intracellular MTX was elevated in CEM/ CI cells introduced with miR-411-3p mimic (Fig. 1B).The cell growth was tested.The results illustrated that after incubation with different concentrations of MTX, elevating miR-411-3p resulted in a clear augmentation in the repression rate of CEM/CI cells (Fig. 1C) and a memorable decline in the number of colonies (Fig. 2A).Considering that apoptosis is a mechanism by which MTX exerts its antitumor effect, the latent function of miR-411-3p in MTX-induced apoptosis in CEM/CI cells was then figured out.It turned out that the apoptosis index of CEM/CI cells in the miR-411-3p mimic introduced with different concentrations of MTX was elevated at all MTX concentrations (Fig. 2B).Moreover, after incubation with different concentrations of MTX, enhancing miR-411-3p could restrain Bcl-2 but accelerate Bax (Fig. 1D-E).These results suggest that elevating miR-411-3p restrains CEM/C1 cell growth by motivating MTX's cellular uptake and cytotoxic influences.

Depressing YY1 motivates MTX's cellular uptake and cytotoxic influences
To figure out YY1's impact on MTX's cellular uptake and cytotoxicity in CEM/C1 cells, si-YY1/NC was transfected into CEM/C1 cells (Fig. 6A).It turned out that intracellular MTX was clearly elevated in CEM/CI cells transfected with si-YY1 (Fig. 6B).It came out that after incubation with different concentrations of MTX, repressing YY1 resulted in a clear elevation in the inhibition rate of CEM/CI cells (Fig. 6C) and a distinct decline in the number of colonies (Fig. 7A).It turned out that the apoptosis index of CEM/CI cells in the si-YY1 treated with different concentrations of MTX was elevated at all MTX concentrations (Fig. 7B).Moreover, after incubation with different concentrations of MTX, depressing YY1 could restrain Bcl-2 but motivate Bax (Fig. 6D/E).The above results illustrate that repressive YY1 can accelerate MTX's cellular uptake and cytotoxicity, thereby refraining CEM/C1 cell growth.

Enhancing YY1 restrains MTX's cellular uptake and cytotoxic influences
To further testify YY1's role, it was transfected oe-YY1/NC into CEM/C1 cells and successful transfection was verified (Fig. 8A).It turned out that intracellular    MTX was clearly declined in CEM/CI cells transfected with oe-YY1 (Fig. 8B).It came out that after incubation with different concentrations of MTX, strengthening YY1 resulted in a clear decline in the repression rate of CEM/CI cells (Fig. 8C) and a distinct elevation in the number of colonies (Fig. 9A).It turned out that the apoptosis index of CEM/CI cells in the si-YY1 treated with different concentrations of MTX was declined at all MTX concentrations (Fig. 9B).Moreover, after incubation with different concentrations of MTX, depressing YY1 could motivate Bcl-2 but restrain Bax (Fig. 8DE).Overall, elevating YY1 can restrain MTX's cellular uptake and cytotoxicity, thereby motivating CEM/C1 cell growth.

DISCUSSION
MTX is a chemotherapeutic drug for ALL's, which majorly exerts its therapeutic impact in ALL by impairing the synthesis of DNA in cells.The ability of leukemic blasts to uptake MTX and MTX-polyglutamate has been reported to be vital for the survival of children with ALL.However, MTX has a narrow therapeutic range, its pharmacokinetics and therapeutic response display great variability in ALL's, and it exhibits serious toxicity in plentiful patients, often resulting in an interruption or discontinuation of chemotherapy (Hu et al., 2019).Recent studies have exhibited that miRNAs are implicated in the concentration of MTX in the blood of ALL patients (Wang et al., 2014), suggesting that miR-NAs are likely to participate in controlling the uptake of MTX by ALL cells.This research illustrated originally that elevation of miR-411-3p could motivate MTX's cellular uptake and cytotoxicity in CEM/C1 cells, and restrain cell growth, whereas repressing miR-411-3p did the opposite.Meanwhile, it was also testified that miR-411-3p functioned by targeting YY1.Multiple studies have clarified that miR-411-3p takes part in various human diseases.For example, miR-411-3p was reduced in the tumor tissues of patients with multiple myeloma, and mediated LncRNA ANRIL dysregulation through hypoxia-inducible factor 1α to suppress the malignant proliferation and tumor growth of multiple myeloma stem cell-like properties (Wang et al., 2020)].(Fu et al., 2020) testified that miR-411-3p was reduced in oral squamous cell carcinoma cells, and elevating miR-411-3p restrained cell growth by targeting NFAT5.miR-411-3p mitigated silica-induced pulmonary fibrosis (Gao et al., 2020).Meanwhile, miR-411-3p was linked with the improved overall survival in lung cancer patients treated with nivolumab (Halvorsen et al., 2018).However, miR-411-3p's impacts on the efficacy of MTX treatment in ALL patients are uncertain.In this research, elevation of miR-411-3p memorably enhanced MTX concentration, cell repression rate and apoptosis index, reduced colony number and suppressed Bcl-2 but motivated Bax in CEM/C1 cells treated with disparate concentrations of MTX, whereas depressing miR-411-3p had the exact opposite effect.These results conveyed that miR-411-3p could control MTX sensitivity in CEM/C1 cells.
YY1 is an extensively expressed multifunctional transcription factor affiliated with the GLI-Krüppel class of zinc finger proteins (Zhang et al., 2021), which takes on momentous roles in diversified biological processes and is linked with tumorigenesis (Li et al., 2020).discovered that YY1-mediated elevation of lncRNA MCM3AP-AS1 motivated lung cancer angiogenesis and progression (Chen et al., 2019).confirmed that YY1 targeted tubulin polymerization-promoting protein and restrained pancreatic cancer migration, invasion, and angiogenesis through p38/MAPK and PI3K/AKT pathways (Li et al., 2021).verified that YY1-induced elevation of LncRNA SNHG5 motivated angiogenesis in AML through the miR-26b/ CTGF/VEGFA axis.Recently (Antonio-Andres et al., 2021).confirmed that YY1 was elevated in peripheral blood leukemia cells of children with ALL with Pro-B and T phenotypes, and its elevation is clearly linked with poor survival.Meanwhile, YY1 has also been shown to be linked with drug resistance to multiple leukemia chemotherapeutics.For example (Zhang et al., 2021).demonstrated that in chronic myeloid leukemia, KDM6A motivated imatinib resistance through YY1-mediated transcriptional elevation of TRKA.The present study discovered that repressing YY1 motivated MTX's cellular uptake and cytotoxic impacts, but repressed CEM/ C1 cell growth, while enhancing YY1 turned around strengthening miR-411-3p's impacts on MTX cell uptake and cytotoxicity in CEM-C1 cells, and enhanced CEM/ C1 cell growth.
However, this research still has several limitations.First, animal experiments were not performed to clarify miR-411-3p's impacts on MTX sensitivity.Secondly, it only figured out the regulatory effect of miR-411-3p on MTX sensitivity in a single ALL cell line (CEM/C1 cells) and not in other leukemia cell lines.Finally, further exploration of the downstream mechanisms of YY1 was not conducted.It is hoped that in future studies, the regulatory mechanism of miR-411-3p in leukemia MTX resistance can be further improved.

CONCLUSION
In short, this research demonstrated that miR-411-3p motivated MTX's cellular uptake and cytotoxic influences by targeting YY1 in ALL cells.The findings not only offer new insights into the mechanisms of variable MTX responses noticed in ALL patients, but also new references for addressing MTX chemotherapy resistance in ALL patients.

Figure 2 .
Figure 2. Elevating miR-411-3p motivates MTX's cellular uptake and cytotoxic influences.(A) Clone formation assay detection of cell proliferation; (B) Flow cytometry detection of cell apoptosis; The data in the figures were all measurement data, and the values were clarified as mean ± S.D. (N=3); *vs. the mimic NC, P<0.05.

Figure 7 .
Figure 7. Depressing YY1 motivates the cellular uptake and cytotoxic effects of MTX.(A) Clone formation assay detection of cell proliferation; (B) Flow cytometry detection of cell apoptosis; the values were clarified as mean ± S.D. (N=3); *vs. the si-NC, P<0.05.

Figure 9 .
Figure 9. Enhancing YY1 restrains the cellular uptake and cytotoxic effects of MTX.(A) Clone formation assay detection of cell proliferation; (B) Flow cytometry detection of cell apoptosis; The data in the figures were all measurement data, and the values were clarified as mean ± S.D. (N=3); *vs. the oe-NC, P<0.05.

Figure 10 .
Figure 10.Elevation of YY1 could turn around elevating miR-411-3p's impacts on MTX cellular uptake and cytotoxicity in CEM-C1 cells.(A) RT-qPCR and Western blot verification of successful transfection; (B) ELISA detection of intracellular MTX concentration; (C) CCK-8 detection of cell viability; (D/E) RT-qPCR and Western blot detection of apoptosis-linked genes (Bcl-2 and Bax); The data in the figures were all measurement data, and the values were clarified as mean ± S.D. (N=3); *vs. the mimic NC group, P<0.05; #vs. the miR-411-3p mimic+oe-NC, P<0.05.

Figure 11 .
Figure 11.Elevation of YY1 could turn around elevating miR-411-3p's impacts on MTX cellular uptake and cytotoxicity in CEM-C1 cells.(A) Clone formation assay detection of cell proliferation; (B) Flow cytometry detection of cell apoptosis; The data in the figures were all measurement data, and the values were clarified as mean ± S.D. (N=3); *vs. the mimic NC group, P<0.05, #vs. the miR-411-3p mimic+oe-NC, P<0.05.