Introduction
In recent years, nanomaterials have been at the center of attention of many researchers due to their unique and wonderful cancer applications, such as cancer detection, anticancer drug delivery, anticancer agents, anti-inflammatory activities and cancer therapeutic [1, 2]. Several studies have reported various effect of the nanoparticles (NPs) on DNA damages, inflammatory response, apoptosis, oxidative stress, immunotoxicity, reproductive toxicity and epigenetic mechanisms on both normal and cancer cells [3-5]. 
Zinc-oxide (ZnO) NPs with different sizes, surface charge, shapes, and morphology showed an oxidative stress and genotoxic effect [6]. ZnO-NPs have exhibited a high degree of selective cytotoxicity and anticancer performance towards the cancer cells [7, 8]. Several studies show that ZnO-NPs a promising new class of anticancer agents [8, 9]. Also, ZnO-NPs has the potential to alter cellular metabolism, genetic and epigenetic patterns, such as aberrant DNA methylation, non-coding RNA (ncRNA) expression and histone modification [10-12]. The most important mechanisms of ZnO-NPs on cancer cell include increasing levels of cellular reactive oxygen species  (ROS) and subsequent induce intrinsic apoptosis and autophagy (ATG) [9, 13, 14]. 
ATG and apoptosis are highly conserved pathway and regulated cellular process, which is crucial for response to metabolic stress [15]. The targets of ATG include degraded and recycled proteins and organelles under stressful conditions [16]. In breast cancer cells, core proteins can be identified for regulated apoptosis (caspase-3, caspase-9, Bax/Bcl-2), and ATG (LC3II/LC3I ratio, Atg5, Beclin-1) [17]. Recently studies have shown that ZnO-NPs can induce ATG and apoptosis in several cells and tissues [18-20]. The results of recent study show that ncRNA generally control ATG and apoptosis via regulating the expression of related genes [21, 22].
ncRNA is a powerful tool for controlling the vital function of cells by interacting with macro-molecules [23]. Long ncRNA (lncRNAs) are longer than 200 nucleotides with ability encoding peptide (<100 amino acids) or proteins as promising candidates in biomarkers and antitumor drugs [24]. Similar to pre-mRNA, lncRNAs are mainly transcribed by RNA polymerase II but in contrast, expression and conservation in exons are fewer in the lncRNA [23]. lncRNAs are involved in many critical aspects of cell function, such as epigenetic regulation, gene expression regulation metabolism, cell cycle, apoptosis, ATG and RNA splicing [25]. The aberrant lncRNAs expression has been detected in physiological and pathological conditions in various cancers, especially breast cancer occurrence, progression and development [23, 26]. Recent studies identified several lncRNAs in tight connection with breast cancer [23, 27, 28]. A common property of cancer cells is their ability to overcome environmental stresses. lncRNA acts as major regulator of cellular and environmental stress in cancer [29]. In other words, lncRNAs can sensitize and resist cells to response to drug and oxidative stress. 
The first aim of this study was to provide evidence that ZnO-NPs could induce ATG in breast cancer. The second aim of the present study was to determine lncRNAs associated with ZnO-induced ATG and apoptosis in breast cancer cells. Finally, we evaluated genetic and epigenetic alteration in ATG and apoptosis related lncRNA in breast cancer cell. Our results will provide fundamental role of lncRNA in the mechanism of cellular response to oxidative stress and drug in breast cancer.

Materials and Methods 
Study chemicals
Dulbecco’s modified eagle’s medium (DMEM), penicillin–streptomycin and fetal bovine serum were purchased from Life Technologies (Waltham, MA, USA) Invitrogen (Carlsbad, CA, USA). Then, (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, M5655) hydrocortisone was purchased from Sigma-Aldrich (India). Also, 0.25% trypsin–ethylenediaminetetraacetic acid was purchased from Corning Incorporated (Corning, NY, USA). Insulin and horse serum were purchased from Gibco (Grand Island, NY, USA).

Synthesis of ZnO-NPs
The ZnO-NPs have been synthesized through the modified solvothermal method [30]. During this procedure, two solutions were prepared. For the solution A, 2.86 g Zn (CH3COO)2.2H2O was dissolved in 63 mL ethanol (99%) and was heated to 60 °C while stirring. Solution B, which contains KOH (0.75 g) and 35 mL ethanol (99%) is a precursor solution to the growth of ZnO-NPs. After the complete dissolve of chemicals in ethanol, solution B added dropwise to solution A stirring 100 rpm at 60 °C. In the next step, the final mixture was kept in room temperature for an hour. The solution was poured in a stainless-steel vessel (autoclave) and placed in a furnace at 150 °C for 18 h. After cooling down the sample to room temperature, the formed white precipitates were collected by centrifuging at 4000 rpm for 10 min. To remove the impurities, the collected white precipitates were washed with acetone and pure deionized water three times. Subsequently, the white precipitates were dried using the freeze drier overnight. The obtained dried product was ground two powder for later applications.

Database search and lncRNA signature selection
Selection, expression evaluation, and DNA methylation status of lncRNA consist of 4 phases.
Screening phase (I) is to select ATG/apoptosis related lncRNA for investigation in breast cancer according to the following items. We manually searched and retrieved paper list for additional studies and review articles. The inclusion criteria were as follows: ATG/apoptosis related lncRNA in human cancer analyzed using RNA analysis methods, such as quantitative PCR, RNA sequencing and microarrays; published between August 1, 2013 to June 30, 2022 as original and review articles without language restriction; and selective ATG/apoptosis related lncRNA expressed in breast cancer cells. The exclusion criteria were as follows: animal studies or the articles that could not be found in full. 
Training phase (II) included downloading the differentially expressed genes in 1048 breast cancer sample from The Cancer Genome Atlas (TCGA) using cBioPortal. Top 12 significantly differentially expressed lncRNAs in breast cancer from both apoptosis and ATG groups were selected as candidates. The selected lncRNAs promoter were obtained from the Genome Browser or Eukaryotic Promoter Database.
Validation phase (III) consists of two steps: a) the lncRNA levels in breast cancer and normal MCF7 cell lines before and after exposure to ZnO-NPs was analyzed via real-time polymerase chain reaction (RT-PCR), b), the methylation status of lncRNA promoters and CpG island were investigated via MeDIP-RT-PCR and chromatin immunoprecipitation (ChIP).

Cell culture and treatment with ZnO-NPs
MCF-7 cells were obtained from the National Cell Bank of Iran (Pasteur Institute of Iran). MCF-7 cells were cultured in DMEM supplemented containing horse serum (5%), EGF (100 mg/mL), hydrocortisone (0.5 mg/ml), insulin (10 mg/mL) and 1% penicillin-streptomycin. Cells were grown under conditions of 5% CO2 and 37 °C in a controlled humidified incubator. ZnO-NPs stock solution (2 mg) was suspended in 800 µL of distilled water and sonicated in an ice-water bath for 30 min at 30 W. Immediately, after sonication, 100 µL of BSA (15%) and 100 µL of PBS (10X) added to 800 µL distilled water of suspension. The dilutions of ZnO-NPs were vigorously vortexed for 30 s before cell exposure to avoid NPs agglomeration. The cell exposed to ZnO-NPs were done when the cells had grown to about 80% confluence.

Cytotoxicity assay 
The cell viability of the MCF-7 cells after treatment with ZnO-NPs was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To evaluate the proliferative activity, MCF-7 counted and added to 4×104 cells per mL each well and exposed to various concentrations (range 1–100 μg/mL) and then incubated for 24-72 h. Wells containing medium but no cells were used as the blank, and wells containing culture medium without ZnO-NPs treatment were used as the control. The amount of 20 μL of MTT solution at 5 (mg/mL) was added to each well and incubated for 4 h at 37 °C. The MTT medium was discarded and the cells were lysed in 100 μL of DMSO. Afterwards, 60 μL of the DMSO was added to each well and incubated in room temperature (RT) for 30 min. The resulting Formosan crystals were solubilized and quantified by measuring absorbance at 570 nm by a multiwell-plate reader (Flex station III, Molecular Devices, Sunnyvale, CA, USA). 
The viability was calculated as the ratio of exposure cell absorbance to control cell absorbance (OD ZnO-NPs−OD blank)/(OD control – OD blank)×100%). This experiment was carried out in triplicate. The DMSO (0.1%) was used as negative control.

Colony-forming assay 
A colony-forming assay is used to determine the cells’ re-proliferation capacity and disseminated tumor cells after treatment with NPs. The breast cancer cells (50 cells/well) were seeded into a 96-well plate and incubated for 24 h. The MCF-7 cells were treated to concentrations ranging from 1-100 μg/mL for 10 days in the CO2 incubator. Cells were stained with 1% crystal violet (w/v) in 10% ethanol for 30 min. Percent viability was calculated by dividing the number of colonies formed by the number of cells plated per 100.

Determination of cytochrome c
In the intrinsic apoptotic pathway, cytochrome c (Cyt c) release from mitochondria was the main marker inactivates cell death. The concentrations of Cyt c (Cat. No. RBMS263R) in cell apoptosis were determined by using a sandwich enzyme-linked immunosorbent assay (ELISA) kit (BioVender Laboratory Medicine, Inc., Czech Republic). Antibody specific for human Cyt c (1 μg/mL) was coated onto a microplate well. Standards, samples, and controls were added into designated microplate wells and specific biotin-conjugated antibody was also pipetted to all wells and incubated at 37 °C for 120 min. After washing the plates 3 times with 1× PBST (phosphate-buffered saline with Tween 20), streptavidin-HRP was added into each well and incubated at room temperature for 1 h. In the next wash step, TMB substrate was added to the microplate wells. The reaction was terminated by addition of 20 µL of sulfuric acid solution (1 M) and absorbance was measured at the wavelength of 450 nm using the microplate reader. The experiment was repeated three times in triplicate wells.

ROS
Intracellular mitochondrial ROS were measured by 2′, 7′- dichlorodihydrofluorescein diacetate (Sigma-Aldrich Co.). after cell-treated with ZnO-NPs 10-100 µg/mL for 24-48 h, cells washed with PBS and then cells were incubated at 3 7 °C with 2′,7′- dichlorodihydrofluorescein diacetate for 20 minutes. The amount of fluorescence was determined using excitation at 485 nm and emission at 530 nm. Fluorescence intensity is related to the amount of ROS formed intracellularly.

Apoptosis detected by flow cytometry
Cells were made into 1×106/mL suspension after being digested with 0.25% trypsin, cleaned twice with PBS, mixed with 100 μL binding buffer, etc. After that, 10 μL of Annexin V-FITC and 10 μL of propidium iodide (PI) were supplemented to the suspension. The suspension was incubated 5 min at room temperature in the dark and detected by NovBCyte flow cytometry (Derica Biotechnology Co., Ltd., Beijing, China, DLK0002051). The test was repeated 3 times and results were averaged.

Analysis of genomic DNA methylation
DNA genomic was extracted using THPG methods according to the method described previously. The percentages of DNA methylation levels were calculated by a MethylFlash Methylated DNA Quantification Kit, an ELISA-based colorimetric assay (Epigentek, Brooklyn, NY, USA) according to the manufacturer’s instructions [31]. 

Total RNA extraction and RT-PCR 
To study gene expression, MCF-7 cells were exposed to ZnO-NPs for 48 h. The control group contained MCF-7 cells with no NP exposure. Total RNA was extracted according to the manufacturer’s instructions for RNA extraction kit (Zistabzar pajoohan, Iran) and spectrophotometrically assessed for its quality. cDNA was prepared using BioFact™ RT Series (Korea). To evaluate the expression of selected lncRNA, synthesized cDNAs were used for RT-PCR experiments. Genes were analyzed by SYBR Green fluorescence real-time RT-PCR with a Rotor-Gene Q instrument (Qiagen, Hilden, Germany). Primers stated in Table 1 were designed using AlleleID software version 7.5 (Premier Biosoft, USA).

The RT-PCR reactions were used for RT-PCR according to the corresponding protocol (Takara, Japan). The RT-PCR reactions were set up in similar conditions Real-time qPCR. The relative expression of genes was calculated using the (2−ΔΔCt) method and normalized using the ACTB as the internal control.

MeDIP-RT-PCR
According to the manufacturer’s instructions, we used MeDIP kit (ab117133, Abcam, USA) for the analysis of methylation of the promoter and CpG island region of selected lncRNA along with enrichment and capture from the total methylation regions on genome DNA. Real-time qPCR was performed to evaluate the methylation status of promoter and CpG island region genes before and after ZnO-NPs treatment. The qPCR reactions were set up in a volume of 20 μL containing SYBR Green (RealQ plus Master Mix Green without ROX, Ampliqon, Denmark.) with GC buffer (Kawsar, Iran). The qPCR reactions were set up in a volume of 20 μL containing SYBR Green (RealQ plus Master Mix Green without ROX, Ampliqon, Denmark.) with GC buffer (Kawsar, Iran) Specific primers and methylated DNA. The amplification program consisted of an initial denaturation of 95 °C for 15 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 15 s. All sample reactions were performed in triplicates and the mean of the three reactions was considered as a representative value for each sample.  Melting curve analysis was carried out following each reaction to verify the specificity of the products and the absence of primer dimers.

Preparation of standard curve
The standard curves were established using serially diluted concentrations of the selected lncRNA for an absolute quantification according to the method as described [31]. Thus, the nested PCR reaction was performed to amplify the selected regions using 4 specific primer sets. Both PCR reaction solution contained 5 μL DNA, 12.5 μL Taq DNA polymerase master mix RED (Ampliqon, Denmark), 10 pM of each primer, GC buffer and ddH2O per 25 μL total reaction volume. Each PCR reaction was carried out using a thermal cycler gradient PCR system (Eppendorf, Germany). The first amplification program included predenaturation at 95 °C for 10 min, followed by 40 cycles of 94 °C for 30 s, 57 °C for 30 s, 72 °C for 1 min and final extension 72 °C for 10 min; and the second amplification program were predenaturation at 95 °C for 10 min, followed by 40 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min and final extension 72 °C for 10 min. The concentration of selected lncRNA in the stock solution was analyzed using a fluorospectrometric technique. The Finnzymes program was used to calculate the initial copy number of the calibrator dilution in the standard series. The standard series of 6.6 pg was used to convert the copy number to genome equivalents (GEq).

ChIP assay
Histone modification includes cytosine methylation and histone tail methylation which regulate chromatin structure, function and gene expression [32]. Epigenetic marks induced by ZnO-NPs on histone modification in promoter and CpG island of selected lncRNA genes were quantified with ChIP assay, followed by RT-PCR. To perform ChIP assays, exposed and not-exposed MCF-7 cells were lysed. Subsequently, cross-linked chromatin was sonicated and incubated with 3 µg antibody against H3K4me3 (Abcam: ab1012, broadly distributed on active enhancers and transcription start site (TSS)) and H3K27me3 (enriched around inactive transcription start site (TSS)) (Abcam, USA) at 4 °C overnight. The immune-complex was precipitated with protein A-agarose (Upstate, Charlottesville, VA), and the obtained beads were washed and proteins were removed with 75 μg of proteinase K (45 °C for 4 h). The complex was incubated at 65 °C overnight, and then DNA was recovered by triton/heat/phenol/glycogen (THPG) extractions [31], and DNA dissolved in 50 μL of TE buffer. The RT-PCR assays were performed by employing the primers in Table 1.

Statistical analysis
Data analysis of qPCR was performed with Prism, version 9.0.0 (GraphPad Software, Inc., La Jolla, CA, USA). Data were expressed as Mean±SD, and statistical significance was tested among and between groups using one-way and 2-way ANOVA, respectively, followed by post hoc Tukey’s multiple-comparison or Bonferroni tests when appropriated. Differences with P<0.001 were considered to be statistically significant. All experiments were done at least three times unless otherwise indicated.

Results 
Characterization of ZnO-NPs 
The synthesized ZnO-NP in this study was characterized by a variety of techniques and the results were reported in this section. Transmission electron microscopy (TEM) (Figure 1) shows homogenous hexanol shape ZnO-NPs as a piece of evidence for the good quality of the NPs.

 Furthermore, the scanning electron microscope (SEM) micrograph (Figure 1) is used to study the physical characterization and morphological structure of NPs and confirm the homogenous shape and structure of synthesized ZnO-NPs. The size histogram of synthesized ZnO-NPs (presented in (Figure 1) indicates that the average size of NPs is about 25±6 nm. 
The UV-vis absorption spectra of dispersed ZnO-NPs in ethanol 30% (0.1% v/w) is presented in Supplementary Figure 2.

 The ZnO-NPs characteristic absorption peak at wavelength 357 nm is highlighted accordingly [33]. The sharp peak indicates that the particle size is narrow. The XRD pattern of synthesized ZnO-NPs is presented in Supplementary Figure 2 and all highlighted peaks indexed to ZnO-NPs structure. Figure 2 shows high purity of NPs with intense, narrow and diffraction peaks. Brunauer-Emmett-Teller (BET) test was carried out by N2 adsorption temperature of 77k.
Figure 2C represents the nitrogen (N2) adsorption-desorption isotherms of ZnO-NP from BET study. It is a relatively flat typical type 4 adsorption similar to the results obtained by Zhou et al. [34]. This finding confirms that the mean particle size obtained from BET agrees with TEM and XRD particle size finding (25±6 nm). Fourier Transform Infrared (FTIR) spectroscopy was performed to analyses and determine the active functional group of solvothermal method synthesized ZnO-NPs (Figure 2). A broad absorption band at 370 cm-1 attributes to Zn-O stretching vibration and correspond to E2 mode of hexagonal ZnO. Furthermore, other small absorption band at 930, 1050, and 3400 cm-1 are determined. These absorption band were corresponded to symmetric O-C-O and O-H stretching vibration of adsorbed CO2 and H2O from the air which can be abandoned. These results indicate the high purity of synthesized ZnO-NPs.

Cytotoxicity of ZnO NPs
To evaluate the viability of MCF-7 cells following exposure to ZnO-NPs (1–100 μg/mL), an MTT assay was performed after 24, 48, and 72 h of treatment. The results demonstrated a significant dose- and time-dependent cytotoxic effect. Increasing concentrations of ZnO-NPs and longer exposure times markedly reduced cell viability. Based on these findings, 15 μg/mL at 48 h was selected for subsequent experiments, as this condition induced moderate cytotoxicity without causing excessive cell death that could obscure subtle epigenetic alterations (Figure 3A).

The long-term proliferative capacity of treated cells was further assessed using a colony-forming assay (Figure 3B). ZnO-NP exposure significantly reduced colony formation in a concentration-dependent manner, indicating effective inhibition of cell proliferation in MCF-7 cells

Determination of Cyt c
Cyt c release from mitochondria is a key event in the intrinsic apoptotic pathway. In this study, the level of Cyt c significantly increased following treatment with ZnO‑NPs in both a dose‑ and time‑dependent manner (Figure 4A).

The highest Cyt c release was observed after exposure to 100 μg/mL ZnO‑NPs for 72 h compared with the control group. These findings indicate that ZnO‑NP treatment promotes mitochondrial membrane disruption and triggers the mitochondrial apoptotic pathway. Consistently, MCF‑7 breast cancer cells exposed to increasing concentrations of ZnO‑NPs exhibited progressively higher Cyt c release over time, further supporting the role of ZnO‑NPs in inducing apoptosis through mitochondrial signaling.

ZnO-NP exposure on genomic DNA methylation
Global genomic DNA methylation levels in MCF‑7 cells were evaluated after exposure to different concentrations of ZnO‑NPs (1, 5, 10, 15, 25, 50, and 100 μg/mL) for 24 and 72 h (Figure 4B). The results demonstrated that ZnO‑NP treatment significantly altered the genomic DNA methylation profile. A clear dose‑dependent decrease in DNA methylation levels was observed compared with the untreated control cells. The methylation percentage progressively declined from approximately 85% in control cells to about 42% in cells treated with higher concentrations of ZnO‑NPs. These findings indicate that exposure to ZnO‑NPs induces global DNA hypomethylation in MCF‑7 cells, suggesting that ZnO‑NPs may influence epigenetic regulation in breast cancer cells

ZnO-NPs induced production of ROS in MCF-7
ROS generation in MCF-7 cells treated with ZnO-NPs increased in a dose- and time-dependent manner. The highest level of ROS production was observed after treatment with 100 μg/mL ZnO-NPs for 72 h, which was significantly greater than that observed at lower concentrations and shorter exposure times. A marked increase in fluorescence intensity was also detected after treatment with 25 μg/mL for 48 h compared with the control group. In contrast, the increase in ROS at lower concentrations such as 5–10 μg/mL was relatively modest (Figure 5).

Screening of BC-associated candidate lncRNA
To investigate the effect of ZnO-NPs on lncRNA expression in breast cancer, the top 10 differentially expressed lncRNAs were selected from the TCGA cBioPortal database. These lncRNAs included PVT1, NORAD, GAS5, CASC15, ZFAS1, TUG1, DANCR, XIST, HOTAIR, and MALAT1, and were used as candidate targets for further analysis in this study (Figure 6).

MeDIP- RT-PCR
The methylation status of promoter and CpG island selected lncRNAs was evaluated by MeDIP-real time quantitative PCR (primers in Table 1) in MCF-7 cells after exposure with ZnO-NPs. The results for the analysis lncRNAs are shown in Table 2.

After exposed with ZnO-NPS, the promoter methylation patterns showed that the lncRNA including GAS5 exhibited significantly decreased methylation in their promoters. Also, our result showed that MALAT1, TUG1, and XIST exhibited significantly increased methylation in their promoters. However, PVT1, DANCR, CASC15, HOTAIR, NORAD, and ZFAS do not show a significant alteration in methylation status. 

Histones modification analysis
H3K27me3 and H3K4me3 histone modification marks at the promoter and CpG island regions of selected lncRNAs were evaluated to investigate histone tail modification status following ZnO‑NP exposure in MCF‑7 cells. As shown in Figure 7, ZnO‑NP treatment significantly decreased H3K27me3 levels while increasing H3K4me3 enrichment at the GAS5 locus.

 In contrast, MALAT1, XIST, and TUG1 exhibited a significant increase in the repressive H3K27me3 mark accompanied by a decrease in the activating H3K4me3 mark. However, no significant changes in either H3K4me3 or H3K27me3 enrichment were observed at the promoter or CpG island regions of PVT1, DANCR, CASC15, HOTAIR, NORAD, or ZFAS1. These findings indicate that ZnO‑NP exposure modulates the epigenetic landscape of specific lncRNAs by altering activating and repressive histone marks, which may contribute to the regulation of their transcriptional activity.

Gene expression analysis
Relative expression levels of the selected lncRNAs were evaluated in control and ZnO‑NP–treated MCF‑7 cells (15 μg/mL, 48 h) using qRT‑PCR (Figure 8).

Expression values were normalized to ACTB and calculated relative to untreated control cells. ZnO‑NP exposure resulted in a significant upregulation of GAS5. In contrast, MALAT1, XIST, and TUG1 were significantly downregulated following treatment. Although PVT1, DANCR, CASC15, HOTAIR, NORAD, and ZFAS1 exhibited a slight increase in expression, these changes were not statistically significant compared with the control group. Data are presented as Mean±SE from three independent biological replicates.

Discussion
Human exposure to NPs including engineered and natural ones may happen in different ways throughout the lifetime. Thus, a range of associated adverse effects on human health may be expected from NPs side. The toxicity of NPs is commonly generated by the generation of free radicals and oxidative stress [35]. Previous studies showed that NPs toxicity depends on their dose and exposure time [36]. Therefore, synthesized NPs have been proposed for epigenetic therapy. The major observed effect of ZnO-NPs exposure is reduced the level of global DNA methylation [37]. The most important epigenetic modification, including DNA methylation patterns, histone modification, and ncRNA profiles. However, the effect of ZnO-NP on epigenetics, including ncRNA expression is not been investigated. This work represents the first study to investigate the effect of nanoparticle-induced epigenetic effects on breast cancer MCF-7 cells and to evaluate this effect on ROS-regulator lncRNA. 
Treatment with ZnO-NPs was found to reduce not only the amount of genomic DNA methylation, but also the cell viability in breast cancer cell. In our study, we observed increase in genomic DNA hypomethylation (Figure 4) and ROS level (Figure 5) along with corresponding alteration in lncRNA expression at the 15 µg/ml of ZnO-NPs for 48 h (Figure 8). We compared 10 top lncRNA in treated and untreated conditions. Our study was based on quantitative analysis of lncRNA expression (quantitative real time PCR) and determination of promoter status of selected lncRNA using MeDIP-real time and ChIP assay. After exposure of MCF-7 to ZnO-NPs, we investigated whether changes in the expression of lncRNA could correlate with changes in promoter methylation and histone modification. The result reveal that the exposure to ZnO-NPs resulted in 4 lncRNA (GAS5, TUG1, XIST, and MALAT1) with significant altered expression levels in MCF-7 cells (Figure 8). Expression of MALAT1, XIST, and TUG1 were decreased at 15 µg/mL for ZnO-NPs at 48 h. In contrast, TUG1 expression significantly increased under the same conditions. Also, the results of DNA methylation and histones modification were consistent with the lncRNA expression profiles. It seems that increased cell death and ROS level in ZnO-NPs exposed cells are associated with more epigenetic alterations of lncRNA involved in the regulation of intracellular ROS.
Several studies showed that ZnO-treat of cancer cells increase cellular ROS and cell death [38-40]. Also, the recently studies reveal that lncRNA acts as major regulators of cell stress in cancer [29, 41]. In this study, we have hypothesized that crosstalk between ZnO-NPs and ROS leads to altered epigenetic of lncRNA, which regulate ROS generation and removal. The results indicate a significant relationship between ROS production and changes in the expression of ROS regulator lncRNA.
Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) located at 11q13, also known as (NEAT2) with a size of about 8 kb, is highly conserve and abundant lncRNAs in normal mamalian tissues. Knockdown of MALAT1 suppressed proliferation, motility, and increased apoptosis in vitro [42]. 

Conclusion
Previous studies have reported that global DNA hypomethylation leads to overexpression of genes that is important in carcinogenesis. However, in this study, we hypothesized that increased global DNA hypomethylation that was associated with ZnO-NPs leads to regulated lncRNA expression involve in regulation ROS generation. In conclusion, our data suggest strong correlation between ZnO-NPs exposure, lncRNA (ZFAS1, MALAT1, and PVT1) expression, and amount of viability and apoptosis in MCF7 cells. The present study provides new insights into the mechanism of genotoxic effects and epigenetic of ZnO-NPs on lncRNA profile in breast cancer cells. It is important to assay the toxicity of NPs before use of them in medicine. To our knowledge, this is the first report on genetic and epigenetic study of ZNO-NPs effect on lncRNA expression in breast cancer. Further researches are needed to fully characterize the relation between observed including the alteration in lncRNA expression and the effect of ZnO-NPs observed at the cellular level. 

Ethical Considerations
Compliance with ethical guidelines
There were no ethical considerations to be considered in this research.

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors.

Authors' contributions
Conceptualization: Ali Saberian and Mostafa Akbariqomi; Funding acquisition: Mostafa Akbariqomi and Sara Keshtkari; Resources: Mostafa Akbariqomi and Sara Keshtkari; Software: Reza Heidari; Validation: Mostafa Akbariqomi and Reza Heidari; Formal analysis: Reza Heidari and Sara Keshtkari; Data curation: Ali Saberian and Reza Heidari; Visualization: Sara Keshtkari and Reza Heidari; Writing the original draft: Ali Saberian; Methodology, investigation, review, editing and final approval: All authors; Supervision and project administration: Mostafa Akbariqomi.

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
The authors would like to express their sincere gratitude to the Comprehensive Laboratory of AJA University of Medical Sciences for providing the laboratory facilities and technical support required to conduct this study. 



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