Background
CcRCC (Clear cell renal cell carcinoma) is the most common histological subtype of kidney cancers, affecting more than 403,000 individuals and causing over 175,000 deaths in 185 countries worldwide per year [
1]. At present, radical surgical operation is the main treatment for patients with early ccRCC, and targeted therapy may prolong the survival time of patients with advanced or metastatic ccRCC [
2‐
4]. Unfortunately, the prognosis of ccRCC patients is still poor, especially for patients with advanced and metastatic disease, the 5-year survival rate after diagnosis is only 12% [
4‐
6]. Therefore, finding more effective and safer therapeutic targets has great potential value for improving the prognosis of ccRCC patients.
Although ncRNAs (non-coding RNAs) account for more than 90% of human genomic RNA, most of the > 50,000 ncRNAs have only been discovered in the past 10 years and remain largely unstudied [
7,
8]. As a new class of ncRNAs, lncRNAs (long non-coding RNAs) are characterized as non-coding transcripts greater than 200 base pairs in length transcribed by RNA Pol II from independent promoters [
9]. In many cases, lncRNAs have been proven to be the main regulator of gene expression, thus they can play key roles in a variety of biological functions and disease processes including cancers [
10]. Recent accumulating evidence has indicated that lncRNAs, such as OTUD6B-AS1, URRCC, HOTAIRM1 and MRCCAT1, play important regulatory roles in diverse biological processes in ccRCC [
11‐
14].
Epigenetic alterations have been considered as one of the hallmarks of tumorigenesis [
15], and emerging evidence suggests that epigenetic modification is one of the main mechanisms regulating lncRNA expression and tissue specificity [
16,
17]. For instance, DNA methylation-mediated activation of lncRNA SNHG12 increases temozolomide resistance in glioblastoma [
18]; aberrant methylation-mediated downregulation of lncRNA SSTR5-AS1 promotes the progression and metastasis of laryngeal squamous cell carcinoma [
19]; LOC134466 methylation accelerates oncogenesis of endometrial carcinoma through LOC134466/hsa-miR-196a-5p/TAC1 axis [
20]. Previous studies have reported that ZNF582-AS1 is a novel lncRNA with diagnostic and prognostic values in RCC based on TCGA (The Cancer Genome Atlas) data [
21], and it is epigenetically silenced by aberrant DNA methylation in colorectal cancer [
22]. However, the specific clinical significance of ZNF582-AS1 in ccRCC and its DNA methylation status and molecular mechanism remain unknown.
In this study, we found that lncRNA ZNF582-AS1 expression was significantly downregulated in ccRCC tissues than that in the adjacent normal renal tissues, and decreased ZNF582-AS1 expression was significantly correlated with advanced tumor stage, higher pathological stage, distant metastasis, and poor prognosis. Besides, we identified that ZNF582-AS1 was epigenetically deactivated by DNA methylation at the CpG islands within its promoter, which played an important role in decreased ZNF582-AS1 transcription in ccRCC. Moreover, we showed that ZNF582-AS1 overexpression inhibited cell proliferative, migratory and invasive ability, and increased cell apoptosis in vitro and in vivo. Mechanistically, we demonstrated that ZNF582-AS1 overexpression suppressed the N(6)-methyladenosine modification of MT-RNR1 by reducing rRNA adenine N(6)-methyltransferase A8K0B9 protein level, resulting in the decrease of MT-RNR1 expression, followed by the inhibition of MT-CO2 protein expression. Furthermore, we confirmed that MT-RNR1 overexpression reversed the decreased MT-CO2 expression and phenotype inhibition of ccRCC induced by increased ZNF582-AS1 expression. Collectively, our findings demonstrate that ZNF582-AS1 is a powerful tumor biomarker, which highlights its potential clinical value as a promising prognostic and therapeutic target of ccRCC.
Methods
Clinical sample collection
62 fresh ccRCC tissue samples and pair-matched adjacent normal tissue samples were obtained from patients who underwent surgery. After resection, fresh ccRCC and pair-matched adjacent normal renal tissues obtained from the same patient were snap-frozen in liquid nitrogen immediately. This study was approved by the Biomedical Research Ethics Committee of Peking University First Hospital (Beijing, China, IRB00001052–18004), and each patient included in this study signed an informed consent form.
Cell culture and transfection
The normal human renal tubular epithelial cell line HK2, human embryonic kidney cell line HEK293 and 5 ccRCC cell lines OSRC2, 786-O, Caki-1, 769-P and A498 were used in this study. HK2, HEK293, Caki-1 and A498 cells were cultured in DMEM supplemented with 10% foetal bovine serum, while the OSRC2, 786-O and 769-P cells were cultured in RPMI 1640 supplemented with 10% FBS.
For overexpression of ZNF582-AS1, A8K0B9, B4DRY2 and MT-RNR1, recombinant pLV-EF1a-hluc-P2A-Puro-WPRE-CMV-ZNF582-AS1, pLV-hef1a-mNeongreen-P2A-Puro-WPRE-CMV-A8K0B9-3Xflag, pLV-hef1a-mNeongreen-P2A-Puro-WPRE-CMV-B4DRY2-3Xflag, pLV-hef1a-Bla-WPRE-CMV-MT-RNR1 and their corresponding control plasmid vectors were constructed by the SyngenTech Company (SyngenTech Co. Ltd., Beijing, China). Cells were transfected with the corresponding vector using Lipofectamine 3000 Transfection Reagent (Invitrogen, USA) according to the manufacturer’s instructions. After 48 h, cells transfected with the corresponding vector were harvested for qRT-PCR. The stable cell line was established by lentivirus infection accordingly. Lenti-virus was produced using three vectors system: transfer vector, viral packaging (psPAX2) and viral envelope (pMD2G) at 6:3:1 ratio transfected into 293 T cells. Then, the cells were infected by lentiviruses according to the MOI value (the number of lentiviruses per number of cells). The ZNF582-AS1, A8K0B9 and B4DRY2 overexpressed stable cell lines were selected with puromycin (5 μg/mL) and MT-RNR1 overexpressed stable cell lines were selected with blasticidin (10 μg/mL).
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from the tissue samples or the transfected cells using the TRIzol reagent (Invitrogen; USA). cDNA was generated using reverse transcription (Invitrogen; USA). qRT-PCR was performed according to the manufacturer’s instructions, and normalized to GAPDH. All experiments were repeated at least three times. The detailed primer sequences included in this study are shown in Additional file
1: Table S1.
RNA fish
RNA FISH was performed using a fluorescent in situ hybridization kit (RiboBio, China) following the manufacturer’s instructions. The lncRNA ZNF582-AS1, U6 and 18S FISH probes were also designed and synthesized by the RiboBio Company. Fluorescence detection was performed with a confocal laser-scanning microscope (Leica, Germany).
Methylation-specific PCR (MSP)
MethPrimer 2.0 (
http://www.urogene.org/methprimer2/) was used to predict the CpG island of ZNF582-AS1 and designing MSP primers. Genomic DNA was extracted from ccRCC and adjacent normal renal tissues. The purified DNA was exposed to bisulfite with a DNA Bisulfte Conversion Kit (Tiangen, China) according to the manufacturer’s protocol. The methylation-specific PCR (MSP) of bisulfite-transformed DNA was carried out with a nested, two-stage PCR method. Amplified PCR products were separated by 1.5% agarose gel electrophoresis and visualized with GelRed. The specific primers used for MSP were listed in Additional file
1: Table S1.
Sequenom MassARRAY quantitative DNA methylation analysis
Genomic DNA was extracted from ccRCC and pair-matched adjacent normal tissues, and the bisulfite conversion reaction was performed according to the manufacturer’s instructions. The PCR mixtures were pre-heated for 4 min at 94 °C, followed by 45 cycles of 94 °C for 20 s, 56 °C for 30 s and 72 °C for 1 min, the final extension at 72 °C for 3 min. PCR products were incubated with Shrimp Alkaline Phosphatase following the manufacturer’s protocol. After in vitro transcription and RNase A digestion, small RNA fragments with CpG sites were acquired for the reverse reaction. The methylation ratios of the products were calculated using Epityper software Version 1.0 (Sequenom, San Diego, CA, USA). The Sequenom MassARRAY platform (Oebiotech, Shanghai, China) was utilized to quantitatively analyze the DNA methylation status of ZNF582-AS1 DNA. PCR primers were designed using EpiDesigner, and their sequences were listed in Additional file
1: Table S1.
Demethylation analysis
ccRCC cells were seeded in six-well plates at a concentration of 1 × 105 cells per well, grown for 24 h, and then treated with 5 μM 5-Aza-2′-deoxycytidine (5-Aza-dC, A, Sigma-Aldrich) for 4 days. Cells were cultured with or without 100 Nm Trichostatin A (TSA, T, Sigma-Aldrich) for the final 24 h. RNA was isolated for qRT-PCR analysis and DNA was extracted for ZNF582-AS1 MSP.
Ethynyl-2-deoxyuridine (EdU) incorporation and CCK-8 assays
Cell proliferation was determined by an ethynyl-2-deoxyuridine (EdU) incorporation assay using an EdU Apollo DNA in vitro kit (RiboBio, Guangzhou, China) and BeyoClick™ EdU Cell Proliferation Kit with DAB (Beyotime, China) following the manufacturer’s instructions. ccRCC cells were seeded in 96-well plates and cell viability was evaluated with the Cell Counting Kit 8 (TransGen Biotech, Beijing, China). Absorbance was measured (OD value) at a wavelength of 450 nm.
TUNEL and flow cytometry assays
Cells apoptosis was detected by TUNEL staining using One Step TUNEL Apoptosis Assay Kit (Beyotime, China) and Colorimetric TUNEL Apoptosis Assay Kit (Beyotime, China) according to the manufacturer’s instructions. Cell apoptosis was also assayed by staining with Annexin V-FITC and PI (KeyGEN BioTECH) following manufacturer’s instructions and detected by a flow cytometer (FACSCalibur, Becton Dickinson, New Jersey, USA).
Wound healing assay
Cell migration was determined via a wound-healing assay. Briefly, approximately 3 × 105 cells were seeded in 6-well plates at equal densities and grown to 80% ~ 90% confluency. Artificial gaps were generated by a 1 ml sterile pipette tip after transfection. Wounded areas were marked and photographed with a microscope.
Transwell migratory and invasive assays
For the transwell migration assay, 2000 cells were plated into the upper chambers (24-well insert, pore size 8 μm, Corning) with 100 μL serum-free PRIM-1640. The lower chambers were filled with 600 μL PRIM-1640 containing 10% FBS. 48 h later, cells under the surface of the lower chamber were washed with PBS and stained with 0.5% crystal violet for 10 min. For the invasion assay, 2000 cells were seeded on transwells coated with 100 μL Matrigel (1:4 dilution in PBS, Corning Inc., USA). The culture conditions were the same as described for the transwell migration assay. After 48 h, adherent cells on the lower surface were stained with 0.5% crystal violet. The number of cells on the lower surface was photographed with a microscope.
iTRAQ (isotope tagging for relative and absolute protein quantitation)
Protein extraction was carried out using the RIPA buffer (Applygen, China). The BCA protein assay Kit (Applygen, China) was used to quantitate total protein levels. The protein (20 μg) from each sample was mixed with 5X loading buffer and separated on a 12.5% SDS-PAGE gel and visualized by Coomassie Blue R-250 staining. A filter-aided sample preparation (FASP) was used to remove the detergent, DTT, and other low molecular weight components and digest the proteins. One hundred micrograms of each peptide mixture was labeled using an iTRAQ reagent 8-plex kit (SCIEX, Framingham, MA) according to the manufacturer’s instructions. iTRAQ-labeled peptides were fractionated by Phenomenex Luna Strong Cation Exchange (SCX) chromatography. Each fraction was injected for nano-LC-MS/MS analysis. High-resolution LC-MS/MS analysis was performed on a Q Exactive mass spectrometer (Thermo Fisher Scientific) operated in a positive ion mode that was coupled to an EASY-nLC liquid chromatograph (Thermo Fisher Scientific). The MS data were acquired in a data-dependent acquisition mode. The top 20 precursor ions were selected from each MS full scan in the HCD collision cell. The instrument was run with the peptide recognition mode enabled. The raw files were processed using Proteome Discoverer 1.4 (Thermo Scientific) and searched using the Mascot search engine (version 2.2, Matrix Science) against the UniProt protein human database (134,919 sequences). The results were evaluated for difference significance using ANOVA analysis. It is recommended to select proteins with p value less than 0.05, ratio ≥ 1.2 or ratio ≤ 0.83 as differential proteins.
Western blot analysis
Protein extraction was carried out using the RIPA buffer, and the BCA protein assay Kit was used to quantitate total protein levels. Protein (40 μg per lane) was separated by SDS-PAGE. Proteins were transblotted to PVDF membranes, and membranes were blocked in 5% nonfat milk powder and incubated overnight at 4 °C with anti-FLAG (1:1000; CST, 14793S), anti-TFB1M (1:1000; Sigma-Aldrich, HPA029428), anti-TFB1M (1:1000; Abcam, ab236901), anti-MT-CO2 (1:1000; Abcam, ab79393), Bcl-2 (1:1000, Abcam, ab32124), Cleaved Caspase-3 (1:1000, Affinity, AF7022), E-cadherin (1:1000, CST, 3195 T) and N-cadherin (1:1000, CST, 13116 T). After incubated with horseradishperoxidase-conjugated goat anti-rabbit IgG, membranes were resolved by chemiluminescence. All membranes were stripped and reprobed with anti-GAPDH antibody (1:8000, Proteintech, China) as a loading control.
Immunohistochemistry staining
The paraffin sections of mice pulmonary metastasis samples were used to perform immunohistochemical staining to measure the protein expression levels of E-cadherin and N-cadherin. The specific primary antibody information is as follows: anti-E-cadherin (1:400, CST, 3195 T) and anti-N-cadherin (1:125, CST, 13116 T).
RNA pull-down assay
The ZNF582-AS1-binding proteins were examined using RNA pull-down assays according to the instructions of the Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, 20,164, USA). Biotin-labeled RNAs were transcribed in vitro with the Biotin RNA Labeling Mix and T7 RNA polymerase (RiboBio, China). Biotinylated RNAs were mixed with streptavidin magnetic beads (Thermo Fisher Scientific, 20,164, USA) at 4 °C overnight. Total cell lysates were freshly prepared and added to each binding reaction with Protease/Phosphatase Inhibitor Cocktail and RNase inhibitor, and then the mixture was incubated with rotation for 1 h at 4 °C. After washing thoroughly three times, the RNA–protein binding mixture was boiled in SDS buffer and the eluted proteins were detected by western blot.
RNA immunoprecipitation (RIP) assay
The RIP experiments were performed with a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (17–700, Millipore, USA) according to the manufacturer’s protocol. Cell lysates were prepared and incubated with RIP buffer containing magnetic beads conjugated with human anti-Flag antibody (Sigma Aldrich). Normal mouse IgG (17–700, Millipore) functioned as the negative control. The RNA fraction precipitated by RIP was analyzed by qRT-PCR.
rRNA MeRIP-seq and MeRIP-qRT-PCR
Total RNAs were extracted by TRizol from the stable ZNF582-AS1 overexpressed and control OSRC2 cells. RNA was tested for quality using nanodrop and gel electrophoresis. Chemically fragmented RNA (100 nucleotides) was incubated with m6A antibody for immunoprecipitation according to the standard protocol of Magna methylated RNA immune-precipitation (MeRIP) m6A Kit (17–10,499, Millipore, USA). Enrichment of m6A containing rRNA was analyzed either by high-throughput rRNA sequencing or by qRT-PCR with the primers listed in Additional file
1: Table S1.
Mouse model experiments
Animal experiments were conducted in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals with the approval of the Review Board of Peking University First Hospital, Beijing. Mice were maintained under pathogen-free conditions with regulated temperature and humidity levels. Mice were randomly assigned to cages in groups of 5 and fed ad libitum under controlled light/dark cycles.
Twenty-four 5-week-old male BALB/c nude mice were purchased from Vitalriver, Beijing, China. Approximately 5 × 106 ZNF582-AS1-overexpressed, MT-RNR1-overexpressed-ZNF582-AS1-overexpressed and control OSRC2 cells suspended in 100 μL Hank’s Balanced Salt Solution (Thermo Fisher Scientific, USA) were mixed with Matrigel (1:1, Corning Inc., USA). Then, 200 μL tumor cells were subcutaneously implanted on the right flank of 6-week BALB/c nude mice using a 28-gauge needle (Thermo Fisher Scientific, USA). Tumor size was measured every week and calculated using the formula: (length × width2)/2. For cell proliferation assay, ethynyl-2-deoxyuridine (EdU, 50 mg/kg; Beyotime, China) was intraperitoneally injected 4 h before mice were euthanized.
For the metastasis experiment, twenty 5-week-old male B-NDG mice (NOD- Prkdcscid IL2rgtm1/Bcgen) that lacked mature T cells, B cells, and natural killer (NK) cells, were purchased from BIOCYTOGEN, Beijing, China. Approximately 5 × 105 ZNF582-AS1- -Luc, MT-RNR1-ZNF582-AS1-Luc and or CON-Luc Caki-1 cells were suspended in 150 ul PBS and injected into the lateral tail veins of each unanesthetized B-NDG mouse at five-weeks-old. Thirty days after injection, mice were anesthetized with isoflurane (YIPIN Pharmaceutical CO., LTD, Hebei, China). Ten minutes after D-Luciferin, sodium salt (150 mg/kg) was injected intraperitoneally, and cancer cells were detected with an in vivo imaging system, Xenogen IVIS (PerkinElmer, MA, USA). The total flux in photons per second were calculated and reported for each mouse’s lung and liver region using Living Image 4.3.1 (PerkinElmer/Caliper).
Statistical analyses
Non-parametric Mann-Whitney test was used to detect differences in continuous variables. Survival curves for patients were plotted using the Kaplan-Meier method, with log-rank tests for statistical significance. The correlation between ZNF582-AS1 expression and MT-RNR1 expression, MT-RNR1 expression and MT-CO2 mRNA expression in ccRCC was examined using Pearson’s correlation analysis. All data were analyzed using Graphpad prism 7.0 and R language. A p value of < 0.05 was regarded as statistical difference.
Discussion
LncRNAs with a length of more than 200 nucleotides have been shown to function as oncogenes or tumor suppressor genes in the process of tumorigenesis [
29]. In addition to their functional effects on tumor progression, lncRNAs also display various regulatory effects through different mechanisms, including regulation of mRNA processing and translation, epigenetic transcriptional modulation, remodeling of and interactions with chromatin, genome defense or RNA turnover [
30]. Besides, lncRNAs expression has been quantitatively studied in several tumor cell types and tissues by high-throughput RNA sequencing (RNA-seq) and have commonly been found to be more tumor cell type specific than the expression of protein-coding genes [
31]. Moreover, many studies have also suggested that lncRNAs abnormal expression is responsible for drug resistance, which is a major obstacle for tumor treatment [
18,
32]. Therefore, lncRNAs have been considered not only as potential molecular biomarkers, but also as significantly therapeutic targets for tumor treatment.
Recent advancements in the rapidly evolving field of cancer epigenetics have shown extensive reprogramming of every component of the epigenetic machinery in RCC, including DNA methylation, histone modification and nucleosomal localization [
33]. DNA methylation was one of the first modes of epigenetic regulation to be discovered, and alterations in DNA methylation have also been examined in ccRCC [
34]. Numerous tumor suppressor genes have been reported to be partially or completely silenced due to hypermethylation of their promoters in single-locus studies, and the use of hypomethylating agents has been shown to restore the expression of many of these genes in vitro [
35]. ZNF582-AS1 is a newly identified lncRNA that has been mapped to the human chromosome 19q13.43, and it is differentially expressed in ccRCC and exhibit diagnostic and prognostic values in ccRCC [
21]. Besides, study indicated that ZNF582-AS1 is epigenetically silenced by aberrant DNA methylation in colorectal cancer [
22]. However, most of these results are obtained by analyzing the TCGA data, and no experimental study has explored the specific role and mechanism of ZNF582-AS1 in ccRCC so far.
During our study, we found that ZNF582-AS1 expression was significantly downregulated in ccRCC tissues and cells, and decreased ZNF582-AS1 expression was significantly associated with advanced tumor stage, higher pathological stage, distant metastasis, and poor prognosis. Besides, the RNA FISH results showed that ZNF582-AS1 was distributed mostly in the cytoplasm of ccRCC cells. Bioinformatic data mining found DNA hypermethylation in the promoter ZNF582-AS1 DNA, and the expression of ZNF582-AS1 was negatively correlated with the methylation status of its CpG sites. Meanwhile, higher methylation levels of these CpG sites were significantly associated with ccRCC progression and poor prognosis. In addition, our MSP and Sequenom MassARRAY results confirmed the hypermethylation status of ZNF582-AS1 in ccRCC tissues and cells, and treatment with 5-Aza and TSA induced demethylation of the ZNF582-AS1 promoter and increased ZNF582-AS1 expression. Furthermore, in vitro and in vivo assays both found that ZNF582-AS1 overexpression significantly inhibited cell proliferation and induced cell apoptosis. Cell migratory and invasive abilities were also suppressed by ZNF582-AS1 overexpression. Interestingly, previous studies indicated that ZNF582-AS1 does not affect cell viability or proliferation, the cell cycle or apoptosis in CRC cell lines [
22]. Therefore, ZNF582-AS1 may act in different ways in different tumors, and its role in other tumors needs further study.
To explore the molecular mechanisms of the tumor suppressive function of ZNF582-AS1 in ccRCC, iTRAQ analysis and RNA pull down assays were performed to identify the downstream targets of ZNF582-AS1. Results showed that ZNF582-AS1 was able to bind with A8K0B9 protein (rRNA adenine N(6)-methyltransferase) and caused its degradation, which indicates that ZNF582-AS1 may modulate the N(6)-methyladenosine modification of rRNA in ccRCC. N(6)-methyladenosine is one of the most common RNA modifications in eukaryotes, mainly in mRNA [
36]. Recent studies have discovered a number of lncRNAs modified by N(6)-methyladenosine in multiple cancers, and they can regulate gene expression and function through a series of complex mechanisms [
37‐
42]. In turn, lncRNAs can target or modulate N(6)-methyladenosine regulators to influence the development of cancer [
43]. However, the role of lncRNAs in the N(6)-methyladenosine modification of rRNA remains unknown. In this study, our results showed that the N(6)-methyladenosine modification level of MT-RNR1 was downregulated in ZNF582-AS1-overexpressed OSRC2 cells compare with control OSRC2 cells, and the expression of MT-RNR1 in ZNF582-AS1-overexpressed OSRC2 cells was decreased. Moreover, A8K0B9 protein had a certain binding ability with MT-RNR1 in OSRC2 cells. It was reported that binding of YTHDF2 (the human YTH domain family 2), a N(6)-methyladenosine “reader” protein, results in the localization of bound mRNA from the translatable pool to mRNA decay sites, such as processing bodies, thereby promoting mRNA degradation [
44]. However, in the present study, we found that reduced N(6)-methyladenosine modification level of MT-RNR1 caused a reduction in MT-RNR1 expression. In order to clarify this inconsistency, more studies are needed in the near future to identify the specific effect of N(6)-methyladenosine modification on rRNA expression.
Mitochondria are organelles that perform major roles in cellular operation. Therefore, alterations in mitochondrial genome may lead to mitochondrial dysfunction and cellular deregulation, influencing carcinogenesis [
45]. A recent study indicated that ZCCHC4 is a new human N(6)-methyladenosine methyltransferase, and knockout of ZCCHC4 eliminates the N(6)-methyladenosine modification on 28S rRNA, thereby reducing overall translation activity, which contributes to inhibiting liver cancer cells proliferation and reducing liver tumor size [
46]. In the current study, our results demonstrated that MT-CO2 expression was positively correlated with MT-RNR1 expression, and ZNF582-AS1 overexpression decreased the expression of MT-CO2 protein. Previous studies have shown that elevated mitochondrial protein Lon promotes EMT via reactive oxygen species (ROS)-dependent signaling [
47]. Studies also indicated that MRC proteins including MT-CO2 are induced early before ROS and apoptosis of multiple cell types induced by multiple stimuli [
48,
49]. Consistent with the above findings, our results showed that the expression of Bcl-2 and N-cadherin protein was decreased, while Cleaved Caspase 3 and E-cadherin protein expression and ROS levels were increased in ZNF582-AS1-overexpressed OSRC2 cells. Moreover, our results demonstrated that MT-RNR1 overexpression rescued the decreased expression of MT-CO2, Bcl-2 and N-cadherin and the increased expression of Cleaved Caspase 3 and E-cadherin and ROS levels caused by ZNF582-AS1 overexpression. Furthermore, MT-RNR1 overexpression reversed decreased cell proliferative, migratory and invasive ability and increased cell apoptotic rate caused by ZNF582-AS1 overexpression in vitro and in vivo.
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