Epigenetic regulation of AURKA by miR-4715-3p in upper gastrointestinal cancers

Aurora kinase A (AURKA) is frequently overexpressed in several cancers. miRNA sequencing and bioinformatics analysis indicated significant downregulation of miR-4715-3p. We found that miR-4715-3p has putative binding sites on the 3UTR region of AURKA. Upper gastrointestinal adenocarcinoma (UGC) tissue samples and cell models demonstrated significant overexpression of AURKA with downregulation of miR-4715-3p. Luciferase reporter assays confirmed binding of miR-4715-3p on the 3UTR region of AURKA. miR-4715-3p mediated a reduction in AURKA levels leading to G2/M delay, chromosomal polyploidy, and cell death. We also detected a remarkable decrease in GPX4, an inhibitor of ferroptosis, with an increase in cleaved PARP and caspase-3. Inhibition of AURKA using siRNA produced similar results, suggesting a possible link between AURKA and GPX4. Analysis of UGC samples and cell models demonstrated increased methylation levels of several CpG nucleotides upstream of miR-4715-3p. 5-Aza-2′-deoxycytidine induced demethylation of several CpG nucleotides, restoring miR-4715-3p expression, leading to downregulation of AURKA. In conclusion, our data identified a novel epigenetic mechanism mediating silencing of miR-4715-3p and induction of AURKA in UGCs. Inhibition of AURKA or reconstitution of miR-4715-3p inhibited GPX4 and induced cell death, suggesting a link between AURKA and ferroptosis.


miR-4715-3p is downregulated in upper gastrointestinal cancers (UGCs).
We performed miR-NA-seq using human and mouse tissue samples and identified a conserved miRNA signature in mouse and human gastric cancers 22 . We searched for miRNAs with predicted binding sites on AURKA 3′UTR using three online databases (www.TargetScan.org, www.microRNA.org, and www.miRDB.org). Among the downregulated microRNAs in gastric cancer, we found miR-4715-3p had a predictable target score of 86% for AURKA 3′UTR (Fig. 1A). We investigated the level of AURKA mRNA and miR-4715-3p expression in de-identified normal and tumor tissues. As shown in Fig. 1B, AURKA mRNA was significantly elevated in gastric tumor tissue samples, as miR-4715-3p reconstitution induced downregulation of AURKA by directly targeting its 3′UtR. We next investigated the possible role of miR-4715-3p in regulating AURKA. As shown in Fig. 2A,B, transient reconstitution of miR-4715-3p (50 picomole for 48 h) led to marked downregulation of AURKA protein and mRNA levels in OE33 and MKN45 cells. We constructed and utilized a luciferase reporter assay, using a reporter containing binding sites of miR-4715-3p on AURKA 3 UTR region, to determine if miR-4715-3p acts directly on AURKA. We cloned the AURKA 3′UTR in a luciferase reporter with or without deleting the binding site of miR-4715-3p on AURKA 3′UTR from 508 to 515 (Fig. 2C). DNA fragments were amplified using PCR and cloned into pLenti-Luc to generate a wild-type and mutant AURKA 3′UTR luciferase reporters. Reconstitution of miR-4715-3p with AURKA wild-type 3′UTR-luc led to a significant reduction in luciferase levels (P < 0.001). On the other hand, transfection of miR-4715-3p with mutant 3′UTR luciferase reporter did not cause changes in the luciferase activity (Fig. 2D,E). These results indicated that AURKA 3′UTR region contains functional binding sites for miR-4715-3p. miR-4715-3p reconstitution increasesd polyploidy and decreased spheroid formation. As previously reported, AURKA inhibition induced G2/M delays, polyploidy, and cell death 23,24 . Using the OE33 and MKN45 in vitro models of UGC, we investigated the effects of miR-4715-3p reconstitution on cell cycle. Transient miR-4715-3p reconstitution for 48 h significantly reduced the percentage of cells in G1-phase, increased the percentage of cells in G2/M, and increased polyploidy in cancer cells (Fig. 3A,B, and Supplementary Fig. 2) similar to effects of AURKA inhibition. To investigate if miR-4715-3p reconstitution alters spheroid forming ability of MKN45 and OE33 cells, we reconstituted miR-4715-3p in OE33 and MKN45 UGC cells by using lentivirus miR-4715-3p reconstitution enhanced cisplatin sensitivity. We investigated the effects of miR-4715-3p reconstitution on UGC cells treated with or without cisplatin (CDDP) a standard chemotherapeutic drug in UGC. Transient miR-4715-3p reconstitution for 72 h led to a significant reduction in the survival of OE33 and MKN45 cancer cells (P < 0.001), comparable to the effects of cisplatin treatment (Fig. 4A,B). Interestingly, miR-4715-3p reconstitution significantly enhanced cisplatin effects (P < 0.001) (Fig. 4A,B). Using Annexin V staining to quantify cell death, we found miR-4715-3p reconstitution significantly promoted cisplatin-induced cell death in OE33 and MKN45 cells (Fig. 4C,D).

miR-4715-3p reconstitution increased UGC cell death through inhibition of GPX4.
To determine how miR-4715-3p reconstitution induced cell death, we transiently reconstituted miR-4715-3p in OE33, MKN45 and STKM2 cancer cell lines. miR-4715-3p reconstitution led to marked reduction in AURKA levels in the three cell lines and sensitized cells to cisplatin treatment, showing higher levels of cleaved PARP and cleaved caspase 3 ( Fig. 5A,B, Supplementary Fig. 3). To determine the type of cell death, we investigated the effects of miR-4715-3p on GPX4 levels (inhibitor of ferroptosis) 25,26 . Interestingly, miR-4715-3p reconstitution markedly decreased protein expression of GPX4, suggesting the occurrence of ferroptosis. To determine if miR-4715-3p reconstitution decreased GPX4 through AURKA-dependent mechanism, we knocked down AURKA using siRNA. AURKA knockdown markedly decreased GPX4 expression, similar to the miR-4715-3p reconstitution results (Fig. 5C). These findings identified AURKA as an inhibitor of ferroptosis where its knockdown or downregulation with miR-4715-3p reconstitution promoted ferroptotic cell death via inhibition of GPX4. miR-4715-3p methylation mediated miR-4715-3p downregulation. DNA methylation is an important epigenetic mechanism that controls gene expression. Using human genome browser (https://genome.ucsc. edu/), we analyzed 1,000 bp upstream of miR-4715-3p, as a potential candidate promoter region, and identified multiple CpG nucleotides (Fig. 6A). Therefore, we investigated CpG methylation as a potential regulatory www.nature.com/scientificreports www.nature.com/scientificreports/ mechanism that controls miR-4715-3p expression. Using quantitative pyrosequencing on normal and tumor human tissue samples, we detected increased methylation levels in cancer samples, as compared with normal samples (Fig. 6B,C). We analyzed methylation pattern of the same region in OE33, MKN45 and STKM2 cancer cells treated with or without 5-Aza-2′deoxycytidine (5-aza), a DNA methyl transferase inhibitor. This treatment www.nature.com/scientificreports www.nature.com/scientificreports/ led to a significant reduction in CpG nucleotide methylation levels (P < 0.001) (Fig. 6D,E, Supplementary  Fig. 4A-C) with a significant increase in miR-4715-3p and decrease in AURKA and GPX4 expression levels, as compared with no treatment control (Fig. 6F,G, Supplementary Fig. 4D,E). These results suggested that epigenetic DNA methylation played an important role in regulating miR-4715-3p, AURKA and GPX4 expression levels in UGC cells.
Although genomic amplifications of AURKA gene have been described in cancer 41-45 , these do not fully explain the prevalent frequency of high levels of AURKA protein. In this study, we investigated epigenetic regulation of AURKA in UGC. We previously reported miRNA-seq and identified frequent alterations of miR-NAs in human and mouse gastric cancers 22 . Among the downregulated miRNAs, predicted to bind AURKA 3″ UTR, miR-4715-3p was significantly downregulated in human and mouse gastric cancers. Our molecular analysis suggested that, miR-4715-3p can bind to AURKA 3′UTR to negatively regulate its levels in UGC. This finding provided a plausible explanation for the frequent overexpression of AURKA in UGCs, a finding that may be applicable to several other cancer types. However, this must be confirmed as miRNAs expression and functions are known to be tissue and context specific 46 . Of note, our analysis indicated that AURKA overexpression, as well as miR-4715-3p downregulation, correlated with an overall poor patients' survival rate.
Inhibition of AURKA is known to induce G2/M delays with increase in polyploidy 34,47 . Our analysis demonstrated similar functions following reconstitution of miR-4715-3p. This finding confirmed the functional validity of miR-4715-3p in regulating AURKA levels. We also detected increased sensitivity to cisplatin under the same conditions, a result that was further confirmed in spheroid cell models. Our findings agree with earlier results showing a synergistic effect of alisertib (AURKA inhibitor) and cisplatin in cancer cells 21,48 . www.nature.com/scientificreports www.nature.com/scientificreports/ Protection against ferroptosis, a form of cell death, is recognized as an important molecular mechanism that promotes drug resistance in cancers 49,50 . Glutathione peroxidase-4 (GPX4) is a key regulator of ferroptosis 51,52 . For example, inhibition of cysteine dioxygenase 1 (CDO1) prevents ferroptotic cell death through upregulation of GPX4 53 . Inhibition of AURKA by genetic knockdown or via reconstitution of miR-4715-3p led to a marked reduction of GPX4. These results identified a novel function of AURKA in regulating GPX4 levels, providing a link between overexpression of AURKA and protection against ferroptosis in cancer cells.
Aberrant DNA methylation in human cancers disrupt expression of several tumor suppressor genes and signaling pathways during tumorigenesis and cancer progression 54 . For example, promoter hypermethylation induces downregulation of E-cadherin and chromodomain helicase DNA binding protein 5 (CHD5) promoting gastric tumorigenesis 55,56 . We detected high methylation levels of CpG sites in an upstream predicted promoter of miR-4715-3p in tumor samples and cancer cell lines. The use of 5-Aza-2′deoxycytidine (5 AZA) a demethylation agent, increased expression miR-4715-3p with concomitant decrease in AURKA levels. This finding suggests DNA hypermethylation as an important cause for miR-4715-3p downregulation in UGCs.
In conclusion, this study highlights a novel epigenetic mechanism that mediates AURKA overexpression in cancer. Silencing of miR-4715-3p by DNA methylation promotes high expression levels of AURKA in UGCs. Inhibition of AURKA or reconstitution of miR-4715-3p induced cell death and inhibited GPX4, suggesting a link between high levels of AURKA and protection against ferroptosis.

Material and Methods
tissue samples. All de-identified gastric tissue samples were obtained following written informed consents in accordance with approval from the Institutional Review Board-approved protocols at the University of Miami. The use of archival de-identified human tissues samples was approved as non-human subjects in accordance with the University of Miami's institutional review board. For DNA, mRNA, and miRNA analyses, 72 de-identified archival human gastric tissue samples (49 non-tumor normal stomach and 33 gastric cancer samples) were used. All adenocarcinomas were classified according to the recent guidelines of the International Union against Cancer TNM classification system. www.nature.com/scientificreports www.nature.com/scientificreports/ patients' survival data analysis. We analyzed AURKA and miR-4715-3p relationship to overall survival of 876 and 436 gastric cancer patients, respectively. The data was obtained from an online database (http://kmplot. com/analysis/) 57,58 . Kaplan-Meier survival analysis was performed using SPSS, version 24 (IBM, Armonk, NY).
Western blot. Cells were scraped on ice, centrifuged, and pellets were re-suspended in RIPA lysis buffer (Santa Cruz). Cell lysates were placed on ice. Protein concentration was determined using BCA (ThermoScientific, Waltham, PA). Equal amounts of protein were subjected to SDS/PAGE and transferred onto nitrocellulose membranes using the semi-dry transfer protocol (Bio-Rad Laboratories, Hercules, CA). After transfer, membranes were probed with each respective primary antibody overnight at 4 °C. Following incubation, the membranes were probed with HRP-conjugated secondary antibodies (Bio-Rad). Protein bands were visualized using the commercial Immobile Western Chemiluminescent HRP Substrate Kit (Millipore, Billerica, MA). Images of immunoblots were obtained using the ChemiDoc XRS+ system (Bio-Rad).
celltiter-Glo luminescence assay. Cells were seeded at 2,000 cells/well in a 96-well plate. Following transient transfection with miR-4715-3p with or without cisplatin treatment, cells were seeded at 2,000 cells/well in a 96-well plate. Cells were treated with 5 uM cisplatin treatment in 5% FBS-RPMI 1640. After 3 days, cell viability was calculated using the CellTiter-Glo Luminescence Assay (Promega). GraphPad Prism 9 was used to obtain the curves (GraphPad Software, Inc., San Diego, CA, USA). Three independent experiments were performed to generate data. miR-4715-3p reconstitution and AURKA silencing by small interfering RNA (siRNA). OE33, MKN45 and STKM2 cells were seeded at 60-70% confluency in 10% FBS RPMI 1640 in 6 well plates. Validated AURKA siRNA (Invitrogen) was used to transiently silence AURKA, using LipoJet reagent (SignaGen). Western blot analysis was performed to validate AURKA knockdown. Flow cytometry. For cell cycle analysis, OE33 and MKN45 cells were transiently transfected with miR-4715-3p for 48 h. Cells were harvested and fixed in 70% ethanol. Cells were stained with Propidium Iodide (PI) solution (PI 50 μg/ml and RNase 1 μg/ml dissolved Phosphate Buffered Saline) for 15-30 min. Finally, we analysed the cell cycle using BD LSR III Flow Cytometer (BD Biosciences, San Jose, CA). The data was processed by using the Flowjo software. The FITC Annexin V kit (BD Biosciences) was used to analyze cell death, following miR-4715-3p overexpression. OE33 and MKN45 cells were transfected with 50 picomole miR-4715-3p mimic and/or control (Applied Biological Materials), using the LipoJet transfection reagent (SignaGen Laboratories, Rockville, MD). After 48 h, cells were treated with cisplatin for 24 h. The FITC Annexin V and PI staining were performed according to the manufacturer's instructions. The Flow Cytometry Shared Resources at Sylvester Comprehensive Cancer Center was used for analysis of data.
Luciferase reporter assay. To confirm the binding of miR-4715-3p to the 3′ UTR regions of AURKA, OE33 and MKN45 cells were transfected with miR-4715-3p mimic or control. After 24 h, cells were transfected with 250 ng AURKA 3′-UTR Luciferase/GFP reporter vector, purchased from Abmgood (Applied Biological Materials, Richmond, BC, Canada) and 60 ng of ubiquitin promoter Renilla luciferase plasmid (control), using Polyjet (SignaGen Laboratories). We collected cells for analysis after 48 h. The Firefly luciferase activities were normalized to Renilla luciferase levels. The miR-4715-3p binding site in AURKA 3′ UTR Luciferase/GFP reporter vectors were deleted using the QuikChange Lightning Site-Directed Mutagenesis Kit from Agilent Technologies (Santa Clara, CA). We confirmed the deletion of miR-4715-3p binding site by DNA sequencing (Supplementary Fig. 1). Statistical analysis. GraphPad Prism 8 software (GraphPad Software Inc.) was used for analysis of statistical significance. We applied One-way ANOVA Test to assess the difference between the treatment and control groups. We presented the data as means +/− standard error of mean (SEM). Two-tailed Student's test was used to analyze the correlation between two parameters. A P value ≤ 0.05 was considered significant.