Hypoxia-induced epigenetic regulation of miR-485-3p promotes stemness and chemoresistance in pancreatic ductal adenocarcinoma via SLC7A11-mediated ferroptosis

Hypoxia-induced epigenetic regulation of miR-485-3p promotes stemness and chemoresistance in pancreatic ductal adenocarcinoma via SLC7A11-mediated ferroptosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Hypoxia-induced epigenetic regulation of miR-485-3p promotes stemness and chemoresistance in pancreatic ductal adenocarcinoma via SLC7A11-mediated ferroptosis Yinmo Yang, Xiaodong Tian, xinxin liu, Zhihua Huang, Qiuzheng Chen, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3865266/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 May, 2024 Read the published version in Cell Death Discovery → Version 1 posted 7 You are reading this latest preprint version Abstract The mechanism of hypoxia in chemoresistance of pancreatic ductal adenocarcinoma (PDAC) remains elusive. In this study, we reveled essential role of miR-485-3p in PDAC, particularly its impact on cancer stemness and gemcitabine resistance under hypoxic condition. We found substantial downregulation of miR-485-3p in PDAC tissues, with lower expression correlating to poor patient outcomes. Mechanistically, miR-485-3p influenced stemness characteristics, as evidenced by reduced tumor sphere formation and increased sensitivity to gemcitabine upon overexpression. Moreover, we identified SOX9 and SLC7A11 as two targets of miR-485-3p, which play vital role in stemness and ferroptosis. Under hypoxic condition, DNMT3B expression was upregulated, leading to hypermethylation of miR-485-3p promoter region. the reduced miR-485-3p expression promoted stemness and chemoresistance of PDAC. In conclusion, our findings elucidate the intricate interplay of hypoxia, epigenetic modifications, and ferroptosis in PDAC and shed light on potential avenues for targeted interventions that modulate cancer stemness and chemosensitivity, offering prospects for improved therapeutic strategies for PDAC. Biological sciences/Cancer/Gastrointestinal cancer/Pancreatic cancer Biological sciences/Genetics/Gene regulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive gastrointestinal malignancy with a dismal prognosis 1 . PDAC is currently the 4th leading cause of cancer-related death, and a 5-year survival rate is only approximately 11% 2 . Gemcitabine is still the cornerstone of chemotherapeutic agent for PDAC 3 . However, the response to chemotherapy agents is hampered by drug resistance of PDAC. Hypoxia is a common occurrence in PDAC due to poor vascularization, uncontrolled growth, and fibrotic stroma 3 . Accumulating evidence has revealed that hypoxia is associated with stemness maintenance, gemcitabine resistance, and pathological angiogenesis 4 , 5 . Our recent study demonstrated that hypoxic conditions in PDAC promote tumor angiogenesis by suppressing GJA1 expression 6 . However, molecular mechanisms modulating PDAC cells chemoresistance in hypoxic conditions remain unclear. Recent studies have underscored important role of microRNAs (miRNAs) in regulating proliferation, migration, invasion, stemness, and chemotherapy resistance in various tumors, including pancreatic cancer 7 – 10 . MiRNAs are a class of endogenous small noncoding RNAs typically composed of 19–24 nucleotides and exert regulatory effects by inhibiting mRNA translation or inducing mRNA degradation 11 . Moreover, hypoxic environments can affect the expression of miRNAs 12 , 13 . MiR-485-3p, derived from the MIR485 gene located on chromosome 14q32.31, has been implicated in breast cancer, osteosarcoma, prostate cancer, gastric cancer, hepatocellular carcinoma, and other tumors due to its aberrant expression 14 – 16 . In our previous study, aberrant expression of miR-485-3p inhibited the migration and invasion of PDAC cells 17 , but the mechanism underlying its aberrant expression remains unclear. In this study, we found that SOX9 and SLC7A11 are the downstream targets of miR-485-3p and DNMT3B downregulates miR-485-3p expression. DNMT3B/miR-485-3p/SLC7A11 axis promotes stemness maintenance and induces gemcitabine resistance in PDAC cells under hypoxic condition. Results 1. miR-485-3p is inhibited in PDAC cell lines under hypoxia conditions and associated with good clinical prognosis. To evaluate whether hypoxia related signaling pathways are involved in gemcitabine resistance in pancreatic cancer cells, we used GEO database to screen the differential expression genes (DEGs) of gemcitabine-sensitive and -resistant pancreatic cancer cells, and the work flow was presented in Fig. 1 A. The results showed that these DEGs had a correlation with HIF signaling pathway in most datasets, except for the GSE197352 dataset (Fig. 1 B, Supplementary Table 1). Next, we employed CoCl2 induced- or physical- method to imitate hypoxic condition in vitro (Fig. 1 C). While HIF-1α was upregulated under hypoxia (Supplementary Fig. 1A, B), we found a significant downregulation of miR-485-3p expression (Fig. 1 D-F). We evaluated the impact of miR-485-3p on the chemosensitivity of PDAC cells. The results showed that miR-485-3p overexpression under normoxia or hypoxia (Fig. 1 G, H), increased the sensitivity of PDAC cells to gemcitabine and partially reversed hypoxia-induced resistance of PDAC to gemcitabine (Fig. 1 I, J). Moreover, we established drug-resistant cell lines and observed lower miR-485-3p expression in these cells (Fig. S2A, C). Overexpression of miR-485-3p in drug-resistant cell lines reversed the resistance of PDAC cells to gemcitabine (Fig. S2B, D). Furthermore, lower miR-485-3p expression was closely correlated with reduced overall survival in PDAC (Fig. 1 K). Collectively, these data indicated that miR-485-3p exhibited aberrant expression patterns in PDAC tissues and cells, and was downregulated under hypoxic condition. 2. Hypoxia upregulates SOX9 and SLC7A11 expression by downregulating the expression of miR-485-3p To elucidate the molecular mechanism underlying the effects of miR-485-3p on gemcitabine resistance in PDAC cells under hypoxic condition, we utilized the “Starbase” to predict potential targets of miR-485-3p (Fig. 2 A, Supplementary Fig. 2A). Comparing the PDAC GEO datasets of DEGs between wild-type and drug-resistant cells, we found that SOX9 and SLC7A11were potential targets of miR-485-3p (Fig. 2 B). Moreover, we analyzed the TCGA-PDAC dataset to confirm that both SOX9 and SLC7A11 expression were negatively correlated with miR-485-3p expression (Supplementary Fig. 3B, C). Luciferase assays further revealed that miR-485-3p overexpression inhibited the luciferase activity of the wild-type containing SOX9 (Fig. 2 C, D). Moreover, there were three potential sites of miR-485-3p binding within the 3'-UTR region of SLC7A11 (Fig. 2 E) and the luciferase activity of the binding site 3 was the lowest (Fig. 2 F). MiR-485-3p overexpression inhibited the expression of SOX9 and SLC7A11, while silencing miR-485-3p had the opposite effects (Fig. 2 G, Supplementary Fig. 3D, E). Additionally, miR-485-3p could reverse hypoxia-induced SOX9 and SLC7A11 expression (Fig. 2 H). Taken together, these results suggested that miR-485-3p could target SOX9 and SLC7A11 under hypoxic condition. 3. miR-485-3p suppresses stemness of PDAC cells under hypoxic condition Accumulating evidence has revealed that SOX9 is a master regulator of pancreatic progenitor cells and plays an important role in pancreatic endocrine and ductal cell differentiation during pancreatic development 18 , 19 . SOX9 is also recognized as a marker of PDAC cancer stem cells (CSCs), contributing to their stemness properties 20 . Therefore, we investigated the role of miR-485-3p in PDAC CSC stemness. Hypoxia enhanced the tumor sphere formation ability in MIA PaCa-2 and PANC-1, which could be reversed by miR-485-3p overexpression (Fig. 3 A). PDAC cells exposed to hypoxia had higher expression of CD24 and CD44 than those exposed to normoxia, while miR-485-3p overexpression inhibited the proportion of CD24 + CD44 + PDAC stem cells (Fig. 3 B) and CD133 + cells (Fig. 3 C). In addition, overexpression of miR-485-3p downregulated the expression of stemness marker SOX2 in MIA PaCa-2 (Supplementary Fig. 4A) and PANC-1 (Supplementary Fig. 4B). In vivo, miR-485-3p overexpression in PANC-1 decreased the tumor initiating cell frequency to nearly 1/16 (from 1/25140 to 1/ 417215), which indicated that the frequency of CSCs in the miR-485-3p overexpression group was significantly lower than that in the control group (Fig. 3 D). These data suggested that hypoxia increases PDAC cell stemness by suppressing miR-485-3p. 4. miR-485-3p promoted ferroptosis SLC7A11 is involved in ferroptosis, a form of iron-dependent programmed cell death characterized by intracellular lipid peroxidation induced by excess oxygen free radicals generated via the Fenton reaction during iron metabolism. One essential component of the classic antioxidant systems is the glutathione antioxidant system mediated by the SLC7A11-GPX4 axis (Fig. 4 A). In PDAC tissues from TCGA, SLC7A11 expression was correlated with ferroptosis pathway (Fig. 4 B). Therefore, we hypothesized that miR-485-3p may regulate ferroptosis pathway. Changes in the sensitivity to ferroptosis inducers, ROS, MDA, and intracellular GSH levels, as well as the restoration of these indicators by ferroptosis inhibitors, serve as markers for ferroptosis regulation (Fig. 4 C). MiR-485-3p overexpression increased the sensitivity of MIA PaCa-2 and PANC-1 cells to ferroptosis inducers Erastin and RSL3, and this effect was attenuated by ferroptosis inhibitors (Fig. 4 D, E, Table S3). Additionally, ferroptosis inducers significantly elevated intracellular ROS (Fig. 4 F, G) and MDA (Fig. 4 H, I) levels in PDAC cells overexpressing miR-485-3p, while ferroptosis inhibitor Fer-1 effectively reduced ROS and MDA levels. Furthermore, intracellular glutathione content significantly decreased upon miR-485-4p overexpression, but could be restored by Fer-1 (Fig. 4 J, K). These findings suggested that miR-485-3p plays a role in regulating ferroptosis. 5. miR-485-3p regulates stemness and chemosensitivity of PDAC cells through SLC7A11-mediated ferroptosis The expression of stemness markers SOX2 and ALDH1A significantly increased in response to miR-485-3p silencing in MIA PaCa-2 and PANC-1 cells, while SLC7A11 knockdown significantly attenuated their expression. Treatment with ferroptosis inhibitors significantly upregulated the expression of stemness markers (Fig. 5 A). Knockdown of SLC7A11 inhibited drug resistance induced by miR-485-3p silencing, and these effects were restored by Fer-1 (Fig. 5 B, Supplementary Fig. 5A, B). Notably, silencing miR-485-3p in MIA PaCa-2 and PANC-1 cells led to an increase in tumor cell sphere formation, while knockdown of SLC7A11 reversed this effect (Fig. 5 C). Furthermore, treatment with a ferroptosis inhibitor partially restored the stemness sphere formation inhibited by miR-485-3p knockdown (Fig. 5 C). Flow cytometry showed that silencing miR-485-3p increased the proportion of CD24 + CD44 + cells, which was mitigated by SLC7A11 knockdown (Fig. 5 D). A similar trend was observed for CD133 + cells (Fig. 5 E). Furthermore, miR-485-3p inhibition decreased MDA levels (Supplementary Fig. 6A, B) and increased GSH concentration (Supplementary Fig. 6C, D), and these effects were reversed by SLC7A11 knockdown and subsequently restored by Fer-1. These results suggested that miR-485-3p regulates PDAC stemness partially through SLC7A11-mediated ferroptosis pathway. 6. The methylation of miR-485-3p promoter region was mediated by DNMT3B under hypoxia To explore molecular mechanism by which hypoxia downregulates miR-485-3p expression in PDAC cells, we considered the role of DNA methylation in regulating transcription of non-coding RNA. In PDAC cells treated with the DNA methyltransferase inhibitor 5-azacytidine (5-AZA), miR-485-3p expression restored in hypoxic condition (Fig. 6 A). Subsequently, we overexpressed DNMTs in MIA PaCa-2 and PANC-1 cells and found that DNMT3B significantly inhibited miR-485-3p expression (Fig. 6 B). PDAC cells with DNMT3B knockdown exhibited lower expression of SLC7A11 compared to scramble group under hypoxia (Fig. 6 C). Hypoxia-induced upregulation of SLC7A11 expression was antagonized by DNMT3B knockdown, suggesting that hypoxia elevates SLC7A11 expression by promoting DNMT3B expression (Fig. 6 C). Furthermore, miR-485-3p inhibitor could upregulate SLC7A11 expression, indicating that DNMT3B regulates SLC7A11 expression through miR-485-3p (Fig. 6 D). We used TransmiR ( www.cuilab.cn/transmir ), mirTrans (mcube.nju.edu.cn/jwang/lab/soft/mirtrans/) and geneXplain (platform.genexplain.com/) to screen regions of miR-485-3p promoter and identified three possible sites responsible for miR-485-3p transcription (Fig. 6 E). Promoter3 showed strong transcriptional activity, suggesting that Promoter3 may contain the promoter region for miR-485-3p (Fig. 6 F). Methprimer tool predicted CpG island in this region (Fig. 6 G). We found higher level of methylation in the miR-485-3p promoter region in cells exposed to hypoxia compared to cells exposed to normoxia (Fig. 6 H). These data indicated that miR-485-3p promoter is methylated by DNMT3B under hypoxic condition, resulting in miR-485-3p downregulation and subsequent upregulation of SLC7A11 expression. 7. DNMT3B regulates stemness and chemosensitivity through SLC7A11-mediated ferroptosis in PDAC cells To confirm whether DNMT3B play an oncogenic role in PDAC cells by upregulating SLC7A11 expression and inhibiting ferroptosis process, we assessed the effect of DNMT3B overexpression and SLC7A11 knockdown on PDAC cell sensitivity to gemcitabine. Overexpression of DNMT3B reduced the sensitivity of PDAC cells to gemcitabine, while SLC7A11 knockdown significantly reversed this effect (Fig. 7 A). Similar changes were observed for stemness marker expression (Fig. 7 B). PDAC cells with both DNMT3B overexpression and SLC7A11 silencing had significantly less tumor sphere formation compared to cells with only DNMT3B overexpression (Fig. 7 C). Meanwhile, overexpression of DNMT3B under hypoxic condition increased the proportion of CD24 + CD44 + (Fig. 7 D) and CD133 + cells (Fig. 7 E). However, SLC7A11 knockdown reduced the proportion of CD24 + CD44 + and CD133 + cells. These data indicate that SLC7A11 knockdown inhibits stemness maintenance induced by DNMT3B. We further examined biochemical indicators of ferroptosis, such as ROS, MDA, and intracellular GSH content (Supplementary Fig. 7A). PDAC cells exposed to gemcitabine, DNMT3B overexpression significantly inhibited ACSL4 expression, reduced ROS and MDA levels and increase GSH levels (Supplementary Fig. 7B-F). Silencing SLC7A11 could reverse these effects, which was then partially restored by Fer-1 (Supplementary Fig. 7B-F). These results demonstrate that ferroptosis mediated by DNMT3B-SLC7A11 axis regulates the levels of ROS and lipid peroxidation in PDAC cells exposed to gemcitabine. 8. miR-485-3p sensitizes PDAC cells to gemcitabine mediated by ferroptosis in vivo and DNMT3B is correlated with SLC7A11 expression in PDAC patients To confirm that miR-485-3p sensitizes PDAC cells to gemcitabine in vivo, we found that tumors with miR-485-3p overexpression group grew much slower than those with pLV, and miR-485-3p overexpression enhanced the responsiveness of PDAC to gemcitabine (Fig. 8 A). Moreover, miR-485-3p overexpression inhibited SOX9, SLC7A11 and SOX2 expression, while upregulated ACSL4 expression (Fig. 8 B). The results indicate that miR-485-3p decreases PDAC cell stemness and increases sensitivity to gemcitabine mediated by ferroptosis in vivo. We further assessed DNMT3B expression by IHC in 31 human PDAC tissues. The expression of DNMT3B was positively correlated with SLC7A11 expression (Fig. 8 C). Our results demonstrated that the expression level of SLC7A11 was significantly higher in DNMT3B high-expression group compared to DNMT3B low-expression group (Fig. 8 D). Taken together, the data suggest the important pathological role of DNMT3B-SLC7A11 axis in pancreatic cancer. Discussion In this study, we reported that miR-485-3p was downregulated in PDAC cells exposed to hypoxia and further explored the role of miR-485-3p in PDAC chemoresistance. Overexpression of miR-485-3p markedly decreased the tumor-sphere formation ability and gemcitabine resistance in PDAC cells. In addition, miR-485-3p inhibited PDAC in vivo model. Mechanistically, miR-485-3p downregulated SOX9 and SLC7A11 expression by directly binding to mRNAs and promoting degradation. Furthermore, miR-485-3p regulated ferroptosis pathway via SLC7A11. We also demonstrated that miR-485-3p promoter is hypermethylated by DNMT3B, contributing to miR-485-3p aberrant expression under hypoxia. Moreover, DNMT3B-SLC7A11 decreased the tumor-sphere formation ability and gemcitabine resistance in PDAC cells with regulation of ferroptosis (Fig. 8 E). Hypoxia is a common characteristics of PDAC microenvironment, with a median tissue partial oxygen pressure (pO2) of 0–5.3 mmHg (0–0.7%) compared to the adjacent normal pancreas pO2 at 24.3–92.7 mmHg (3.2–12.3%) 21 . Hypoxic areas within tumors provide well-established niches for cancer cells to acquire and maintain stem-like properties, referred to as CSCs phenotype 22 . Pancreatic CSCs harbor high heterogeneity in terms of surface and intracellular markers, such as CD24, CD44, CD133, SOX2 and ALDH1A, as well as their response to chemotherapy and hypoxia 22 , 23 . Besides, Chen et al. demonstrated that HIF-1α induced by P4HA1 contributed to PDAC stemness and chemoresistance 24 . However, few studies have determined the mechanisms by which miRNAs regulate the stemness and chemoresistance of PDAC. Our discovery that miR-485-3p targets SOX9 and SLC7A11 to inhibit stemness-like properties and gemcitabine resistance provides new insights into the regulation of hypoxia, CSCs and chemoresistance in pancreatic cancer. SLC7A11 is a key component of the cystine/glutamate transporter and plays important role in cell metabolism and ferroptosis regulation 25 . Ferroptosis is a form of regulated cell death characterized by iron-dependent lipid peroxidation. SLC7A11 negatively regulates ferroptosis by facilitating cystine uptake, which, in turn, leads to glutathione synthesis and scavenging of reactive oxygen species 25 . Ferroptosis has been implicated in stemness maintenance and chemotherapy resistance in various cancers, and is an attractive target for therapeutic intervention 26 , 27 . Targeting SLC7A11 can inhibit the proliferation ability of various tumor cells such as colorectal cancer, breast cancer and pancreatic cancer. MiR-375 inhibited the stemness characteristics of gastric cancer cells by targeting SLC7A11-mediated ferroptosis 9 . SLC7A11-mediated ferroptosis in glioma cells was involved in regulating the stemness maintenance of glioma cells 28 . In addition, ferroptosis significantly inhibited the growth of PDAC in SLC7A11-deficient genetically engineered mice 29 . However, the role of SLC7A11 in PDAC CSCs remains unclear. In this study, we found that miR-485-3p directly targets and significantly inhibits the expression of SLC7A11. Furthermore, silencing miR-485-3p promoted stemness marker expression and sphere formation. Importantly, these effects could be attenuated by ferroptosis inhibitor Fer-1. These results suggest that miR-485-3p may modulate stemness maintenance and chemotherapy resistance through SLC7A11-mediated ferroptosis pathway. Notably, significant downregulation of miR-485-3p in PDAC tissues, was correlated with poor patient prognosis, consistent with previous studies identifying miR-485-3p as a tumor suppressor in breast cancer, colorectal cancer and prostate cancers 15 , 16 , 30 . However, the mechanism of miR-485-3p downregulation in PDAC cells, especially under hypoxia, is still unclear. DNA methylation, one of epigenetic modifications, plays a central role in regulating gene expression 31 , 32 . We found that hypoxia in PDAC cells led to the upregulation of DNMT3B, which subsequently promoted the hypermethylation of the miR-485-3p promoter region. Epigenetic modification resulted in reduced miR-485-3p expression and, in turn, upregulated the expression of SOX9 and SLC7A11, contributing to stemness maintenance and gemcitabine resistance. This study has several limitaions. First, while our findings strongly suggest that the identified region is the promoter region of miR-485-3p, we did not confirm it. Chromatin immunoprecipitation or reporter gene assays are needed to provide conclusive evidence. Second, while we found a positive correlation between HIF-1α and DNMT3B expression, the detailed mechanisms require deeper exploration. Dissection of the pathways that modulate DNMT3B expression in hypoxic environments could shed light on potential therapeutic targets. Conclusion In summary, miR-485-3p is downregulated in PDAC tissues and cells, and its aberrant expression predicts a poor prognosis in PDAC patients. MiR-485-3p acts as a tumor suppressor to inhibit stemness and gemcitabine resistance in PDAC cells. Mechanistically, the region of miR-485-3p promoter is hypermethylated by DNMT3B induced by hypoxia, resulting in the upregulation of SOX9 and SLC7A11 to hinder ferroptosis process. Materials and methods 1. Tissue Specimens A total of 31 PDAC tissue samples, along with their matched adjacent non-PDAC tissues, were retrospectively collected from patients who underwent R0 resection at Peking University First Hospital from January 2013 to December 2017 and with comprehensive follow-up data. This study was conducted in accordance with the Declaration of Helsinki and approved by the local ethics committee of Peking University First Hospital (No. 201933) 17 . 2. Cell culture, Plasmid construction and Transfection Human PDAC cell lines, including Mia PaCa-2, PANC-1, and 293T, were cultured in the general surgery laboratory of Peking University First Hospital and tested for mycoplasma contamination. Gemcitabine-resistant (GR) cell lines Mia PaCa-2 and PANC-1 were maintained in complete medium containing 200 nM gemcitabine (Gemzar, Eli Lilly). Lentivirus plasmids pLV-485-3p and pLV-Inh-485-3p were purchased from GenePharma (Shanghai, China). miR-485-3p mimics (miR10002176) and inhibitors (miR20002176) were obtained from Ribobio (Guangzhou, China). Lentiviral vectors for SLC7A11 shRNA or DNMT3B shRNA were constructed based on the Plko.1 plasmid. Human DNMT1, DNMT3A, and DNMT3B coding sequences with flag-tags, amplified from a cDNA library, were cloned into pITA vector. Sequences for cloning are detailed in Supplementary Table S1 . Transfection was performed following the Vigofect (Vigorous Biotechnology, Beijing, China) instructions. 3. Q-PCR and Western blot analysis Total RNA from cells was extracted using the TRIzol kit (Invitrogen, #15596018, USA). Reverse transcription into cDNA was accomplished using a SYBR Kit (Vazyme Biotech, #P611, Nanjing, China). The 7500 equipment was employed to measure mRNA expression levels of target genes normalized to β-actin (ACTB). qPCR primer sequences are listed in Supplementary Table S2. Cells were lysed on ice for 30 minutes using cell lysis buffer (Beyotime, China) supplemented with proteinase inhibitor Cocktail (Roche, USA). The lysateds were centrifuged at 4°C (12,000 ×g, 10 minutes) and proteins were separated by SDS-PAGE (Biotides, China) and transferred to PVDF membranes (Bio-Rad, USA). The membranes were blocked with 5% skim milk powder in TBST and primary antibodies anti-HIF-1α (Abcam, #ab279654, USA), anti-SOX9 (Abclonal, #A19710, China), anti-SLC7A11 (Abclonal, #A2413, China), anti-ALDH1A (Abclonal, #A0517, China), anti-SOX2 (Abcam, #Ab92494, USA), anti-β-actin (MBL, #PM053, Japan), anti-ACSL4(Abclonal, #A20414, China), anti-flag HRP (Abmart, #PA9020), and anti-DNMT3B (Abclonal, #A7239, China) at 4 ℃ overnight. The membranes were washed with TBST for 5 minutes (3 times) and incubated with HRP-labeled goat anti-rabbit IgG(H + L) (Earthox, #E030120, USA) or -mouse IgG(H + L) (Earthox, #E030110) for 40 minutes. ECL substrate (Millipore, #WBULS0500, USA) was applied to develop the membranes. 4. In Vivo Tumorigenicity Assay BALB/c nude mice were randomized into different groups. Cancer cells at different dilutions were subcutaneously injected into the flanks of the mice (n = 10) using random number method with no blinding. Stem cell frequency was calculated using the website ( http://bioinf.wehi.edu.au/software/elda/ ). For in vivo tumor estimation, total 1 × 10 7 PANC-1 cells were inoculated subcutaneously into the right flanks of nude mice (n = 6) using random number method with no blinding. The mice were treated with 50 mg/kg gemcitabine or equal volumes of PBS. The tumor size was calculated and the mice were sacrificed at the indicated time. The resected tumor was kept in liquid nitrogen. 5. Tumor Sphere Formation Assay Cells were cultured in ultralow adhesion plates at 1 000 cells/ per well in serum-free medium with B27 (1:50), EGF (20ng/ml), bFGF (100ng/ml) and LIF (10ng/ml). After culture for 2 weeks, tumor spheres with a diameter of > 100 µm were counted. 6. CCK-8 Assay Cells were seeded into 96-well plates (3000 cells/well) with 100 µl medium containing 10% FBS and treated with gemcitabine for 72 hours or RSL3 or Erastin for 48 hours. Next, cells were treated with 100 µl of DMEM and CCK-8 (Dojindo, Japan) mixture (90 µl:10 µl) at 37°C for 1 hour, and absorbance was measured at 450 nm using a 96-well plate reader. 7. Flow cytometry Cells were stained with anti-hCD24 (Biolegend, #311118, USA), anti-hCD44(Biolegend, #338816, USA), anti-hCD133(Biolegend, #393906, USA), or appropriate control antibodies, with isotype controls used as negative controls or ROS probes DCFH-DA (Beyotime, #S0033S, China). Data analysis was performed using FlowJo V.10.0. 8. Luciferase assay Following transfection of luciferase reporter plasmids and mutant sequences containing the 3'-UTR of SLC7A11 and SOX9 with miR-485-3p binding sites into cells for 24 hours, Dual-Luciferase® Reporter Assay System (Promega, E1910) was employed to measure firefly luciferase activity and renilla luciferase activity. The ratio of the two was calculated and compared with the control group to determine relative luciferase activity. 9. Malondialdehyde (MDA) Assay The level of MDA was evaluated using the MDA detection kit (Beyotime, #S0131S, China) following the instructions. 10. GSH Assay The reduced glutathione content was evaluated by using the reduced glutathione detection kit (Solarbio, BC1170, China) following the instructions. 11. Bisulfite Sequencing PCR (BSP) Genomic DNA was extracted from cells using the DNA extraction Kit (TIANGEN, #DP304-02, China). DNA was denatured by incubation with NaOH for 10 minutes at 37°C, followed by bisulfite modification, PCR amplification, cloning to the pGM-T vector (TIANGEN, #VT402 and #VT202-01, China) and then sequencing by Beijing Tsingke Biotech Company. 12. Immunohistochemistry IHC scoring was conducted by two independent pathologists who were blinded to patients' clinicopathological features and prognosis. 13. Bioinformation analysis The differential expression genes (DEGs) of gemcitabine-sensitive and -resistant pancreatic cancer cells in GEO datasets were analyzed by GEO2R tool ( https://www.ncbi.nlm.nih.gov/geo/geo2r ). KEGG enrichment of DEGs was analyzed by DAVID online tool ( https://david.ncifcrf.gov/tools.jsp ) and visualized by bioinformatics tool ( https://www.bioinformatics.com.cn ). The Starbase database ( https://starbase.sysu.edu.cn/ ) was used to predict the targets of miR-485-3p. 14. Statistical analysis Data were analyzed using SPSS 27.0 software. For continuous variables with homogeneous variances, t-test was performed, and results were presented as the mean ± SD. χ 2 test was used for categorical variables. Un-paired Student’s t-test was used for in vivo experiments. Survival analysis was conducted using Kaplan-Meier survival curves and Log-rank tests. Difference was considered statistically significant at P < 0.05. Declarations Conflict of interest: The authors declare no competing interests. Author Contributions: The conception and design of the study: Yinmo Yang, Xiaodong Tian. The acquisition of data:Xinxin Liu, Zhihua Huang, Qiuzheng Chen. Analysis and interpretation of data: Xinxin Liu, Zhihua Huang, Qiuzheng Chen, Kai Chen, Guangnian Liu, Xiangyu Chu, Dongqi Li, Long Ma. Drafting the article or revising it: Xinxin Liu, Zhihua Huang, Yinmo Yang and Xiaodong Tian. All authors read and approved the final paper. Funding: This study was supported by National Natural Science Foundation of China (NO. 82171722, 82271764, and 81871954), Beijing Municipal Natural Science Foundation (7212111), Peking University Medicine Sailing Program for Young Scholars’Scientific & Technological Innovation (BMU2022MX020), the National Key Research and Development Program of China (2021YFA0909900) and National High Level Hospital Clinical Research Funding (Interdepartmental Research Project of Peking University First Hospital 2023IR23) Data Availability Statement: The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author. Ethics approval and consent to participate: All procedures performed in studies involving human participants were in accordance with Declaration of Helsinki and the research protocols were approved by the Research Ethics Committees of Peking University First Hospital. 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Cancer Res 81, 5217–5229 (2021). https://doi.org:10.1158/0008-5472.CAN-21-0567 Zhao, X., Zhou, M., Yang, Y. & Luo, M. The ubiquitin hydrolase OTUB1 promotes glioma cell stemness via suppressing ferroptosis through stabilizing SLC7A11 protein. Bioengineered 12, 12636–12645 (2021). https://doi.org:10.1080/21655979.2021.2011633 Badgley, M. A. et al. Cysteine depletion induces pancreatic tumor ferroptosis in mice. Science 368, 85–89 (2020). https://doi.org:10.1126/science.aaw9872 Zhao, K. et al. Exosome-Mediated Transfer of circ_0000338 Enhances 5-Fluorouracil Resistance in Colorectal Cancer through Regulating MicroRNA 217 (miR-217) and miR-485-3p. Mol Cell Biol 41 (2021). https://doi.org:10.1128/MCB.00517-20 D'Anna, F. et al. DNA methylation repels binding of hypoxia-inducible transcription factors to maintain tumor immunotolerance. Genome Biol 21, 182 (2020). https://doi.org:10.1186/s13059-020-02087-z Ma, L. et al. The Mechanism of DNA Methylation and miRNA in Breast Cancer. Int J Mol Sci 24 (2023). https://doi.org:10.3390/ijms24119360 Additional Declarations (Not answered) Supplementary Files sumpplementaryInformation.pdf Cite Share Download PDF Status: Published Journal Publication published 29 May, 2024 Read the published version in Cell Death Discovery → Version 1 posted Editorial decision: revise 04 Mar, 2024 Review # 1 received at journal 25 Feb, 2024 Reviewer # 1 agreed at journal 07 Feb, 2024 Reviewers invited by journal 29 Jan, 2024 Submission checks completed at journal 15 Jan, 2024 First submitted to journal 14 Jan, 2024 Editor assigned by journal 14 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3865266","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":269893188,"identity":"6ff9db15-dd9d-4775-9af0-496282ccb753","order_by":0,"name":"Yinmo Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYBACgwNgEoj5Dz/+8LGBDSwqQZwWiTSDgzOJ0SLZAGNJ8Agc5oXy8GrhZz97+DVPwZ1ofgkehsO2O/iiDQ4wH7zNw2CXh0sLG09emjWPwbPcmf1nGA7nnmHL3XCALdmahyG5GKcWhhwzYx6Dw0CVOUAtbSAtPGbSPAwHEhtwaeF/g6TFEqyF/xt+LRI5xo+hWgQOM0JsYSOg5Y0Z4xyglpkzgIHcC/TLzMNsxpZzDJLxOCzH+MObP4dz+0FR+XPHsdy+480Pb7ypsMOpBaRLigfBOcbAwAyiDXCrBwLmjz8QnBq8SkfBKBgFo2BkAgB8L13F7kCHJQAAAABJRU5ErkJggg==","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yinmo","middleName":"","lastName":"Yang","suffix":""},{"id":269893189,"identity":"252c4c2c-f029-4d94-95ba-85154e811119","order_by":1,"name":"Xiaodong Tian","email":"","orcid":"","institution":"Peking Universtiy First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaodong","middleName":"","lastName":"Tian","suffix":""},{"id":269893190,"identity":"5c531b8e-360d-4990-bef3-ace5ff49daf2","order_by":2,"name":"xinxin liu","email":"","orcid":"","institution":"Beijing University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"xinxin","middleName":"","lastName":"liu","suffix":""},{"id":269893191,"identity":"3b8d4265-2aab-44a4-8007-f368d996bd2f","order_by":3,"name":"Zhihua Huang","email":"","orcid":"","institution":"School of Basic Medicine Science, Peking University","correspondingAuthor":false,"prefix":"","firstName":"Zhihua","middleName":"","lastName":"Huang","suffix":""},{"id":269893192,"identity":"cf6ce600-9a56-4bff-a2ea-13587cce394f","order_by":4,"name":"Qiuzheng Chen","email":"","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qiuzheng","middleName":"","lastName":"Chen","suffix":""},{"id":269893193,"identity":"07572240-29dd-4d4f-a24f-18b871cc4d7f","order_by":5,"name":"Kai Chen","email":"","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Chen","suffix":""},{"id":269893194,"identity":"d9caccb4-213e-49a8-908e-67ef98163081","order_by":6,"name":"Weikang Liu","email":"","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Weikang","middleName":"","lastName":"Liu","suffix":""},{"id":269893195,"identity":"58495735-1aea-4872-a341-3160c05d00b1","order_by":7,"name":"Guangnian Liu","email":"","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Guangnian","middleName":"","lastName":"Liu","suffix":""},{"id":269893196,"identity":"8a20d1d6-5e5c-4ad7-aae1-7cddeee02d8f","order_by":8,"name":"Xiangyu Chu","email":"","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiangyu","middleName":"","lastName":"Chu","suffix":""},{"id":269893197,"identity":"0a5912cb-77ea-4ebb-9a3a-d1f012f547b8","order_by":9,"name":"Dongqi Li","email":"","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Dongqi","middleName":"","lastName":"Li","suffix":""},{"id":269893198,"identity":"2e853219-c1a9-42a3-8dea-d73c1c6f9ea1","order_by":10,"name":"Yongsu Ma","email":"","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yongsu","middleName":"","lastName":"Ma","suffix":""}],"badges":[],"createdAt":"2024-01-15 03:10:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3865266/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3865266/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41420-024-02035-x","type":"published","date":"2024-05-29T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50442943,"identity":"b98a2e85-6e17-4bf5-add2-fa95deb45926","added_by":"auto","created_at":"2024-01-31 15:24:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":475595,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-485-3p is associated with good clinical prognosis and downregulated in PDAC cells under hypoxia. A.\u003c/strong\u003e Schematic of the screening strategy to identify the differential expression genes (DEGs) of gemcitabine -sensitive and -resistant pancreatic cancer cells in GEO datasets.\u003cstrong\u003e B.\u003c/strong\u003e Bubble Chart was shown to visualize KEGG enrichment of DEGs.\u003cstrong\u003e C.\u003c/strong\u003e Hypoxic chamber or 200 uM CoCl2 method was used to induce hypoxic condition for PDAC cells in vitro\u003cstrong\u003e. D-H. \u003c/strong\u003eqPCR was used to detect miR-485-3p expression in MIA PaCa-2 and PANC-1 cells. \u003cstrong\u003eI, J. \u003c/strong\u003eGemcitabine dose-response curve showed the effect of miR-485-3p overexpression on the chemosensitivity of Mia Paca-2 (I) and PANC-1 cells (J) under normoxic or hypoxic conditions. \u003cstrong\u003eK. \u003c/strong\u003eKaplan-Meier survival curve analysis of miR-485-3p expression and the prognosis of PDAC patients. Data are expressed as mean ± SD from three or more than three independent experiments. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3865266/v1/cfa5dff8576eb2b8cbef1905.png"},{"id":50442941,"identity":"4855ebc2-be9e-456c-bc57-3080f2583ac6","added_by":"auto","created_at":"2024-01-31 15:24:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":700013,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSOX9 and SLC7A11 are targets of miR-485-3p. A. \u003c/strong\u003eSchematic of the screening strategy to identify targets of miR-485-3p in Starbase source. \u003cstrong\u003eB. \u003c/strong\u003eVenn diagram showing intersection genes (SOX9, SLC7A11) of wild-type and drug-resistant and predicted targets from Starbase.\u003cstrong\u003e C. \u003c/strong\u003eThe predicted binding sites of miR-485-3p in the 3’UTR of SOX9 and corresponding mutant sites of SOX9. \u003cstrong\u003eD. \u003c/strong\u003eRelative luciferase activity was determined in Mia Paca-2 and PANC-1 cells transfected with wild-type or mutant 3′-UTR of SOX9.\u003cstrong\u003e E.\u003c/strong\u003e Three predicted binding sites of miR-485-3p in the 3’UTR of SLC7A11 and corresponding mutant sites of SLC7A11. \u003cstrong\u003eF. \u003c/strong\u003eRelative luciferase activity was determined in Mia Paca-2 and PANC-1 cells transfected with the wild-type or mutant 3′-UTR of SLC7A11. \u003cstrong\u003eG. \u003c/strong\u003emiR-485-3p affected SOX9 and SLC7A11 mRNA and protein expression in Mia PaCa-2 and PANC-1 cells. \u003cstrong\u003eH.\u003c/strong\u003e miR-485-3p overexpression affected SOX9 and SLC7A11 mRNA and protein expression in Mia PaCa-2 and PANC-1 cells under normoxia or hypoxia conditions.\u003cstrong\u003e \u003c/strong\u003eData are expressed as mean ± SD from three independent experiments. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3865266/v1/d4f3e166734386b8ea8ec452.png"},{"id":50444116,"identity":"bfd4be4d-9fe0-4dbb-b0b0-dae2c7f8873a","added_by":"auto","created_at":"2024-01-31 15:32:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1487340,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMiR-485-3p suppresses stemness of PDAC cells under hypoxic condition.\u003c/strong\u003e \u003cstrong\u003eA. \u003c/strong\u003eEffects of miR-485-3p overexpression on the tumor sphere formation of PDAC cells MIA PaCa-2 and PANC-1 under normoxic or hypoxic conditions. Scale bar, 100 μm. \u003cstrong\u003eB.\u003c/strong\u003e Effects of miR-485-3p overexpression on the proportion of CD24\u003csup\u003e+\u003c/sup\u003e CD44\u003csup\u003e+\u003c/sup\u003e cells in PDAC cells MIA PaCa-2 and PANC-1 under normoxic or hypoxic conditions. \u003cstrong\u003eC.\u003c/strong\u003e Effects of miR-485-3p overexpression on the proportion of CD133\u003csup\u003e+\u003c/sup\u003e cells in PDAC cells MIA PaCa-2 and PANC-1 under normoxic or hypoxic conditions. \u003cstrong\u003eD, E.\u003c/strong\u003e Subcutaneous tumor formation rate of overexpressed miR-485-3p and its control group in nude mice. Data are expressed as mean ± SD from at least three independent experiments. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3865266/v1/6219dadcaa18b5285925e292.png"},{"id":50442948,"identity":"36bc5464-0ae0-4a79-95b4-a20640034a8f","added_by":"auto","created_at":"2024-01-31 15:24:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":607892,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-485-3p promotes ferroptosis. A.\u003c/strong\u003e Schematic illustration of SLC7A11-GPX4 mediated ferroptosis pathway.\u003cstrong\u003e B.\u003c/strong\u003e GSEA showed that differentially expressed genes identified following SLC7A11 high and low (median) in TCGA-PDAC were enriched in the ferroptosis pathway.\u003cstrong\u003e C.\u003c/strong\u003e The schematic illustration for human PDAC cells ferroptosis evaluation. \u003cstrong\u003eD, E. \u003c/strong\u003eSensitivity to ferroptosis inducers Erastin and RSL3 of MIA PaCa-2 (D) and PANC-1 (E) overexpressing miR-485-3p. \u003cstrong\u003eF-K. \u003c/strong\u003echanges of intracellular ROS (F, G), MDA (H, I) and GSH (J, K) levels in MIA PaCa-2 (F, H, J) and PANC-1 (G, I, K), treated with miR-485-3p combined with ferroptosis inducers (Erastin or RSL3) and or Fer-1. Data are expressed as mean ± SD from at least three independent experiments. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3865266/v1/0dca47419cad61dcb163f6a8.png"},{"id":50444117,"identity":"88f0c0c8-3234-4337-bab9-35f325f30641","added_by":"auto","created_at":"2024-01-31 15:32:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1228067,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-485-3p regulated stemness and chemosensitivity of PDAC cells through SLC7A11-mediated ferroptosis. A. \u003c/strong\u003eExpression levels of PDAC CSCs markers in cells with knockdown of miR-485-3p and/or SLC7A11, combined with the treatment with ferroptosis inhibitor Fer-1.\u003cstrong\u003e B. \u003c/strong\u003eThe chemosensitivity of PDAC cells with knockdown of miR-485-3p and/or SLC7A11, combined with treatment with Fer-1.\u003cstrong\u003e C. \u003c/strong\u003eThe stemness and sphere formation of PDAC cells MIA PaCa-2 and PANC-1 with knockdown of miR-485-3p and/or SLC7A11, combined with treatment with Fer-1. Scale bar, 100 μm. \u003cstrong\u003eD.\u003c/strong\u003e The proportion of CD24+CD44 stem cells in PDAC cells MIA PaCa-2 and PANC-1 with knockdown of miR-485-3p and/or SLC7A11, combined with treatment with Fer-1. \u003cstrong\u003eE. \u003c/strong\u003eThe proportion of CD133\u003csup\u003e+\u003c/sup\u003e stem cells in PDAC cells MIA PaCa-2 and PANC-1 with knockdown of miR-485-3p and/or SLC7A11, combined with treatment with Fer-1. Data are expressed as mean ± SD from at least three independent experiments. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3865266/v1/934a49cbd2da4f1a6109e657.png"},{"id":50442945,"identity":"696739d5-969e-4bf6-9726-571b5bead371","added_by":"auto","created_at":"2024-01-31 15:24:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":925690,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe methylation of miR-485-3p promoter region was mediated by DNMT3B under hypoxia. A. \u003c/strong\u003eThe expression of miR-485-3p in cells exposed to hypoxia and/or treated with 5-AZA . \u003cstrong\u003eB.\u003c/strong\u003e The expression of miR-485-3p in cells with the overexpression of DNMTs. \u003cstrong\u003eC. \u003c/strong\u003eThe expression of miR-485-3p in cells with knockdown of DNMT3B. \u003cstrong\u003eD. \u003c/strong\u003eThe expression of SLC7A11 in cells exposed to hypoxia and/or with the knockdown of DNMT3B and/or treatment with miR-475-3p inhibitor. \u003cstrong\u003eE. \u003c/strong\u003eOnline prediction of the possible promoter region of miR-485-3p. \u003cstrong\u003eF. \u003c/strong\u003eDual-luciferase reporter assay of transcription activity of the three promoter regions. \u003cstrong\u003eG. \u003c/strong\u003eMethprimer predicts the distribution of CpG islands in the promoter region. \u003cstrong\u003eH. \u003c/strong\u003eBSP identification of the CpG methylation level in the miR-485-3p promoter of the intracellular genome after hypoxia exposure, the black circle represents the presence of methylation modification, and the white circle represents the absence of methylation modification in the CpG where it is located. Data are expressed as mean ± SD from at least three independent experiments. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3865266/v1/9b0750745c6e392af4695787.png"},{"id":50442946,"identity":"d7d3af51-8497-4bcd-9cae-966b4bf235aa","added_by":"auto","created_at":"2024-01-31 15:24:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1065775,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDNMT3B-SLC7A11 regulates stemness and chemosensitivity in PDAC cells. A. \u003c/strong\u003eIC50 of gemcitabine in MIA PaCa-2 and PANC-1 with DNMT3B overexpression and/or SLC7A11 knockdown under hypoxia condition.\u003cstrong\u003e B. \u003c/strong\u003eThe expression levels of stem cell markers in PDAC cells with DNMT3B overexpression and/or SLC7A11 knockdown under hypoxia condition.\u003cstrong\u003e C. \u003c/strong\u003eSphere formation of PDAC cells with DNMT3B overexpression and/or SLC7A11 knockdown under hypoxia condition. Scale bar, 100 μm. \u003cstrong\u003eD.\u003c/strong\u003e The proportion of CD24\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003e cells in PDAC cells with DNMT3B overexpression and/or SLC7A11 knockdown under hypoxia condition. \u003cstrong\u003eE.\u003c/strong\u003eThe proportion of CD133\u003csup\u003e+\u003c/sup\u003e cells in PDAC cells with DNMT3B overexpression and/or SLC7A11 knockdown under hypoxia condition. Data are expressed as mean ± SD from at least three independent experiments. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3865266/v1/be98825a63240156a4807d3d.png"},{"id":50442947,"identity":"135eceb7-21a7-4ab2-bb65-be69b8984033","added_by":"auto","created_at":"2024-01-31 15:24:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":4726955,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMiR-485-3p increased chemosensitivity in vivo and the correlation between DNMT3B and SLC7A11 expression in patients with pancreatic cancer. A. \u003c/strong\u003eTumor formation in groups of PDAC cell lines with control or miR-485-3p overexpression after treatment with gemcitabine. The volume growth and tumor weight of subcutaneous tumors were recorded. \u003cstrong\u003eB.\u003c/strong\u003e The levels of SOX9, SLC7A11, SOX2, and ACSL4 were assessed in different groups by IHC. Scale bar, 50 μm.\u003cstrong\u003e C.\u003c/strong\u003e Representative IHC staining for DNMT3B and SLC7A11 in PDAC patients. CASE1 and CASE2 refer to two representative samples categorized by low and high DNMT3B expression. Scale bar, 50 μm. \u003cstrong\u003eD. \u003c/strong\u003eThe percentage of specimens with low or high SLC7A11 expression in the low or high DNMT3B expression groups. \u003cstrong\u003eE. \u003c/strong\u003eGraphical abstract highlighting the role of hypoxia/DNMT3B/miR-485-3p/SLC7A11 and SOX9 axis in regulating stemness and chemoresistance of PDAC cells.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-3865266/v1/8eefa60b9286b5eba99c20e2.png"},{"id":57396804,"identity":"ae271749-8447-45a0-a58d-30184ba552ca","added_by":"auto","created_at":"2024-05-30 07:11:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11961626,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3865266/v1/1dac0ce2-6d91-401f-a164-d80626fa3ba0.pdf"},{"id":50442950,"identity":"446b9b84-b07c-4ac5-88bd-e4d7f43f7801","added_by":"auto","created_at":"2024-01-31 15:24:31","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":1482542,"visible":true,"origin":"","legend":"","description":"","filename":"sumpplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3865266/v1/59fd5e6154605acd2851685a.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"Hypoxia-induced epigenetic regulation of miR-485-3p promotes stemness and chemoresistance in pancreatic ductal adenocarcinoma via SLC7A11-mediated ferroptosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePancreatic ductal adenocarcinoma (PDAC) is a highly aggressive gastrointestinal malignancy with a dismal prognosis\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. PDAC is currently the 4th leading cause of cancer-related death, and a 5-year survival rate is only approximately 11%\u003csup\u003e2\u003c/sup\u003e. Gemcitabine is still the cornerstone of chemotherapeutic agent for PDAC\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, the response to chemotherapy agents is hampered by drug resistance of PDAC. Hypoxia is a common occurrence in PDAC due to poor vascularization, uncontrolled growth, and fibrotic stroma\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Accumulating evidence has revealed that hypoxia is associated with stemness maintenance, gemcitabine resistance, and pathological angiogenesis\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Our recent study demonstrated that hypoxic conditions in PDAC promote tumor angiogenesis by suppressing GJA1 expression\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. However, molecular mechanisms modulating PDAC cells chemoresistance in hypoxic conditions remain unclear.\u003c/p\u003e \u003cp\u003eRecent studies have underscored important role of microRNAs (miRNAs) in regulating proliferation, migration, invasion, stemness, and chemotherapy resistance in various tumors, including pancreatic cancer\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. MiRNAs are a class of endogenous small noncoding RNAs typically composed of 19\u0026ndash;24 nucleotides and exert regulatory effects by inhibiting mRNA translation or inducing mRNA degradation\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Moreover, hypoxic environments can affect the expression of miRNAs\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. MiR-485-3p, derived from the MIR485 gene located on chromosome 14q32.31, has been implicated in breast cancer, osteosarcoma, prostate cancer, gastric cancer, hepatocellular carcinoma, and other tumors due to its aberrant expression\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In our previous study, aberrant expression of miR-485-3p inhibited the migration and invasion of PDAC cells\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, but the mechanism underlying its aberrant expression remains unclear.\u003c/p\u003e \u003cp\u003eIn this study, we found that SOX9 and SLC7A11 are the downstream targets of miR-485-3p and DNMT3B downregulates miR-485-3p expression. DNMT3B/miR-485-3p/SLC7A11 axis promotes stemness maintenance and induces gemcitabine resistance in PDAC cells under hypoxic condition.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003e1. miR-485-3p is inhibited in PDAC cell lines under hypoxia conditions and associated with good clinical prognosis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate whether hypoxia related signaling pathways are involved in gemcitabine resistance in pancreatic cancer cells, we used GEO database to screen the differential expression genes (DEGs) of gemcitabine-sensitive and -resistant pancreatic cancer cells, and the work flow was presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. The results showed that these DEGs had a correlation with HIF signaling pathway in most datasets, except for the GSE197352 dataset (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Supplementary Table\u0026nbsp;1). Next, we employed CoCl2 induced- or physical- method to imitate hypoxic condition in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). While HIF-1α was upregulated under hypoxia (Supplementary Fig.\u0026nbsp;1A, B), we found a significant downregulation of miR-485-3p expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe evaluated the impact of miR-485-3p on the chemosensitivity of PDAC cells. The results showed that miR-485-3p overexpression under normoxia or hypoxia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, H), increased the sensitivity of PDAC cells to gemcitabine and partially reversed hypoxia-induced resistance of PDAC to gemcitabine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, J). Moreover, we established drug-resistant cell lines and observed lower miR-485-3p expression in these cells (Fig. S2A, C). Overexpression of miR-485-3p in drug-resistant cell lines reversed the resistance of PDAC cells to gemcitabine (Fig. S2B, D). Furthermore, lower miR-485-3p expression was closely correlated with reduced overall survival in PDAC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK). Collectively, these data indicated that miR-485-3p exhibited aberrant expression patterns in PDAC tissues and cells, and was downregulated under hypoxic condition.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2. Hypoxia upregulates SOX9 and SLC7A11 expression by downregulating the expression of miR-485-3p\u003c/h2\u003e \u003cp\u003eTo elucidate the molecular mechanism underlying the effects of miR-485-3p on gemcitabine resistance in PDAC cells under hypoxic condition, we utilized the \u0026ldquo;Starbase\u0026rdquo; to predict potential targets of miR-485-3p (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;2A). Comparing the PDAC GEO datasets of DEGs between wild-type and drug-resistant cells, we found that SOX9 and SLC7A11were potential targets of miR-485-3p (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Moreover, we analyzed the TCGA-PDAC dataset to confirm that both SOX9 and SLC7A11 expression were negatively correlated with miR-485-3p expression (Supplementary Fig.\u0026nbsp;3B, C). Luciferase assays further revealed that miR-485-3p overexpression inhibited the luciferase activity of the wild-type containing SOX9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). Moreover, there were three potential sites of miR-485-3p binding within the 3'-UTR region of SLC7A11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) and the luciferase activity of the binding site 3 was the lowest (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). MiR-485-3p overexpression inhibited the expression of SOX9 and SLC7A11, while silencing miR-485-3p had the opposite effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, Supplementary Fig.\u0026nbsp;3D, E). Additionally, miR-485-3p could reverse hypoxia-induced SOX9 and SLC7A11 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Taken together, these results suggested that miR-485-3p could target SOX9 and SLC7A11 under hypoxic condition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3. miR-485-3p suppresses stemness of PDAC cells under hypoxic condition\u003c/h2\u003e \u003cp\u003eAccumulating evidence has revealed that SOX9 is a master regulator of pancreatic progenitor cells and plays an important role in pancreatic endocrine and ductal cell differentiation during pancreatic development\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. SOX9 is also recognized as a marker of PDAC cancer stem cells (CSCs), contributing to their stemness properties\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Therefore, we investigated the role of miR-485-3p in PDAC CSC stemness. Hypoxia enhanced the tumor sphere formation ability in MIA PaCa-2 and PANC-1, which could be reversed by miR-485-3p overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). PDAC cells exposed to hypoxia had higher expression of CD24 and CD44 than those exposed to normoxia, while miR-485-3p overexpression inhibited the proportion of CD24\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003e PDAC stem cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and CD133\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In addition, overexpression of miR-485-3p downregulated the expression of stemness marker SOX2 in MIA PaCa-2 (Supplementary Fig.\u0026nbsp;4A) and PANC-1 (Supplementary Fig.\u0026nbsp;4B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn vivo, miR-485-3p overexpression in PANC-1 decreased the tumor initiating cell frequency to nearly 1/16 (from 1/25140 to 1/ 417215), which indicated that the frequency of CSCs in the miR-485-3p overexpression group was significantly lower than that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These data suggested that hypoxia increases PDAC cell stemness by suppressing miR-485-3p.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e4. miR-485-3p promoted ferroptosis\u003c/h2\u003e \u003cp\u003eSLC7A11 is involved in ferroptosis, a form of iron-dependent programmed cell death characterized by intracellular lipid peroxidation induced by excess oxygen free radicals generated via the Fenton reaction during iron metabolism. One essential component of the classic antioxidant systems is the glutathione antioxidant system mediated by the SLC7A11-GPX4 axis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In PDAC tissues from TCGA, SLC7A11 expression was correlated with ferroptosis pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Therefore, we hypothesized that miR-485-3p may regulate ferroptosis pathway. Changes in the sensitivity to ferroptosis inducers, ROS, MDA, and intracellular GSH levels, as well as the restoration of these indicators by ferroptosis inhibitors, serve as markers for ferroptosis regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMiR-485-3p overexpression increased the sensitivity of MIA PaCa-2 and PANC-1 cells to ferroptosis inducers Erastin and RSL3, and this effect was attenuated by ferroptosis inhibitors (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E, Table S3). Additionally, ferroptosis inducers significantly elevated intracellular ROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, G) and MDA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, I) levels in PDAC cells overexpressing miR-485-3p, while ferroptosis inhibitor Fer-1 effectively reduced ROS and MDA levels. Furthermore, intracellular glutathione content significantly decreased upon miR-485-4p overexpression, but could be restored by Fer-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ, K). These findings suggested that miR-485-3p plays a role in regulating ferroptosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e5. miR-485-3p regulates stemness and chemosensitivity of PDAC cells through SLC7A11-mediated ferroptosis\u003c/h2\u003e \u003cp\u003eThe expression of stemness markers SOX2 and ALDH1A significantly increased in response to miR-485-3p silencing in MIA PaCa-2 and PANC-1 cells, while SLC7A11 knockdown significantly attenuated their expression. Treatment with ferroptosis inhibitors significantly upregulated the expression of stemness markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Knockdown of SLC7A11 inhibited drug resistance induced by miR-485-3p silencing, and these effects were restored by Fer-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, Supplementary Fig.\u0026nbsp;5A, B). Notably, silencing miR-485-3p in MIA PaCa-2 and PANC-1 cells led to an increase in tumor cell sphere formation, while knockdown of SLC7A11 reversed this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Furthermore, treatment with a ferroptosis inhibitor partially restored the stemness sphere formation inhibited by miR-485-3p knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Flow cytometry showed that silencing miR-485-3p increased the proportion of CD24\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003e cells, which was mitigated by SLC7A11 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). A similar trend was observed for CD133\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, miR-485-3p inhibition decreased MDA levels (Supplementary Fig.\u0026nbsp;6A, B) and increased GSH concentration (Supplementary Fig.\u0026nbsp;6C, D), and these effects were reversed by SLC7A11 knockdown and subsequently restored by Fer-1. These results suggested that miR-485-3p regulates PDAC stemness partially through SLC7A11-mediated ferroptosis pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e6. The methylation of miR-485-3p promoter region was mediated by DNMT3B under hypoxia\u003c/h2\u003e \u003cp\u003eTo explore molecular mechanism by which hypoxia downregulates miR-485-3p expression in PDAC cells, we considered the role of DNA methylation in regulating transcription of non-coding RNA. In PDAC cells treated with the DNA methyltransferase inhibitor 5-azacytidine (5-AZA), miR-485-3p expression restored in hypoxic condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Subsequently, we overexpressed DNMTs in MIA PaCa-2 and PANC-1 cells and found that DNMT3B significantly inhibited miR-485-3p expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). PDAC cells with DNMT3B knockdown exhibited lower expression of SLC7A11 compared to scramble group under hypoxia (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Hypoxia-induced upregulation of SLC7A11 expression was antagonized by DNMT3B knockdown, suggesting that hypoxia elevates SLC7A11 expression by promoting DNMT3B expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Furthermore, miR-485-3p inhibitor could upregulate SLC7A11 expression, indicating that DNMT3B regulates SLC7A11 expression through miR-485-3p (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe used TransmiR (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.cuilab.cn/transmir\" target=\"_blank\"\u003ewww.cuilab.cn/transmir\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.cuilab.cn/transmir\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), mirTrans (mcube.nju.edu.cn/jwang/lab/soft/mirtrans/) and geneXplain (platform.genexplain.com/) to screen regions of miR-485-3p promoter and identified three possible sites responsible for miR-485-3p transcription (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Promoter3 showed strong transcriptional activity, suggesting that Promoter3 may contain the promoter region for miR-485-3p (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Methprimer tool predicted CpG island in this region (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). We found higher level of methylation in the miR-485-3p promoter region in cells exposed to hypoxia compared to cells exposed to normoxia (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). These data indicated that miR-485-3p promoter is methylated by DNMT3B under hypoxic condition, resulting in miR-485-3p downregulation and subsequent upregulation of SLC7A11 expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e7. DNMT3B regulates stemness and chemosensitivity through SLC7A11-mediated ferroptosis in PDAC cells\u003c/h2\u003e \u003cp\u003eTo confirm whether DNMT3B play an oncogenic role in PDAC cells by upregulating SLC7A11 expression and inhibiting ferroptosis process, we assessed the effect of DNMT3B overexpression and SLC7A11 knockdown on PDAC cell sensitivity to gemcitabine. Overexpression of DNMT3B reduced the sensitivity of PDAC cells to gemcitabine, while SLC7A11 knockdown significantly reversed this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Similar changes were observed for stemness marker expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). PDAC cells with both DNMT3B overexpression and SLC7A11 silencing had significantly less tumor sphere formation compared to cells with only DNMT3B overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Meanwhile, overexpression of DNMT3B under hypoxic condition increased the proportion of CD24\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD) and CD133\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). However, SLC7A11 knockdown reduced the proportion of CD24\u003csup\u003e+\u003c/sup\u003eCD44\u003csup\u003e+\u003c/sup\u003e and CD133\u003csup\u003e+\u003c/sup\u003e cells. These data indicate that SLC7A11 knockdown inhibits stemness maintenance induced by DNMT3B.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further examined biochemical indicators of ferroptosis, such as ROS, MDA, and intracellular GSH content (Supplementary Fig.\u0026nbsp;7A). PDAC cells exposed to gemcitabine, DNMT3B overexpression significantly inhibited ACSL4 expression, reduced ROS and MDA levels and increase GSH levels (Supplementary Fig.\u0026nbsp;7B-F). Silencing SLC7A11 could reverse these effects, which was then partially restored by Fer-1 (Supplementary Fig.\u0026nbsp;7B-F). These results demonstrate that ferroptosis mediated by DNMT3B-SLC7A11 axis regulates the levels of ROS and lipid peroxidation in PDAC cells exposed to gemcitabine.\u003c/p\u003e \u003cp\u003e \u003cb\u003e8. miR-485-3p sensitizes PDAC cells to gemcitabine mediated by ferroptosis in vivo and DNMT3B is correlated with SLC7A11 expression in PDAC patients\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo confirm that miR-485-3p sensitizes PDAC cells to gemcitabine in vivo, we found that tumors with miR-485-3p overexpression group grew much slower than those with pLV, and miR-485-3p overexpression enhanced the responsiveness of PDAC to gemcitabine (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Moreover, miR-485-3p overexpression inhibited SOX9, SLC7A11 and SOX2 expression, while upregulated ACSL4 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). The results indicate that miR-485-3p decreases PDAC cell stemness and increases sensitivity to gemcitabine mediated by ferroptosis in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further assessed DNMT3B expression by IHC in 31 human PDAC tissues. The expression of DNMT3B was positively correlated with SLC7A11 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Our results demonstrated that the expression level of SLC7A11 was significantly higher in DNMT3B high-expression group compared to DNMT3B low-expression group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). Taken together, the data suggest the important pathological role of DNMT3B-SLC7A11 axis in pancreatic cancer.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we reported that miR-485-3p was downregulated in PDAC cells exposed to hypoxia and further explored the role of miR-485-3p in PDAC chemoresistance. Overexpression of miR-485-3p markedly decreased the tumor-sphere formation ability and gemcitabine resistance in PDAC cells. In addition, miR-485-3p inhibited PDAC in vivo model. Mechanistically, miR-485-3p downregulated SOX9 and SLC7A11 expression by directly binding to mRNAs and promoting degradation. Furthermore, miR-485-3p regulated ferroptosis pathway via SLC7A11. We also demonstrated that miR-485-3p promoter is hypermethylated by DNMT3B, contributing to miR-485-3p aberrant expression under hypoxia. Moreover, DNMT3B-SLC7A11 decreased the tumor-sphere formation ability and gemcitabine resistance in PDAC cells with regulation of ferroptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eHypoxia is a common characteristics of PDAC microenvironment, with a median tissue partial oxygen pressure (pO2) of 0\u0026ndash;5.3 mmHg (0\u0026ndash;0.7%) compared to the adjacent normal pancreas pO2 at 24.3\u0026ndash;92.7 mmHg (3.2\u0026ndash;12.3%)\u003csup\u003e21\u003c/sup\u003e. Hypoxic areas within tumors provide well-established niches for cancer cells to acquire and maintain stem-like properties, referred to as CSCs phenotype\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Pancreatic CSCs harbor high heterogeneity in terms of surface and intracellular markers, such as CD24, CD44, CD133, SOX2 and ALDH1A, as well as their response to chemotherapy and hypoxia\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Besides, Chen et al. demonstrated that HIF-1α induced by P4HA1 contributed to PDAC stemness and chemoresistance\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. However, few studies have determined the mechanisms by which miRNAs regulate the stemness and chemoresistance of PDAC. Our discovery that miR-485-3p targets SOX9 and SLC7A11 to inhibit stemness-like properties and gemcitabine resistance provides new insights into the regulation of hypoxia, CSCs and chemoresistance in pancreatic cancer.\u003c/p\u003e \u003cp\u003eSLC7A11 is a key component of the cystine/glutamate transporter and plays important role in cell metabolism and ferroptosis regulation\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Ferroptosis is a form of regulated cell death characterized by iron-dependent lipid peroxidation. SLC7A11 negatively regulates ferroptosis by facilitating cystine uptake, which, in turn, leads to glutathione synthesis and scavenging of reactive oxygen species\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Ferroptosis has been implicated in stemness maintenance and chemotherapy resistance in various cancers, and is an attractive target for therapeutic intervention\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Targeting SLC7A11 can inhibit the proliferation ability of various tumor cells such as colorectal cancer, breast cancer and pancreatic cancer. MiR-375 inhibited the stemness characteristics of gastric cancer cells by targeting SLC7A11-mediated ferroptosis\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. SLC7A11-mediated ferroptosis in glioma cells was involved in regulating the stemness maintenance of glioma cells\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In addition, ferroptosis significantly inhibited the growth of PDAC in SLC7A11-deficient genetically engineered mice\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. However, the role of SLC7A11 in PDAC CSCs remains unclear. In this study, we found that miR-485-3p directly targets and significantly inhibits the expression of SLC7A11. Furthermore, silencing miR-485-3p promoted stemness marker expression and sphere formation. Importantly, these effects could be attenuated by ferroptosis inhibitor Fer-1. These results suggest that miR-485-3p may modulate stemness maintenance and chemotherapy resistance through SLC7A11-mediated ferroptosis pathway.\u003c/p\u003e \u003cp\u003eNotably, significant downregulation of miR-485-3p in PDAC tissues, was correlated with poor patient prognosis, consistent with previous studies identifying miR-485-3p as a tumor suppressor in breast cancer, colorectal cancer and prostate cancers \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. However, the mechanism of miR-485-3p downregulation in PDAC cells, especially under hypoxia, is still unclear. DNA methylation, one of epigenetic modifications, plays a central role in regulating gene expression\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. We found that hypoxia in PDAC cells led to the upregulation of DNMT3B, which subsequently promoted the hypermethylation of the miR-485-3p promoter region. Epigenetic modification resulted in reduced miR-485-3p expression and, in turn, upregulated the expression of SOX9 and SLC7A11, contributing to stemness maintenance and gemcitabine resistance.\u003c/p\u003e \u003cp\u003eThis study has several limitaions. First, while our findings strongly suggest that the identified region is the promoter region of miR-485-3p, we did not confirm it. Chromatin immunoprecipitation or reporter gene assays are needed to provide conclusive evidence. Second, while we found a positive correlation between HIF-1α and DNMT3B expression, the detailed mechanisms require deeper exploration. Dissection of the pathways that modulate DNMT3B expression in hypoxic environments could shed light on potential therapeutic targets.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, miR-485-3p is downregulated in PDAC tissues and cells, and its aberrant expression predicts a poor prognosis in PDAC patients. MiR-485-3p acts as a tumor suppressor to inhibit stemness and gemcitabine resistance in PDAC cells. Mechanistically, the region of miR-485-3p promoter is hypermethylated by DNMT3B induced by hypoxia, resulting in the upregulation of SOX9 and SLC7A11 to hinder ferroptosis process.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e1. Tissue Specimens\u003c/h2\u003e \u003cp\u003eA total of 31 PDAC tissue samples, along with their matched adjacent non-PDAC tissues, were retrospectively collected from patients who underwent R0 resection at Peking University First Hospital from January 2013 to December 2017 and with comprehensive follow-up data. This study was conducted in accordance with the Declaration of Helsinki and approved by the local ethics committee of Peking University First Hospital (No. 201933)\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2. Cell culture, Plasmid construction and Transfection\u003c/h2\u003e \u003cp\u003eHuman PDAC cell lines, including Mia PaCa-2, PANC-1, and 293T, were cultured in the general surgery laboratory of Peking University First Hospital and tested for mycoplasma contamination. Gemcitabine-resistant (GR) cell lines Mia PaCa-2 and PANC-1 were maintained in complete medium containing 200 nM gemcitabine (Gemzar, Eli Lilly). Lentivirus plasmids pLV-485-3p and pLV-Inh-485-3p were purchased from GenePharma (Shanghai, China). miR-485-3p mimics (miR10002176) and inhibitors (miR20002176) were obtained from Ribobio (Guangzhou, China). Lentiviral vectors for SLC7A11 shRNA or DNMT3B shRNA were constructed based on the Plko.1 plasmid. Human DNMT1, DNMT3A, and DNMT3B coding sequences with flag-tags, amplified from a cDNA library, were cloned into pITA vector. Sequences for cloning are detailed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Transfection was performed following the Vigofect (Vigorous Biotechnology, Beijing, China) instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3. Q-PCR and Western blot analysis\u003c/h2\u003e \u003cp\u003eTotal RNA from cells was extracted using the TRIzol kit (Invitrogen, #15596018, USA). Reverse transcription into cDNA was accomplished using a SYBR Kit (Vazyme Biotech, #P611, Nanjing, China). The 7500 equipment was employed to measure mRNA expression levels of target genes normalized to β-actin (ACTB). qPCR primer sequences are listed in Supplementary Table S2.\u003c/p\u003e \u003cp\u003eCells were lysed on ice for 30 minutes using cell lysis buffer (Beyotime, China) supplemented with proteinase inhibitor Cocktail (Roche, USA). The lysateds were centrifuged at 4\u0026deg;C (12,000 \u0026times;g, 10 minutes) and proteins were separated by SDS-PAGE (Biotides, China) and transferred to PVDF membranes (Bio-Rad, USA). The membranes were blocked with 5% skim milk powder in TBST and primary antibodies anti-HIF-1α (Abcam, #ab279654, USA), anti-SOX9 (Abclonal, #A19710, China), anti-SLC7A11 (Abclonal, #A2413, China), anti-ALDH1A (Abclonal, #A0517, China), anti-SOX2 (Abcam, #Ab92494, USA), anti-β-actin (MBL, #PM053, Japan), anti-ACSL4(Abclonal, #A20414, China), anti-flag HRP (Abmart, #PA9020), and anti-DNMT3B (Abclonal, #A7239, China) at 4 ℃ overnight. The membranes were washed with TBST for 5 minutes (3 times) and incubated with HRP-labeled goat anti-rabbit IgG(H\u0026thinsp;+\u0026thinsp;L) (Earthox, #E030120, USA) or -mouse IgG(H\u0026thinsp;+\u0026thinsp;L) (Earthox, #E030110) for 40 minutes. ECL substrate (Millipore, #WBULS0500, USA) was applied to develop the membranes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4. In Vivo Tumorigenicity Assay\u003c/h2\u003e \u003cp\u003eBALB/c nude mice were randomized into different groups. Cancer cells at different dilutions were subcutaneously injected into the flanks of the mice (n\u0026thinsp;=\u0026thinsp;10) using random number method with no blinding. Stem cell frequency was calculated using the website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinf.wehi.edu.au/software/elda/\u003c/span\u003e\u003cspan address=\"http://bioinf.wehi.edu.au/software/elda/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor in vivo tumor estimation, total 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e PANC-1 cells were inoculated subcutaneously into the right flanks of nude mice (n\u0026thinsp;=\u0026thinsp;6) using random number method with no blinding. The mice were treated with 50 mg/kg gemcitabine or equal volumes of PBS. The tumor size was calculated and the mice were sacrificed at the indicated time. The resected tumor was kept in liquid nitrogen.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e5. Tumor Sphere Formation Assay\u003c/h2\u003e \u003cp\u003eCells were cultured in ultralow adhesion plates at 1 000 cells/ per well in serum-free medium with B27 (1:50), EGF (20ng/ml), bFGF (100ng/ml) and LIF (10ng/ml). After culture for 2 weeks, tumor spheres with a diameter of \u0026gt;\u0026thinsp;100 \u0026micro;m were counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e6. CCK-8 Assay\u003c/h2\u003e \u003cp\u003eCells were seeded into 96-well plates (3000 cells/well) with 100 \u0026micro;l medium containing 10% FBS and treated with gemcitabine for 72 hours or RSL3 or Erastin for 48 hours. Next, cells were treated with 100 \u0026micro;l of DMEM and CCK-8 (Dojindo, Japan) mixture (90 \u0026micro;l:10 \u0026micro;l) at 37\u0026deg;C for 1 hour, and absorbance was measured at 450 nm using a 96-well plate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e7. Flow cytometry\u003c/h2\u003e \u003cp\u003eCells were stained with anti-hCD24 (Biolegend, #311118, USA), anti-hCD44(Biolegend, #338816, USA), anti-hCD133(Biolegend, #393906, USA), or appropriate control antibodies, with isotype controls used as negative controls or ROS probes DCFH-DA (Beyotime, #S0033S, China). Data analysis was performed using FlowJo V.10.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e8. Luciferase assay\u003c/h2\u003e \u003cp\u003eFollowing transfection of luciferase reporter plasmids and mutant sequences containing the 3'-UTR of SLC7A11 and SOX9 with miR-485-3p binding sites into cells for 24 hours, Dual-Luciferase\u0026reg; Reporter Assay System (Promega, E1910) was employed to measure firefly luciferase activity and renilla luciferase activity. The ratio of the two was calculated and compared with the control group to determine relative luciferase activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e9. Malondialdehyde (MDA) Assay\u003c/h2\u003e \u003cp\u003eThe level of MDA was evaluated using the MDA detection kit (Beyotime, #S0131S, China) following the instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e10. GSH Assay\u003c/h2\u003e \u003cp\u003eThe reduced glutathione content was evaluated by using the reduced glutathione detection kit (Solarbio, BC1170, China) following the instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e11. Bisulfite Sequencing PCR (BSP)\u003c/h2\u003e \u003cp\u003eGenomic DNA was extracted from cells using the DNA extraction Kit (TIANGEN, #DP304-02, China). DNA was denatured by incubation with NaOH for 10 minutes at 37\u0026deg;C, followed by bisulfite modification, PCR amplification, cloning to the pGM-T vector (TIANGEN, #VT402 and #VT202-01, China) and then sequencing by Beijing Tsingke Biotech Company.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e12. Immunohistochemistry\u003c/h2\u003e \u003cp\u003eIHC scoring was conducted by two independent pathologists who were blinded to patients' clinicopathological features and prognosis.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e13. Bioinformation analysis\u003c/h2\u003e \u003cp\u003eThe differential expression genes (DEGs) of gemcitabine-sensitive and -resistant pancreatic cancer cells in GEO datasets were analyzed by GEO2R tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/geo2r\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo/geo2r\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). KEGG enrichment of DEGs was analyzed by DAVID online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david.ncifcrf.gov/tools.jsp\u003c/span\u003e\u003cspan address=\"https://david.ncifcrf.gov/tools.jsp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and visualized by bioinformatics tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.com.cn\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.com.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The Starbase database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://starbase.sysu.edu.cn/\u003c/span\u003e\u003cspan address=\"https://starbase.sysu.edu.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to predict the targets of miR-485-3p.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e14. Statistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using SPSS 27.0 software. For continuous variables with homogeneous variances, t-test was performed, and results were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. χ\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e test was used for categorical variables. Un-paired Student\u0026rsquo;s t-test was used for in vivo experiments. Survival analysis was conducted using Kaplan-Meier survival curves and Log-rank tests. Difference was considered statistically significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe conception and design of the study: Yinmo Yang, Xiaodong Tian. The acquisition of data:Xinxin Liu, Zhihua Huang, Qiuzheng Chen. Analysis and interpretation of data: Xinxin Liu, Zhihua Huang, Qiuzheng Chen, Kai Chen, Guangnian Liu, Xiangyu Chu, Dongqi Li, Long Ma. Drafting the article or revising it: Xinxin Liu, Zhihua Huang, Yinmo Yang and Xiaodong Tian. All authors read and approved the final paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by National Natural Science Foundation of China (NO. 82171722, 82271764, and 81871954), Beijing Municipal Natural Science Foundation (7212111), Peking University Medicine Sailing Program for Young Scholars\u0026rsquo;Scientific \u0026amp; Technological Innovation (BMU2022MX020), the National Key Research and Development Program of China (2021YFA0909900) and National High Level Hospital Clinical Research Funding (Interdepartmental Research Project of Peking University First Hospital 2023IR23)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in studies involving human participants were in accordance with Declaration of Helsinki and the research protocols were approved by the Research Ethics Committees of Peking University First Hospital. All animal experiments were carried out in accordance with the guidelines for the use of laboratory animals and approved by the institutional animal committee of Peking University First Hospital (No. 2022111).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHalbrook, C. J., Lyssiotis, C. A., di Magliano, M. P. \u0026amp; Maitra, A. Pancreatic cancer: Advances and challenges. Cell 186, 1729\u0026ndash;1754 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.1016/j.cell.2023.02.014\u003c/span\u003e\u003cspan address=\"https://doi.org:10.1016/j.cell.2023.02.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiegel, R. L., Miller, K. D., Wagle, N. S. \u0026amp; Jemal, A. Cancer statistics, 2023. 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Int J Mol Sci 24 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org:10.3390/ijms24119360\u003c/span\u003e\u003cspan address=\"https://doi.org:10.3390/ijms24119360\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3865266/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3865266/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe mechanism of hypoxia in chemoresistance of pancreatic ductal adenocarcinoma (PDAC) remains elusive. In this study, we reveled essential role of miR-485-3p in PDAC, particularly its impact on cancer stemness and gemcitabine resistance under hypoxic condition. We found substantial downregulation of miR-485-3p in PDAC tissues, with lower expression correlating to poor patient outcomes. Mechanistically, miR-485-3p influenced stemness characteristics, as evidenced by reduced tumor sphere formation and increased sensitivity to gemcitabine upon overexpression. Moreover, we identified SOX9 and SLC7A11 as two targets of miR-485-3p, which play vital role in stemness and ferroptosis. Under hypoxic condition, DNMT3B expression was upregulated, leading to hypermethylation of miR-485-3p promoter region. the reduced miR-485-3p expression promoted stemness and chemoresistance of PDAC. In conclusion, our findings elucidate the intricate interplay of hypoxia, epigenetic modifications, and ferroptosis in PDAC and shed light on potential avenues for targeted interventions that modulate cancer stemness and chemosensitivity, offering prospects for improved therapeutic strategies for PDAC.\u003c/p\u003e","manuscriptTitle":"Hypoxia-induced epigenetic regulation of miR-485-3p promotes stemness and chemoresistance in pancreatic ductal adenocarcinoma via SLC7A11-mediated ferroptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-31 15:24:26","doi":"10.21203/rs.3.rs-3865266/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-03-04T11:27:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-02-25T05:53:31+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-02-07T05:17:00+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-01-29T13:51:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-15T15:28:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death Discovery","date":"2024-01-15T03:06:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-15T03:06:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dd2b711c-b30e-4ef8-bd11-6ae16b5afb36","owner":[],"postedDate":"January 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28430861,"name":"Biological sciences/Cancer/Gastrointestinal cancer/Pancreatic cancer"},{"id":28430862,"name":"Biological sciences/Genetics/Gene regulation"}],"tags":[],"updatedAt":"2024-05-30T07:10:52+00:00","versionOfRecord":{"articleIdentity":"rs-3865266","link":"https://doi.org/10.1038/s41420-024-02035-x","journal":{"identity":"cell-death-discovery","isVorOnly":false,"title":"Cell Death Discovery"},"publishedOn":"2024-05-29 04:00:00","publishedOnDateReadable":"May 29th, 2024"},"versionCreatedAt":"2024-01-31 15:24:26","video":"","vorDoi":"10.1038/s41420-024-02035-x","vorDoiUrl":"https://doi.org/10.1038/s41420-024-02035-x","workflowStages":[]},"version":"v1","identity":"rs-3865266","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3865266","identity":"rs-3865266","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}