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. 1A. The results showed that these DEGs had a correlation with HIF signaling pathway in most datasets, except for the GSE197352 dataset (Fig. 1B, Supplementary Table 1). Next, we employed CoCl2 induced- or physical- method to imitate hypoxic condition in vitro (Fig. 1C). While HIF-1α was upregulated under hypoxia (Supplementary Fig. 1A, B), we found a significant downregulation of miR-485-3p expression (Fig. 1D-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. 1G, H), increased the sensitivity of PDAC cells to gemcitabine and partially reversed hypoxia-induced resistance of PDAC to gemcitabine (Fig. 1I, 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. 1K). 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. 2A, 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. 2B). 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. 2C, D). Moreover, there were three potential sites of miR-485-3p binding within the 3'-UTR region of SLC7A11 (Fig. 2E) and the luciferase activity of the binding site 3 was the lowest (Fig. 2F). MiR-485-3p overexpression inhibited the expression of SOX9 and SLC7A11, while silencing miR-485-3p had the opposite effects (Fig. 2G, Supplementary Fig. 3D, E). Additionally, miR-485-3p could reverse hypoxia-induced SOX9 and SLC7A11 expression (Fig. 2H). 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 development18,19. SOX9 is also recognized as a marker of PDAC cancer stem cells (CSCs), contributing to their stemness properties20. 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. 3A). 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. 3B) and CD133+ cells (Fig. 3C). 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. 3D). 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. 4A). In PDAC tissues from TCGA, SLC7A11 expression was correlated with ferroptosis pathway (Fig. 4B). 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. 4C).
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. 4D, E, Table S3). Additionally, ferroptosis inducers significantly elevated intracellular ROS (Fig. 4F, G) and MDA (Fig. 4H, 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. 4J, 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. 5A). Knockdown of SLC7A11 inhibited drug resistance induced by miR-485-3p silencing, and these effects were restored by Fer-1 (Fig. 5B, 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. 5C). Furthermore, treatment with a ferroptosis inhibitor partially restored the stemness sphere formation inhibited by miR-485-3p knockdown (Fig. 5C). Flow cytometry showed that silencing miR-485-3p increased the proportion of CD24+CD44+ cells, which was mitigated by SLC7A11 knockdown (Fig. 5D). A similar trend was observed for CD133+ cells (Fig. 5E).
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. 6A). Subsequently, we overexpressed DNMTs in MIA PaCa-2 and PANC-1 cells and found that DNMT3B significantly inhibited miR-485-3p expression (Fig. 6B). PDAC cells with DNMT3B knockdown exhibited lower expression of SLC7A11 compared to scramble group under hypoxia (Fig. 6C). Hypoxia-induced upregulation of SLC7A11 expression was antagonized by DNMT3B knockdown, suggesting that hypoxia elevates SLC7A11 expression by promoting DNMT3B expression (Fig. 6C). Furthermore, miR-485-3p inhibitor could upregulate SLC7A11 expression, indicating that DNMT3B regulates SLC7A11 expression through miR-485-3p (Fig. 6D).
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. 6E). Promoter3 showed strong transcriptional activity, suggesting that Promoter3 may contain the promoter region for miR-485-3p (Fig. 6F). Methprimer tool predicted CpG island in this region (Fig. 6G). 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. 6H). 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. 7A). Similar changes were observed for stemness marker expression (Fig. 7B). PDAC cells with both DNMT3B overexpression and SLC7A11 silencing had significantly less tumor sphere formation compared to cells with only DNMT3B overexpression (Fig. 7C). Meanwhile, overexpression of DNMT3B under hypoxic condition increased the proportion of CD24+CD44+ (Fig. 7D) and CD133+ cells (Fig. 7E). 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. 8A). Moreover, miR-485-3p overexpression inhibited SOX9, SLC7A11 and SOX2 expression, while upregulated ACSL4 expression (Fig. 8B). 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. 8C). Our results demonstrated that the expression level of SLC7A11 was significantly higher in DNMT3B high-expression group compared to DNMT3B low-expression group (Fig. 8D). Taken together, the data suggest the important pathological role of DNMT3B-SLC7A11 axis in pancreatic cancer.