Nigericin Suppresses the Wnt/β-catenin Signaling in Pancreatic Cancer through Targeting pre-miR-374b-PRKCA/HBP1 Axis

The polyether antibiotic nigericin has been demonstrated recently to have anti-tumor activity in multiple cancers. But the biochemical basis for its anti-cancer effects has not been fully elucidated. The objective of this study was to investigate the potential mechanisms of nigericin in pancreatic cancer (PC) cells. PC cells were exposed to increasing concentrations of nigericin at different time periods, and the corresponding IC50 values were calculated. Then the effects on the biological functions of PC cells were evaluated. Subsequent experiments including the high-throughput RNA sequencing, qRT-PCR, western blot, TOP/FOP-Flash reporter, Co-Immunoprecipitation and luciferase report assays were employed to reveal the potential mechanisms of nigericin. In addition, the inhibitory effects of nigericin on PC cells were also accessed in the subcutaneous tumor model.


Results
The data showed that nigericin was extremely sensitive to PC cells, and could in uence the abilities of cell proliferation, colony formation, apoptosis, migration and invasion. The results in vitro implied that nigericin suppressed the Wnt/β-catenin signaling by up-regulating PRKCA and HBP1 mRNA expressions.
Furthermore, the dual strands of pre-miR-374b were proved to down-regulate the PRKCA and HBP1 expressions coordinately, and over-expression of pre-miR-374b partly antagonized the suppressing effects of PC cells induced by nigericin. Meanwhile, the inhibitory effects of nigericin on PC cells were also con rmed in mice.

Conclusion
These ndings demonstrated that suppressing the Wnt/β-catenin signaling pathway by targeting pre-miR-374b-PRKCA/HBP1 axis might represent a novel molecular mechanism of nigericin in PC. Nigericin remained a candidate for a potential pre-clinical application for PC.

Background
Nigericin, an antibiotic derived from Streptomyces hygroscopicus, is an ionophore and acts as an antiporter of K + /H + and raises the pH of acidic compartments. Early in 1987 and 1989, DNA synthesis in Ehrlich ascites tumor cells was found to be inhibited due to the decreased intracellular pH by the exogenous K + /H + antiporter nigericin [1][2]. Then a growing body of evidence supports that nigericin has exhibited a promising anti-cancer activity in various types of cancers, including glioblastoma [3], lung cancer [4][5], breast cancer [6] and colorectal cancer [7]. Though the anti-cancer effects of this drug have been comprehensively discussed, the mechanisms are still largely a mystery. For example, nigericin could selectively target cancer stem cells (CSCs) in nasopharyngeal carcinoma, which could be considered as a candidate CSCs targeting drug for clinical evaluation [8]. In 2012, a study from Zhou et al. demonstrated that nigericin showed toxicity to colorectal cancer cells, and partly reversed the epithelial-mesenchymal transition (EMT) process during cell invasion and metastasis [7]. Mashima et al. revealed that the antibiotic nigericin inhibited AR-mediated activation of the PSA promoter and PSA production in prostate cancer cells [9]. Besides, nigericin might be exploited to enhance drug sensitivity when used in combination with other drugs, such as adriamycin [10], amiloride [11] and melphalan [12]. Our previous study demonstrated that nigericin could signi cantly suppress the tumor growth and metastasis of colorectal cancer cells by inhibiting Wnt/β-catenin signaling pathway and directly targeting the β-catenin destruction complex [13]. Similar reports were also found in ovarian cancer and chronic lymphocytic leukemia, which strongly indicated that the Wnt signaling pathway might be involved in the nigericin damage [14][15].
The Wnt signaling pathway is highly conserved throughout vertebrate species and has critical roles in embryonic development, tissue self-renewal and human diseases [16]. The role of Wnt signaling in carcinogenesis has most prominently been described for colorectal cancer [17], but aberrant Wnt signaling is also observed in many more cancer entities [18][19]. The Wnt signaling pathway is complex and includes canonical and noncanonical branches. The canonical pathway is activated by Wnt family ligands binding to Frizzled/LRP receptor complexes, which leads to the accumulation of nonphosphorylated β-catenin in the cytoplasm. After its translocation to the nucleus, β-catenin forms a complex with members of the TCF/LEF family of transcription factors, and activates target genes such as c-Myc, Cyclin D1 and Survivin [16,20]. In 2018, in the United States, 55,440 new pancreatic cancer cases were diagnosed. By 2030, PC is projected to cause the second highest cancer-related mortality rate in the US. PC is a highly aggressive malignancy with few effective therapies and a very poor prognosis.
The 5-year overall survival rate of PC is less than 5% [21][22]. The role of Wnt signaling in PC carcinogenesis, metastasis, and prognosis has been reported. Its aberrant activation resulting from activating or inactivating mutations in the CTNNB1 gene locus, or in the negative regulators AXIN and APC involving stabilization of β-catenin, and activation of target genes leads to a more aggressive phenotype of PC [23]. Meanwhile, the Wnt signaling drives drug resistance in PC in many ways [24]. Wnt signaling and the nuclear functions of β-catenin have been also reported to be critical for CSCs proliferation, differentiation, and maintenance [25][26].
The aim of this study was to test the anti-cancer activity of nigericin in human PC cells in vitro and in vivo. The drug was sensitive to PC cells, and the effects on the abilities of cell proliferation, colony formation, apoptosis, migration and invasion in PC cells were evaluated. Then the high-throughput RNA sequencing, qRT-PCR, western blot, TOP/FOP-Flash reporter and Co-Immunoprecipitation assays were performed, and the data revealed that nigericin could signi cantly suppress the Wnt/β-catenin signaling by up-regulating PRKCA and HBP1 mRNA expressions. Furthermore, the intramolecular stem-loop structure of pre-miR-374b, which could be spiced into the mature miR-374b-5p and − 3p, might simultaneously target the PRKCA and HBP1 3'-UTR sites. It was also shown that pre-miR-374b could reverse the inactivation of the Wnt/β-catenin pathway induced by nigericin in vitro. In addition, the inhibitory effects of nigericin on PC cells were also accessed in the subcutaneous tumor model.

Methods
Chemicals and cell culture

Migration and invasion assays
The migration and invasion assays were performed with transwell chambers (8 µm pore size, 24-well insert, Corning Lowell, MA, USA). Cells were collected and suspended in the serum-free medium. Then 200 µl serum-free medium containing 1 × 10 4 cells was added to the upper chamber, while the 600 µl medium containing 10% FBS was added to the lower chamber as chemical attractant. Cells were incubated at 37˚C for 48 h, and the non-invasive cells were removed with cotton swabs. Finally, the inserted membrane was xed in methanol for 10 min and stained with 0.1% crystal violet for 20 min, and the in ltrated cells were counted and photographed under the inverted optical microscope IX71 (Olympus, Japan). For cell invasion evaluation, the insert of the chamber was prepared by Matrigel (100 µl, BD Biosciences, USA) covering the upper surface. Other methods were similar to the migration assay. The invaded cells were also counted and photographed under the microscope.

TOP/FOP ash assay
Luciferase reporter assays of the Wnt/β-catenin signaling were performed with the TOP and FOP reporters that contained wild-type and mutated TCF/LEF DNA-binding sites, respectively. TOP/FOP-Flash was co-transfected into cells along with PRKCA (or HBP1) silence or pre-miR-374b overexpression vector.
The TOP/FOP-Flash values were normalized to the Renilla reniformis (Promega) reading and the TOP/FOP ratio was measured, as previously described [13]. Experiments were performed in triplicate.
High-throughput RNA sequencing and bioinformatics analysis PANC-1 cells were exposed to nigericin (5 µmol/L) for different time periods (0, 8, 16 or 32 h), and the high-throughput RNA sequencing was performed as previously described [27]. These mRNAs and miRNAs with statistical signi cance were screened with p-value less than 0.05, false discovery rate (FDR) less than 0.05 and fold change (FC) more than 2.0 (or FC more than 1.5 for miRNA analysis). The common expressed elements among 3 compared groups (0 h vs 8 h, 0 h vs 16 h, 0 h vs 32 h) were analyzed using the venn analysis. The non-supervised hierarchical clustering was used in the form of heat map to present the expression patterns of the differentially expressed cancer-related mRNAs or miRNAs. Based on the results of Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for the common differentially expressed mRNAs, the network between KEGG pathways and their corresponding genes was used to elucidate that these pathways including the Wnt signaling pathway. The common differentially expressed miRNAs and mRNAs were subjected to the analysis and the putative targets of miRNAs were predicted by means of Targetscan (http://www.targetscan.org/). Then the miRNA-mRNA interaction network between up-regulated miRNAs and their corresponding down-regulated mRNAs (or down-regulated miRNAs and their corresponding up-regulated mRNAs) was conducted. High-throughput sequencing data reported herein had been deposited at the NCBI website (https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA543685&go=go) with the accession number PRJNA543685 (SRR9107550, SRR9107551, SRR9107552 and SRR9107553).
MiR-374b-5p (or miR-374b-3p) mimic and inhibitor were also designed and synthesized from GenePharma along with the negative control (NC or anti-NC). Pre-miR-374b over-expression vector (pGCMV/EGFP/pre-miR-374b) was constructed and purchased from Genepharma (Shanghai, China), and a transfectant of the pGCMV/EGFP empty vector was used as a control. At 48 h after transfection, the transfection e ciency was estimated by observing the green uorescent protein (GFP) signal under the inverted optical microscope IX71 (Olympus, Japan), and the PCR analysis was also used to detect the expression of pre-miR-374b in the collected cells. We also obtained stably transfected clones by blasticidin selection (Sigma, USA) for animal observation.
Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from PC cells using TRIzol (Invitrogen, USA) according to the manufacturer instructions, and then cDNA was generated from RNA using SuperScript III (Invitrogen). MRNAs and miRNAs expressions were evaluated using the SYBR green quantitative PCR kit (Life Technologies, USA) based on the manufacturer description. GAPDH and U6 were performed as a control internally for mRNAs and miRNAs respectively. QRT-PCR was performed on the ABI Prism 7500 Fast Sequence Detection System (Applied Biosystems). Levels of relative expression were calculated and quanti ed with the 2 −ΔΔCt method. Primers were presented in Supplementary Table 1.
The immunoprecipitated samples were analyzed by immunoblotting under standard conditions.

Luciferase reporter assay
The full-length of PRKCA (or HBP1) 3'UTR containing the predicted wild-type or mutated miR-374b-5p (or miR-374b-3p) binding sites were ampli ed by PCR and cloned into the downstream of the re y luciferase reporter gene in pMIR (Ambion, USA) vectors to generate recombinant vectors PRKCA-wt and PRKCA-mut. The constructed luciferase reporter plasmids were co-transfected with miR-374b-5p (or miR-374b-3p) mimics or inhibitors or over-expressing pre-miR-374b vectors using Lipofectamine 2000 reagent (Invitrogen). After 48 h of incubation, the re y and Renilla luciferase activities were quanti ed with a dual-luciferase reporter assay (Promega, USA).

Tumor growth in nude mice
Four-week old male BALB/c-nude mice were purchased and maintained in a sterile environment (each group contained 5 mice). PANC-1 cells or that stably expressing pre-miR-374b were injected subcutaneously into the anks of nude mice with a total volume of 200 µl. Nigericin (4 mg/kg) was injected intraperitoneally every 2 days. Mice body weights and tumor volume (TV) were measured weekly, and TV was calculated as follows: TV (mm 3 ) = length x width 2 x 0.5. Bioluminescent imaging was performed on the 28th day after injection at which mice were anaesthetized and given D-luciferin potassium salt (150 mg/kg in phosphate-buffered saline, Sciencelight, Shanghai, China). After 20 min of injection, the bioluminescence was imaged with a charge-coupled device camera (IVIS; Lumina II, PerkinElmer). Signal was displayed as photons/s/cm2/sr. Mice were killed after the bioluminescent assay and the tumor mass were removed and stored at -80 °C for the subsequent PCR analysis. The animal experiment was approved by the Animal Care and Use Committee of the First A liated Hospital of Soochow University.

Statistical analysis
All data analysis was performed using SPSS 21.0 software (SPSS, Inc., Chicago, IL, USA). Measurement data were presented as mean ± standard deviation (SD). Data differences between two groups were compared by the t-test, while differences among multiple groups were compared using one-way analysis of variance (ANOVA). P < 0.05 was considered statistically signi cant.

Results
PC cells were sensitive to nigericin by IC50 comparison PANC-1 and PL-45 cells were exposed to increasing concentrations of nigericin for different time periods (0, 4, 8, 16, 32 or 48 h). As shown in Fig. 1A-B, nigericin treatment signi cantly suppressed the cell viabilities in a concentration dependent manner both in PANC-1 and PL-45 cells. Besides, the corresponding IC50 values were also calculated, and our results showed that nigericin could also inhibit the cell viability in a time-dependent manner (Fig. 1C-D).

Nigericin inhibited the tumorigenic behaviors of PC cells in vitro
Since PC cells were sensitive to nigericin, we next determined whether nigericin exerted inhibitory effects on tumorigenic behaviors of PC cells in vitro. As calculated above, we chose 1 µmol/L as the proper treated concentration of nigericin on PANC-1 and PL-45 cells (less than the IC50 values at 48 h for our subsequent experiments in vitro). We examined the cell proliferation ability using CCK-8 and colony formation assays. As demonstrated in Fig. 2A-D, nigericin signi cantly exerted the similar inhibitory effects on cell proliferation and colony forming abilities of PANC-1 and PL-45 cells. Conversely, nigericin treatment increased the apoptotic rates of PC cells (Fig. 2E-F). Based on the idea that nigericin served as a potential anti-cancer drug of PC, we further explored its effects on cell migration and invasion. We found that the migration and invasive abilities of PANC-1 cells were signi cantly suppressed, and similar results were also observed in PL-45 cells (Fig. 2G-J). These results indicated that nigericin exerted direct and anti-cancer effects on PC cells.

Nigericin suppressed the Wnt/β-catenin signaling in PC cells
To reveal the potential mechanisms of nigericin in PC, we used the high-throughput RNA sequencing analysis to screen the expression pro le of mRNAs in PC cells after nigericin treatment at different time points (0, 8, 16 and 32 h). 172 mRNAs were commonly dys-regulated, and part of cancer-related mRNAs had been described in the hierarchical clustering analysis ( Supplementary Fig. 1A). To ensure that our results were reliable, we assessed the expressions of 5 random mRNAs between the 0 h and 32 h group by qRT-PCR. Consistent with the data of RNA sequencing ( Supplementary Fig. 1B), the results showed that the mRNA expressions of UGCG, HBP1 and MCL1 were signi cantly up-regulated at 32 h, compared to the 0 h group. Oppositely, other 2 mRNAs (CENPC and RPA2) were down-regulated ( Supplementary  Fig. 1C). These data were consistent well with the sequencing data, which demonstrated the high reliability and validity of the sequencing expression results. The network between KEGG pathways and their corresponding genes strongly implied that Wnt/β-catenin signaling was involved in the nigericinmediated effects on PC cells (Supplementary Fig. 1D).
To identify the Wnt signaling that might play a role in nigericin damage, the expressions of downstream effectors of the pathway were determined. The western blot analysis demonstrated that the protein expression levels of total β-catenin, cytosolic β-catenin, nuclear β-catenin, Cyclin D1, AXIN2 and c-Myc were down-regulated after nigericin treatment at 48 h in a concentration-dependent manner. Whereas, the dose-dependent increased protein expressions of p-β-catenin, PRKCA and HBP1 were also observed in PANC-1 and PL-45 cells (Fig. 3A-B). Besides, the transcriptional activities of β-catenin/TCF/LEF using the TOP Flash cell reporter system were evaluated. The data showed that the relative TOP/FOP ratios were signi cantly down-regulated in a dose-dependent manner, compared to the non-nigericin treated PANC-1 and PL-45 cells (Fig. 3C-D). This was the preliminary evidence that nigericin could suppress the Wnt/βcatenin pathway in PC cells.
Nigericin suppressed the Wnt/β-catenin signaling through up-regulating PRKCA/HBP1 expressions PRKCA and HBP1 were two most-regulated and Wnt signaling-related molecules by the high-throughput RNA sequencing analysis. To further identify the potential roles of these two molecules in nigericinmediated process, we rstly determined the mRNA or protein levels of PRKCA and HBP1 after small interfering RNAs respectively. The data showed that nigericin treatment signi cantly up-regulated the mRNA and protein levels of PRKCA or HBP1, and the small interfering RNAs which speci cally targeted PRKCA or HBP1 were effective for the subsequent experiments (Supplementary Fig. 2A-D).
Regarding the inhibiting effects of Wnt/β-catenin pathway by nigericin, we also evaluated the roles of PRKCA or HBP1 in PC cells. The results of western blot analysis demonstrated that si-PRKCA could signi cantly up-regulated the protein expressions of PRKCA downstream effectors such as nuclear βcatenin, Cyclin D1, AXIN2 and c-Myc. Moreover, si-PRKCA treatment could also partly recover the expressions of these PRKCA downstream proteins induced by nigericin damage (Fig. 4A-B). Similar results were also found in HBP1 knocked down cells (Fig. 4C-D). Furthermore, the TOP Flash cell reporter system was also employed, and our data implied that the Wnt/β-catenin signaling was signi cantly inactivated by nigericin. In comparison with nigericin-treated cells, si-PRKCA, si-HBP1 or silencing these two molecules simultaneously could partly activate the pathway induced by nigericin in PANC-1 and PL-45 cells (Fig. 4E-F). These results indicated that PRKCA and HBP1 might be the key molecules in nigericin-mediated damage in PC cells. Besides, the co-IP assays also elucidated that PRKCA mediated βcatenin phosphorylation with β-TrCP was a novel mechanism regulating the Wnt/β-catenin pathway, and HBP1 physically interacted with TCF-4 and served as a negative regulation of Wnt/β-catenin pathway in PC cells. Interfering PRKCA or HBP1 expression presented a re-activated effects of the Wnt/β-catenin pathway induced by nigericin ( Supplementary Fig. 3A-D).

Pre-miR-374b selectively targeted PRKCA and HBP1 in PC cells
Since PRKCA and HBP1 played an essential role in nigericin-mediated damage in the perspective of mRNA levels, we hypothesized that these two molecules might be regulated singly or simultaneously by other potential molecules. Thus, we screened the expression pro le of miRNAs from our high-throughput RNA sequencing at 4 different time points (0, 8, 16 and 32 h) (Fig. 5A). Three comparison groups (0 h vs 8 h, 0 h vs 16 h and 0 h vs 32 h) were set, and the venn analysis was performed to determine the common differentially expressed miRNAs among these 3 different comparison groups. A total of 74 common differentially expressed miRNAs (48 up-and 26 down-regulated) were found throughout the nigericin treatment ( Fig. 5B-C). Then the changes of 5 random common differentially expressed miRNAs including miR-2210, miR-449a, miR-374b-3p, miR-1268b and miR-29c-5p were presented from the high-throughput RNA sequencing in Fig. 5D, and the results of PCR analysis veri ed the reliability of the sequencing data (Fig. 5E). Besides, the interactions between miRNAs and their target genes from the sequencing data were theoretically predicted by conserved seed-matching sequence. The miRNA-mRNA interaction network between up-regulated miRNAs and their corresponding down-regulated mRNAs were presented (Fig. 5F). Similar network between down-regulated miRNAs and their corresponding up-regulated mRNAs was also constructed (Fig. 5G).
Subsequently, the venn analysis among PRKCA-predicted, HBP1-predicted and down-regulated miRNAs identi ed miR-374b as the potential upstream molecule of PRKCA and HBP1 (Fig. 6A). As described in Fig. 6B, has-pre-miR-374b had a stem-loop, which could be spiced into the mature miR-374b-5p and − 3p by the Dicer protein.
Nigericin suppressed the Wnt/β-catenin signaling via a pre-miR-374b-dependent mechanism in vitro Based on the idea that PRKCA and HBP1 served as tumor suppressors in nigericin-mediated damage, and the data also strongly implied that pre-miR-374b could simultaneously regulate PRKCA and HBP1 mRNA expressions, we further performed a series of rescue assays to explore the key role of pre-miR-374b in PC cells. As shown in Supplementary Fig. 4A-J, nigericin treatment signi cantly suppressed the biological functions of PC cells, including the decreased abilities of cell proliferation, colony formation, migration and invasion, and the increased apoptotic rates. However, pre-miR-374b over-expression exerted a tumor promoting effects on PC cells. When compared to the nigericin-treated cells, pre-miR-374b could also effectively antagonize the suppressing effects of biological functions of PC cells induced by nigericin.
Since nigericin was proved to suppress PC cells through the Wnt/β-catenin signaling, we performed the western blot analysis to evaluate the expressions of Wnt//β-catenin downstream proteins in pre-miR-374b over-expression PC cells (Supplementary Fig. 5A-B). Simultaneously, the changes of TOP/FOP ratios were also calculated and presented in Supplementary Fig. 5C-D. These data strongly demonstrated that pre-miR-374b over-expression effectively activated the Wnt/β-catenin pathway in PC cells, and could also counteract the inhibitory effects induced by nigericin. These data provided the strong evidence that nigericin suppressed the Wnt/β-catenin signaling via a pre-miR-374b-dependent mechanism in vitro.

Nigericin inhibited the tumor growth of PC cells in vivo
Given the observed effects of nigericin on PC growth and metastasis in vitro, we subsequently determined whether nigericin suppressed tumorigenicity in vivo. Though the body weights of experimental mice were not in uenced, the tumor mass observed by the bioluminescent assays on day 28 and tumor growth curves all indicated that nigericin could suppress the tumor growth of PC cells in vivo. On the contrary, subcutaneous injection of mice with pre-miR-374b over-expressing PANC-1 cells resulted in larger tumors, compared to the non-miR-374b over-expressing cells. Meanwhile, our results also con rmed that the inhibitory effects of nigericin could be signi cantly antagonized by pre-miR-374b over-expression ( Fig. 7A-C). To further know the regulatory relationships among pre-miR-374b, miR-374b-5p, miR-374b-3p, PRKCA and HBP1, the PCR analysis was employed. The data indicated that nigericin treatment signi cantly reduced the expressions of pre-miR-374b, miR-374b-5p and miR-374b-3p, and up-regulated the PRKCA and HBP1 mRNA expressions in mice tumors. As expected, pre-miR-374b over-expression could antagonize the expressing levels of PRKCA and HBP1 in mice tumors induced by nigericin injection in vivo, compared to the nigericin treated cells (Fig. 7D-H). Thus, we concluded that nigericin might suppress the Wnt/β-catenin signaling in PC through targeting pre-miR-374b-PRKCA/HBP1 axis in vitro and in vivo (Fig. 7I).

Discussion
Our recent study in 2018 proved that nigericin treatment signi cantly reduced tumor cell proliferation in dose-and timedependent manners in colorectal cancer cells [13]. Then the role of nigericin was also investigated in PC cells after the high-throughput RNA sequencing. From the perspective of circRNAs through regulating parental gene transcriptions, we used the bioinformatics analyses to explore the potential mechanism of nigericin in PC cells [27]. In this study, we exposed PC cell lines (PANC-1 and PL-45) to increasing concentrations of nigericin for different time periods and the IC50 values were evaluated. Similar to the results in colorectal cancer cells, PC cells were also sensitive to nigericin in concentration-or time-dependent manners. Choosing a low and suitable concentration, the effects of nigericin on the tumorigenic behaviors of PC cells were evaluated. Our data demonstrated that nigericin exerted the anti-cancer effects on PC cells, and signi cantly in uenced the abilities of cell proliferation, colony formation, apoptosis, migration and invasion.
To reveal the potential mechanisms of nigericin in PC, we used the high-throughput RNA sequencing analysis to screen the expression pro le of mRNAs, and a total of 172 commonly dys-regulated mRNAs were identi ed throughout the nigericin-treated process. The reliability and validity of the sequencing expression results were veri ed, and the network between KEGG pathways and their corresponding genes strongly implied that the Wnt/β-catenin signaling was involved in the nigericin-mediated effects in PC cells. Then the western blot and TOP/FOP-Flash reporter assays con rmed our hypothesis that nigericin could suppress the growth of PC cells by the Wnt/β-catenin pathway. Among these commonly dysregulated mRNAs from the high-throughput RNA sequencing analysis, PRKCA and HBP1 were two key molecules, which might have a role in tumor suppression by inhibiting the Wnt signaling pathway. PKCα, which is encoded by PRKCA gene, is a member of the Protein Kinase C (PKC) family that includes ten serine/threonine kinases. Signi cant evidence indicates that PKCα acts as a tumor suppressor that PKCαinduced the phosphorylation of β-catenin, leading to its degradation by the proteasome [28][29]. In 2010, Lee et al. also reported that PKCα-induced phosphorylation of the orphan receptor RORα results in βcatenin co-transcriptional activity inhibition and in the trans-repression of Wnt/β-catenin target genes [30]. High mobility group box transcription factor 1 (HBP1) belongs to the sequence-speci c, HMG family of transcription factors. As a putative suppressor of the Wnt pathway, HBP1 may inhibit the transcription of TCF4 targets by directly blocking the binding of TCF4 with DNA [31][32]. As to the effects of PRKCA and HBP1 in the repression of the Wnt-β-catenin pathway in cancers, we further investigated the potential role of these two molecules in the nigericin-mediated process. Our data demonstrated that silencing PRKCA or HBP1 by small interfering RNAs could inactivate the Wnt/β-catenin signaling in PC cells. More importantly, silencing PRKCA, HBP1 or these two molecules simultaneously could partly attenuate the inactivation of the Wnt signaling induced by nigericin in PANC-1 and PL-45 cells. Besides, the results of Co-IP assays further con rmed the signi cance of PRKCA and HBP1 in the nigericin damage.
MiRNAs are a class of endogenous noncoding RNAs, which are derived from longer transcripts termed pre-miRNAs, and recognize mainly the 3'-UTR region of messenger RNA (mRNA) and inhibit protein synthesis. Recent reports indicate that dys-regulation of the mature miRNAs and their target genes promote cancer initiation, progression and drug resistance, including PC [33][34]. More interestingly, the dual strands of certain pre-miRNAs, e.g., pre-miR-150 [35], pre-miR-139 [36] and pre-miR-145 [37], can act as anti-cancer miRNAs in several cancers, and each guide strand and passenger strand coordinately regulates oncogenic genes. The pre-miRNA signature led us to focus on the dual strands of pre-miR-374b, i.e., miR-374-5p (the guide strand) and miR-374-3p (the passenger strand). Our data showed that nigericin could coordinately down-regulate the expressions of pre-miR-374b, miR-374b-5p and miR-374b-3p in PC cells in a time-dependent manner. Analyzing the structure of pre-miR-374b and the predicted conserved seed-matching sequences between the miR-374b products and Wnt-related genes, we speculated that miR-374b-5p or -3p might selectively target the PRKCA and HBP1) 3'-UTR sites respectively. Fortunately, the subsequent experiments including the luciferase report assays, PRC and western blot analysis all con rmed our previous hypothesis, that pre-miR-374b might simultaneously regulate PRKCA and HBP1 mRNA expressions by the mature miR-374b-5p and miR-374b-3p respectively. Using a pre-miR-374b overexpressing vector, a series of rescue assays in vitro were also performed and the results demonstrated that pre-miR-374b could effectively antagonize the suppressing effects of biological functions of PC cells induced by nigericin. These ndings provided strong evidence that nigericin might suppress the Wnt/β-catenin signaling through a pre-miR-374b-dependent mechanism. In addition, the suppressing effects of nigericin on tumor growth, and the regulatory relationships between pre-miR-374b and target genes, were also discussed in a subcutaneous tumor model.

Conclusions
In conclusion, the work presented here is the rst to demonstrate the toxicology and anti-cancer effects of nigericin to PC cells. The high-throughput RNA sequencing was performed to screen the expression pro le of mRNAs, and the results in vitro implied that nigericin could signi cantly suppress the Wnt/β-catenin signaling by up-regulating PRKCA and HBP1 mRNA expressions. Furthermore, the dual strands of certain pre-miR-374b were proved to down-regulate the expressions of PRKCA and HBP1 coordinately, and overexpression of pre-miR-374b might partly antagonize the suppressing effects of PC cells induced by nigericin. These ndings demonstrated that suppressing the Wnt/β-catenin signaling pathway by targeting pre-miR-374b-PRKCA/HBP1 axis might represent a novel molecular mechanism of nigericin in PC. Nigericin remained a candidate for a potential pre-clinical application for PC.

Supplementary Files
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