Salmonella typhimurium downregulates circHIPK2 expression via CeRNA mechanism to inhibit cell migration and tumorigenesis
- Open Access
- 01.12.2025
- Research
Erschienen in:
Abstract
Introduction
Salmonella typhimurium (S. typhimurium) infection is a major contributor to gastrointestinal diseases on a global scale [1]. Evidence suggests that around 2–4 million people worldwide suffer from gastroenteritis due to S. typhimurium infection each year, resulting in an estimated 155,000 deaths and an estimated $2 billion economic losses [2]. Recent studies have shown that S. typhimurium infection can lead to intestinal microecological destruction and increased risk of inflammatory bowel disease (IBD) [3].For example, S. typhimurium decreases the viability and colonization of C. albicans, leading to gut microbiota dysbiosis, a common occurrence in patients with IBD [4]. Inversely, short chain fatty acid (SCFA) mitigates intestinal inflammation by S. typhimurium through modulating the composition of the microbiota, enhancing the production of short-chain fatty acids (SCFAs), and modulating inflammatory responses [5]. Moreover, S. typhimurium potentially enhances macrophage secretion of IFN-β by promoting TRIF-dependent inflammation due to impaired autophagy in individuals with Crohn’s colitis or ulcerative colitis [6]. Furthermore, clinical studies and animal models have demonstrated that persistent intestinal infection and colonization by S. typhimurium can promote the onset and progression of colon cancer [7]. S. typhimurium AvrA chronically activates the STAT3 pathway, thereby enhancing β-catenin signaling and promoting colonic tumorigenesis, impacting intestinal renewal in both the small intestine and colon [8, 9]. On the contrary, several studies revealed that S. typhimurium infection could be the tumor immunotherapies through intratumorally injection of attenuated S. typhimurium strains with stimulating localized innate immunity [10‐12]. Hence, investigating and elucidating the molecular mechanisms underlying the interaction between S. typhimurium and its host is crucial for advancing therapeutic and preventive strategies against diseases caused by S. typhimurium and other pathogens [13].
We and other teams have successively found that S. typhimurium infection results in altered gene expression across various signaling pathways in C57BL/6 mouse models of inflammation and colon cancer, encompassing the NF-κB pathway, interleukin signaling like IL-9 and IL-4, epidermal growth factor (EGF) signaling, and Wnt pathways [2, 13], JAK-STAT pathway [9]. For example, in human monocytes, S. typhimurium up-regulates the expression of IL-6 and IFN-γR1, thereby activating the JAK/STAT signaling pathway [14]. These studies indicate that S. typhimurium can affect host signaling pathways by regulating host gene expression, thereby affecting host inflammatory response, immune response, cell proliferation, cell apoptosis and cell cycle. MicroRNAs (miRNAs) are short (~ 22 nucleotide) RNA molecules encoded by the genome, playing critical roles in the eukaryotic host response to viral infections and extracellular pathogens. The Vogel.J team initially documented that the Let-7 miRNA family is downregulated following S. typhimurium infection in RAW264.7 and HeLa cells. This downregulation is hypothesized to occur via lipopolysaccharide (LPS) signaling through TLR4, resulting in decreased transcription of target molecules such as the proinflammatory cytokine IL-6 and the anti-inflammatory cytokine IL-10. This mechanism contributes to modulating the inflammatory response [15]. Maudet et al. documented that S. typhimurium (SL1344) downregulates the expression of the miR-15 family in HeLa cells. These reduced miRNAs sustain host cells in the G1 phase by modulating the overexpression of their target protein, Cyclin D1, thereby promoting the intracellular replication of S. typhimurium [16]. Zhang et al. reported that S. typhimurium induces the expression of miR-128 in intestinal epithelial cells, leading to decreased secretion of M-CSF by these cells [17]. Consequently, this reduction in M-CSF secretion attenuates macrophage infiltration caused by M-CSF [17]. Apart from the miR-15 family, miR-30c and miR-30e have also been implicated in the intracellular persistence of S. typhimurium. Additionally, ubc-9, the target molecule of these miRNAs, participates in the SUMOylation of host proteins [18]. Together, these investigations into miRNAs and pathogens underscore the complexity of the molecular mechanisms underlying the interaction between the enteric pathogen S. typhimurium and its host. Moreover, they suggest that other non-coding RNA molecules may also contribute significantly to host resistance against pathogen infections.
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Circular RNA (circRNA) represents a distinct RNA species characterized by its closed circular structure formed through RNA back-splicing. Unlike linear RNA, circRNA is resistant to RNA exonucleases, rendering it more stable and less prone to degradation, thus maintaining consistent expression levels [19, 20]. A variety of plants and animals have been widely found with abundant content, high conservation, tissue and developmental specificity circRNA [20]. circRNAs can regulate gene expression both transcriptionally and post-transcriptionally [21, 22]. Recent research indicates that circRNAs hold significant promise as novel clinical diagnostic markers for diseases and potential therapeutic targets. This underscores their potential applications in disease prevention and treatment [23, 24]. At present, increasing evidence highlights the crucial roles of circRNAs in diverse diseases, particularly in cancer. Major functions of circRNA have been reported as the sponge effect of miRNA or competing endogenous RNAs (ceRNAs) in cells to inhibit the activity of miRNAs and regulate genes expression [25]. For example, circ_0038718 enhances the malignant progression of colon cancer cells through modulation of the miR-195-5p/Axin2 signaling axis [26]. Furthermore, circRNA binding with proteins could form circRNA-protein structures, such as circHIPK3, which functions as a scaffold to recruit ubiquitin ligase, thereby preventing cardiac senescence by degrading HuR [27]. Moreover, certain circRNAs, such as circFAM53B which contains internal ribosome entry sites and encodes peptides, exhibit high affinity binding to HLA molecules [28]. They efficiently prime naive CD4 + and CD8 + T cells in an antigen-specific manner, thereby inducing antitumor immunity [28].
To date, research has demonstrated that circRNAs are intricately linked to a spectrum of human diseases, encompassing cancer, cardiovascular diseases, diabetes, and neurodegenerative disorders [29‐32]. Moreover, due to their high richness, tissue specificity, conservation and stability, circRNAs could be easily extracted from body fluids, which holds promise as both a biomarker for early disease detection and a novel target for therapeutic intervention [21]. For example, in investigations involving tumors of the digestive system, the dysregulation of circRNA may directly lead to the failure of a miRNA to play a normal regulatory function, resulting in the failure of tumor suppressor genes to play a tumor suppressor role, thus providing a foundation for the genesis and development of cancer. For instance, miR-7 could regulate some oncogenes expression, while circRNA CDRlas could affect tumorigenesis by regulating miR-7 function [33].
In this study, our objective was to elucidate the impact of S. typhimurium on cellular circRNAs. Using RNA sequencing, we identified circRNAs regulated by S. typhimurium in HCT116 cells infected with either S. typhimurium or PBS. Subsequently, we validated and analyzed the dysregulated expression of several circRNAs following S. typhimurium infection using bioinformatics tools. Among these circRNAs, hsa_circ_0001756, referred to as circHIPK2 in this study, originates from the HIPK2 gene locus and exhibits downregulation following S. typhimurium infection. We then further investigate the role of circHIPK2 in inflammation response and CRC progress. Besides, inhibition the expression of circHIPK2 impaired the cell oncogenic phenotypes. Inversely, overexpression circHIPK2 could aggregate the tumor phenotypes. Furthermore, the miR-124-3p was sponged by circHIPK2. Luciferase reporter assays and RNA pull-down experiments confirmed that circHIPK2 directly interacts with miR-125a-3p. Significantly, overexpression miR-124-3p could impaired cell motility induced by circHIPK2. In conclusion, our findings suggest that the S. typhimurium-modulated circHIPK2/miR-124-3p axis plays a pivotal role in colorectal cancer (CRC) pathogenesis, highlighting its potential as a molecular therapeutic target for CRC-associated diseases.
Materials and methods
Bacterial strains and cell lines
The E. coli O157: H7 strain was cultured overnight in LB medium at 37 °C with agitation (200 rpm) until reaching OD 600 nm of 0.6, resulting in density of approximately 5 × 10^8 CFU/mL. The S. typhimurium (SL1344) strain used in this study originated from the laboratory of Jun Sun. Non-agitated cultures were prepared by inoculating monoclonal bacteria into 5 mL of Luria-Bertani broth for 6 h, followed by transferring 0.05 mL of the stationary phase culture into 50 mL of fresh medium and incubating at 37 °C for 18 h, as previously described [8]. Bacterial heat-inactivation was performed according to an established method [34]. Briefly, bacteria were washed three times with sterile PBS, resuspended in the same buffer, and then heat-killed at 100 °C for 20 min to achieve complete inactivation. The inactivated bacterial preparations were aliquoted and stored at −80 °C until use.
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MC38 cells were from provided by Prof. Chuanping Si (Institute of Immunology and Molecular Medicine, Jining Medical College). HEK293T cells (RRID: CVCL_0063) and Human colonic epithelial HCT116 cells (RRID: CVCL_0291) obtained from the American Type Culture Collection (ATCC, Manassas, Virginia). The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, USA) and penicillin-streptomycin, and maintained at 37 °C. Additionally, all experiments were conducted using mycoplasma-free cells. HCT116 cells were treated with LPS (0, 0.5, 1, 10 µg/ml) for 6 h as previously described [35].
Animals and experimental protocol
Male C57BL/6 mice (16–22 g) aged 6–8 weeks were procured from the Animal Core Facility of Nanjing Medical University and housed under specific pathogen-free conditions. Experimental protocols involving mice were approved by the Nanjing Medical University Experimental Animal Welfare Ethics Committee (IACUC-1811064-7). For cell treatments, cells were harvested at sub-confluence, washed with phosphate-buffered saline (PBS), and resuspended in serum-free medium. Subsequently, 5 × 10^5 MC38 cells were subcutaneously injected into the upper left flank of C57BL/6 mice. Tumor size was measured with digital calipers every four days starting 10 days after injection, and tumor volume was calculated using the formula (length × width^2)/2. After five weeks, the mice were euthanized, and the MC38 cell tumors were excised.
RNA extraction and cDNA library construction
HCT116 cells were exposed to S. typhimurium (SL1344) in Hanks’ balanced salt solution (HBSS) at a concentration of 1.6 × 10^10 bacteria/ml or HBSS alone for 30 min. After washing three times with HBSS, cells were incubated at 37 °C for 6 h in media containing gentamicin (100 µg/ml), as previously described [36]. Total RNA was extracted from both S. typhimurium-infected and control cells treated with HBSS using TRIzol reagent (Invitrogen, USA). Each sample utilized 3 mg of RNA. Initially, ribosomal RNA was depleted using the Epicentre Ribo-zero™ rRNA Removal Kit (Epicentre, RZNB1056, USA). Subsequently, the rRNA-depleted RNA was utilized for library construction using the NEBNext® Ultra™ Directional RNA Library Prep Kit (NEB, USA). Finally, the resulting products were purified using the AMPure XP system, and library quality was assessed using the Agilent Bioanalyzer 2100.
RNA sequencing and data processing
The raw data underwent quality control using fastp to filter out low-quality reads. Subsequently, the clean reads were aligned to the hg38 reference genome using TopHat v2.0.9 [37]. Transcripts from each sample were assembled using Cufflinks (v2.1.1) [37]. The Cuffdiff software was employed to compute FPKMs (Fragments Per Kilobase of exon model per Million mapped reads) for circRNAs and coding transcripts in each sample. Differential expression analysis was conducted using Student’s t-test on log-transformed FPKM values. Genes meeting the criteria of FDR < 0.05 and fold change >= 2 were considered statistically significant.
Quantitative real-time PCR
Epithelial cells were lysed, and total RNA was extracted using TRIzol reagent. RNA integrity was confirmed by gel electrophoresis. Subsequently, RNA was reverse transcribed using the HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, R323-01, China) following the manufacturer’s instructions as described before [34]. The resulting cDNA was then subjected to quantitative real-time PCR using the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02, China) [34].
Preparation of RNA followed by treatment with RNase R
Total RNAs were extracted from the specified cells using TRIzol RNA Isolation Reagents (Life Technologies, Grand Island, NY, USA), following the manufacturer’s protocol. Subsequently, 2 µg of RNA was treated with 6 U of RNase R (Epicenter Technologies, USA) for 20 min at 37 °C as described before [38].
Luciferase activity assays
The CircHIPK2 sequence was inserted downstream of the Renilla luciferase gene in the psiCHECK-2 reporter vector (GeneCopoeia). MiR-124-3p, miR-124-3p mut, and negative control (Neg. Ctrl.) mimics were synthesized by GenePharma (Shanghai, China). HEK293T cells were co-transfected with the miRNA mimic and luciferase reporter DNA and harvested 24 h post-transfection. Relative Renilla luciferase activity was quantified using the Duo-Lite luciferase assay kit (Vazyme, China).
RNA pull-down experiments
RNA pull-down experiments were conducted following previously established methods, utilizing biotin-labeled miRNA mimic oligonucleotides [39, 40]. In brief, cells were lysed using a lysis buffer (Thermo Fisher Scientific, USA), followed by incubation with streptavidin magnetic beads (Thermo Fisher Scientific, USA) conjugated with biotin-labeled oligonucleotides. The binding RNAs were detected using RT-qPCR.
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Cancer cell migration and invasion assay
Cells in logarithmic growth phase were detached with trypsin, suspended in serum-free medium, and seeded into polycarbonate membrane chambers (8-mm pore size, Corning, CLS3422, USA). After washing with PBS and counting using a Neubauer hemocytometer, the cell suspension was adjusted to 1 × 10^5 cells/mL. Subsequently, 200 µL of the cell suspension was added to each chamber well and evenly distributed by gentle agitation. The lower chamber was filled with 500 µL of medium containing 10% FBS. Following a 72-hour incubation period, cells on the upper surface of the membrane were removed using a cotton swab. Cells that had migrated to the lower surface were stained with 0.1% crystal violet for 30 min, washed with PBS, and examined under a microscope. Images of three random fields (10× magnification) were captured per membrane, and migratory or invasive cells were quantified. Migration and invasion results were normalized to the initial cell number and treatment conditions. Each experiment was performed in triplicate.
Statistics
Unless specified otherwise, data are expressed as mean ± SEM from a minimum of three independent biological replicates. Differences between two groups of samples were analyzed using the Student’s t-test, while differences among three or more groups were assessed using one-way ANOVA. All statistical tests were two-sided, with P-values less than 0.05 considered statistically significant.
Results
Characterization of circrnas regulated by S. typhimurium through RNA-sequencing
To ascertain the cellular circRNAs influenced by S. typhimurium strain SL1344, we analyzed circRNA profiles in these cells using RNA-sequencing. The RNA-sequencing results identified differential expressed 10776 circRNAs, including 6664 newly discovered circRNAs (Fig. 1A). The large difference length between circRNAs detected in this study revealed that most circRNAs length were concentrated distribution between 100 bp −1000 bp (Fig. 1B). In addition, the detected circRNAs were distributed randomly on all the chromosomes, except for chromosome Y (Fig. 1C-D). As reported in previous studies [19], the circRNAs identified in this study generated by spliceosome-mediated back-splicing from mRNA CDS, antisense, 5’ UTR, 3’UTR and so on (Fig. 1E). Cluster analysis revealed that S. typhimurium-infected cells exhibited a distinct circRNA expression profile compared to those treated with PBS, identifying 190 circRNAs that were differentially expressed in S. typhimurium-infected cells (Fig. 1F-G). The Volcano Plots illustrated distinct distributions of circRNAs in S. typhimurium-infected cells compared to those in the PBS-treated groups (Fig. 1H). To assess the expression of S. typhimurium-regulated circRNAs identified from RNA-sequencing, RT-qPCR was employed for validation (Fig. 1I). We observed the most significant decrease in hsa_circ_0001756 expression in S. typhimurium-infected cells compared to the control (Fig. 1I). Based on the human reference genome, hsa_circ_0001756, positioned at Chr7:139415730–139,416,814, has been designated as “circHIPK2,” originating from the HIPK2 gene locus (Fig. 2A).
Salmonella typhimurium downregulated the expression of circHIPK2 independent on lipopolysaccharide treatment
As annotated in circBase (www.circbase.org), circHIPK2 originates from the HIPK2 gene through head-to-tail splicing of exon 2, spanning 1084 nucleotides (Fig. 2A). The back-splice junctions of circHIPK2 were confirmed by Sanger sequencing (Fig. 2B). To confirm the presence of circHIPK2, we designed specific primers targeting the circular RNA molecule. Circular HIPK2 (CircHIPK2), unlike its linear counterpart HIPK2, demonstrated resistance to RNase R digestion, highlighting its characteristic resilience to degradation by RNase enzymes, which typically target linear RNA molecules (Fig. 2C). Additionally, The RT-qPCR results indicated a decrease in circHIPK2 mRNA levels in S. typhimurium-treated cells, while the infection did not affect the expression of linear HIPK2 mRNA (Figs. 3A). Like most of circRNAs, nucleocytoplasmic separation experiment showed that circHIPK2 is mostly localized to the cytoplasm, not nucleus (Fig. 2D). Furthermore, circHIPK2 was found to downregulated in S. typhimurium-treated mouse intestinal organoids (Fig. 2E-F), indicating it is conserved circRNA in both genome of human and mouse.
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Lipopolysaccharides (LPS) play crucial roles in initiating and advancing inflammation, tissue damage, and tumorigenesis [41]. To investigate whether LPS or other bacterial components influence the microbiota-induced transcriptional changes of CircHIPK2, we exposed HCT116 cells to LPS and heat-treated preparations of gram-negative bacteria such as S. typhimurium and E. coli 0157. As a result, we observed a significant decrease in CircHIPK2 expression in cells treated with S. typhimurium compared to those treated with E. coli or heat-treated gram-negative bacteria (Fig. 2H-J). The data imply that S. typhimurium regulated expression of CircHIPK2 independent on LPS treatment.
Knockdown of circHIPK2 inhibited cytokines expression in HCT116 cells induced by S. typhimurium and inhibited HCT116 cells migration
Recent study showed that circRNAs promoted CRC epithelial cell inflammation, migration, tumor genesis and development [42‐44]. We asked whether downregulation of circHIPK2 was involved in this process. Gangling Tong et al. reported that circHIPK2 expression was elevated in colorectal cancer (CRC) patients compared to adjacent non-cancerous tissues. Furthermore, individuals with elevated circHIPK2 levels exhibited lower overall survival and disease-free survival rates [45]. To eliminate the influence of the parental gene HIPK2 on cellular malignant phenotypes associated with circHIPK2, we assessed the expression of HIPK2 after circHIPK2 knockdown. Our results indicated that silencing circHIPK2 did not alter the mRNA levels of its parental gene HIPK2(Fig. 3A). In addition, loss of circHIPK2 significantly inhibited epithelial cell cytokines expression like IL8, IL6 and GM-CSF induced by S. typhimurium (Fig. 3B-E). Recent study indicated S. typhimurium infection increased the risk of colorectal cancer [7‐9]. We found the inhibition of circHIPK2 expression impaired cancer cells migration (Fig. 3F-G). Together, the above result implicated that down-regulation of circHIPK2 modulated by S. typhimurium infection might influence the development of colorectal cancer.
miR-124-3p binds to circHIPK2 to inhibit cell migration
Circular RNAs (circRNAs) are recognized for their role as competitive endogenous RNAs (ceRNAs) that sequester miRNAs [46]. Considering the abundance subcellular localization of circHIPK2 in the cytoplasm (Fig. 2D), to identify that whether the circHIPK2 to sponge miRNA and further involve in tumor growth. the Basic Local Alignment Search Tool (BLAST) and circbank (http://www.circbank.cn/index.html) were used to predict potential miRNAs binding to circHIPK2. As a result, we identified several miRNAs that showed potential binding to circHIPK2(Fig. 4A). To validate the interaction between miRNAs and circHIPK2, we constructed a luciferase reporter by cloning the circHIPK2 sequence downstream of the Renilla luciferase gene in the psiCHECK-2 plasmid. This reporter contains miRNA-binding sites. Subsequently, we co-transfected the reporter plasmid with each of the three miRNA mimics into 293 T cells. We employed a dual luciferase reporter assay to validate the interaction between miRNA and circHIPK2. Specifically, miR-124-3p significantly reduced circHIPK2 luciferase activity (Fig. 4B). We created mutated miR-124-3p sequences to target the putative binding site within the circHIPK2 sequence for examination (Fig. 4C). The mutation in the seed sequence of miR-124-3p did not suppress the activity of the circHIPK2 reporter (Fig. 4D). We conducted a biotin-coupled RNA pull-down assay using biotin-labeled wild-type (WT) miR-124-3p and mutated miR-124-3p to investigate the interaction between circHIPK2 and miR-124-3p in cells. Results showed that circHIPK2 was enriched in the miR-124-3p WT group but not in the mutated group (Fig. 4E). After that, we investigated the impact of miR-124-3p on circHIPK2-mediated cell migration. Our findings revealed that overexpression of miR-124-3p in circHIPK2-overexpressing cells significantly inhibited circHIPK2-induced cell migration (Fig. 4F). Together, these data suggest that miR-124-3p binds to circHIPK2 to inhibit cell migration.
Overexpression circHIPK2 promoted MC38 cell proliferation, migration and subcutaneous tumor growth
Given that S. typhimurium could downregulated circHIPK2 expression in mouse intestinal organoids (Fig. 2F) and previous study indicated overexpression of miR-124-3 inhibit cancer cell migration, we further verify circHIPK2 tumorigenic function in vivo, we then overexpressed circHIPK2 in MC38 cell (Fig. 5A). As a results, stable expressing circHIPK2 induced cell proliferation and migration of MC38 (Fig. 5B-D). The circHIPK2 was overexpressed for 10 days and were subcutaneously injected into C57BL/6 mice (Fig. 5F). Six weeks post-injection, the tumor dimensions and mass in the stably expressing circHIPK2 groups were significantly greater compared to the control groups (Fig. 5E-G).
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Discussion
In recent decades, due to advancements in high-throughput sequencing technology, the function of circRNA in disease has been rapidly elucidated. Increasing evidence suggests that circRNAs play essential roles in tumorigenesis and development, including the regulation of protein metabolism signaling, recruitment of chromatin modifiers, control of cell cycle progression, and various other cellular functions [21, 47‐49]. As shown in Fig. 5, in this study, we reported that circHIPK2, a 1084 nt exon head-to-tail spliced non-coding circular RNA located at Chr7:139415730–139,416,814, is downregulated in S. typhimurium-infection. circHIPK2 acts as a sponge, binding to miR-124a-3p, thereby influencing cellular processes. Besides, overexpression of circHIPK2 promoted cell migration and viability, and tumor growth. Furthermore, we found the knockdown of circHIPK2 inhibit cytokines expression, such as IL8, IL6 and GM-CSF induced by S. typhimurium which showed high correlation with tumor development [50–52].
Numerous studies have shown that circRNAs could regulates inflammatory response to facilitate tumor development, progression, and metastasis. For instance, circATP5B functions as a miR-185-5p sponge, thereby promoting HOXB5 expression in glioma. This interaction transcriptionally regulates IL6 expression and facilitates the proliferation of GSCs through the JAK2/STAT3 signaling pathway [53]. EBV-encoded circBART2.2 exhibits high expression levels in nasopharyngeal carcinoma, where it enhances PD-L1 transcription by binding to the helicase domain of RIG-I. This interaction activates transcription factors IRF3 and NF-κB [54]. On the other hand, gut microbiota mediates the inflammatory diseases like IBD, CRC, liver inflammation, glucose intolerance induced obesity, and so on through short chain fatty acids (SCFAs), cell components inducing or inhibiting inflammasome activation and disrupt epithelial hypoxia [55‐58]. Diverse functions of circHIPK2 have been found in multiple diseases development and progression, including cancer, neuroinflammatory, pulmonary fibroblasts, and ischaemic stroke [39, 45, 59‐62]. Especially, fecal microbiota transplantation (FMT) from NLRP3-deficient mice markedly improved depressive-like behavior in neuroinflammatory knockout mice. This effect was associated with reduced circHIPK2 expression and suppressed astrocyte activation [59]. Recent study uncovered that circHIPK2 expression was found to be elevated in colorectal cancer (CRC) patients compared to adjacent non-cancerous tissues. Moreover, higher circHIPK2 expression correlated with reduced overall survival and disease-free survival rates in these patients [45]. Furthermore, circHIPK2 exhibits oncogenic properties in non-small cell lung cancer (NSCLC) by sequestering miR-1249-3p, thereby upregulating VEGFA and promoting angiogenesis. Additionally, it enhances the growth of cisplatin-resistant NSCLC cells in vivo [63]. Here, we identified circHIPK2 as a circRNA downregulated in S. typhimurium infected cells. circHIPK2 is the inaugural circRNA identified to be regulated by S. typhimurium-infection. Host responses diminish its expression to combat pathogenic bacterial infections, thereby reducing inflammation and suppressing tumor development, progression, and metastasis. Hence, our findings suggest that host-pathogen interactions could change the progression of tumorigenesis and development.
To investigate circHIPK2’s role in CRC, we identified the miRNAs regulated by circHIPK2. Mir-124-3p was a cancer suppressor miRNA in multiple cancers like hepatocellular carcinoma, cervical cancer, CRC and so on by means of ceRNA function [64‐66]. Down-regulated mir-124-3p in hepatocellular carcinoma (HCC) was abolished its inhibited ability of PRAS40 expression in HCC patients [64]. In cervical cancer, LINC00240 suppressed the activity of MHC class I-related chain (MIC)-A, thereby inhibiting the cytotoxic activity of natural killer T (NKT) cells through miR-124-3p sponging and upregulation of STAT3 expression [65]. Furthermore, subsequent investigations revealed reduced miR-124-3p expression in CRC tissues. Overexpression of miR-124-3p suppressed colorectal tumorigenesis by targeting PD-L1, reducing CRC cell proliferation, and inducing cell cycle arrest at the G1 phase through downregulation of c-Myc [66]. Consistent with previous studies, our findings also demonstrate that miR-124-3p exerts a tumor-suppressive role. However, the total expression level of miR-124-3p did not change significantly post-infection (Supplementary Figure S1), this finding supports our ceRNA hypothesis: S. typhimurium infection enhances the tumor-suppressive activity of miR-124-3p not by upregulating its transcription, but by downregulating circHIPK2, which reduces its sponge effect and consequently liberates more free miR-124-3p.
Our discovery that S. typhimurium infection inhibits colorectal cancer progression by downregulating circHIPK2 reveals circHIPK2 as a previously unexplored therapeutic target. Given the pathogenicity of wild-type S. typhimurium to humans, current research in bacterial-mediated cancer therapy primarily employs attenuated S. typhimurium strains [10, 11]. Our findings suggest a safer and more precise alternative strategy: developing siRNA or antisense oligonucleotide (ASO) therapies targeting circHIPK2 [67]. Such nucleic acid-based therapeutics could mimic the antitumor effects of S. typhimurium while avoiding the safety concerns associated with live bacteria, offering a new direction for precision medicine in colorectal cancer treatment [68].
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The limitation of our study didn’t find LPS, or other bacterial components attribute the microbiota-induced transcriptional alteration of circHIPK2. S. typhimurium type III secretion system (T3SS) will regulate a lot of signaling pathways through secreting effectors into host cells [69]. Hence, additional investigations are warranted to validate the mechanism underlying the reduction in circHIPK2 expression during S. typhimurium infection.
Fig. 1
S. typhimurium infection regulates circRNA expresion in HCT116 cells. (A) HCT116 cells were exposed to S. typhimurium for 6 h prior to RNA sequencing analysis; (B) x-axis: the length of circRNAs detected in this study. y-axis: abundance of circRNAs categorized by their respective lengths; (C and D) The distribution and count of identified circRNAs across chromosomes were analyzed; (E) The count of circRNAs originating from various genomic regions; (F) Number ofdifferential expression circRNAs; (G and H) Heatmap and volcanoplot and of differential expression circRNAs; (I) To confirm the circular RNA expression after S. typhimurium infection in HCT116 by qPCR. * P < 0.05, ** P < 0.01. NS, No significant difference
Fig. 2
circHIPK2 expression after treatment with bacteria (and heat-killed) and LPS. (A) circHIPK2 (hsa_circ_0001756) is transcribed from the second exon from HIPK2 gene; (B) The RT-PCR product of specific divergent primer was consistent with the sequence of circHIPK2 by Sanger sequencing; (C) qPCR analysis for the abundance of circHIPK2 and HIPK2 mRNA in HCT116 cells with or without RNase R; (D) The abundance of circHPK2 in HCT116 cells nuclear and cytoplasm, U6, GAPDH and β-actin as control; (E and F) S. typhimurium induced circHIPK2 downregulation in HCT116 cells (E) and mouse intestinal organoids (F); (G) circHIPK2 expression after treatment with LPS (0, 0.5, 1, 10 µg/ml) for 6 h; (H, I and J) circHIPK2 expression after treatment with E.coli O157:H7 (H), Heat-killed S. typhimurium (I), Heat-killed E.coli O157: H7 (J). *** P < 0.001. NS, No significant difference
Fig. 3
Knockdown of circHIPK2 inhibited cytokines expression in HCT116 cells induced by S. typhimurium and inhibited HCT116 cells migration. (A, B, C, D and E) The expression of HIPK2, circHIPK2, IL8, IL6 and GM-CSF in HCT116 cells after stable transfection of shRNA specifically targeting circHIPK2 or vector, then infected by S. typhimurium. (F and G) Cell migration ability of HCT116 were evalueated by transwell assay after stable transfection of shRNA specifically targeting circHIPK2 or vector. * P < 0.05, ** P < 0.01, *** P < 0.001. NS, No significant difference
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Fig. 4
miR-124-3p binds to circHIPK2 to inhibit cell migration. (A) BLAST and circBank were utilized to predict miRNAs that potentially bind to circHIPK2. (B) The circHIPK2 sequence was inserted downstream of the Renilla luciferase gene in the psiCHECK-2 plasmid to create the psiCHECK-2-circHIPK2 luciferase reporter plasmid. Mimics of three candidate miRNAs (20 nM) predicted by computational analysis, or a miRNA negative control (Neg. Ctrl.), were co-transfected with the psiCHECK-2-circHIPK2 luciferase reporter into HEK293T cells for luciferase reporter assays lasting 24 h. (C) The putative binding site of miR-124-3p within the circHIPK2 sequence, along with the mutagenesis of the target site in miR-124-3p (miR-124-3p mut), is depicted. (D) Luciferase assays were conducted using the psiCHECK-2-circHIPK2 reporter co-transfected with miR-124-3p mimic (miR-124-3p), miR-124-3p mutant mimic (miR-124-3p mut), or miRNA negative control (Neg. Ctrl.) in HEK293T cells for 24 h (E) RNA pull-down analysis was performed in HCT116 cells using biotin-labeled miRNA and its controls, including bio-Neg. Ctrl., bio-miR-124-3p, and bio-miR-124-3p mut. Specific primers were employed to assess the enrichment of circHIPK2. (F) Transwell migration assays were performed on HCT116 cells transduced with lentiviral circHIPK2 or control pCDH, followed by co-transduction with lentivirus-miR-124-3p or control pGLV10. Representative images were captured 24 h post-seeding (original magnification, ×100). (G) The quantification results of Transwell migration assay in (F). ** P < 0.01, *** P < 0.001. NS, No significant difference
Fig. 5
circHIPK2 promoted MC38 cell proliferation and subcutaneous tumor growth. (A) RT-qPCR analysis was conducted to measure circHIPK2 mRNA levels in MC38 cells transduced with circHIPK2 or control pCDH. (B) CCK-8 assay of MC38 treated as in (A). (C) Transwell migration assays were performed on MC38 cells transduced with lentiviral circHIPK2 or control pCDH, followed by co-transduction with lentivirus carrying miR-124a-3p (miR-124a-3p) or control pGLV10. Representative images were captured 24 h after seeding (original magnification, ×100). (D) The quantification results of Transwell migration assay in (C). (E) The quantification results of tumor size measured each 4 days after ten days MC38 subcutaneously injection. (F) Representative image of tumors. (G) The quantification results of tumor wight. (H) A schematic model illustrating the proposed mechanism by which S. typhimurium-regulated circRNAs modulate the circHIPK2/miR-124-3p axis in tumorigenesis. * P < 0.05, * * P < 0.01, * * * P < 0.001. NS, No significant difference
Declarations
Competing interests
The authors declare no competing interests.
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