MiR-326 downregulation is strongly associated with multidrug resistance (MDR) and has been identified as an adverse prognostic biomarker for pediatric acute lymphoblastic leukemia (pALL). The choice to study miR-326 as a tumor suppressor in cancer biology, particularly its regulation of apoptosis, drug resistance, and stemness, stems from its strong association with MDR and potential as a therapeutic target in pALL. The current study aimed to investigate, for the first time, the molecular mechanisms underlying the role of miR-326 in ALL, using Gene Ontology annotation network and multilayer network analysis. Our findings revealed that miR-326 exhibits a multifunctional anti-tumor behavior, affecting various aspects of drug resistance, stemness, and apoptosis in cancer, particularly in the context of ALL. Quantitative real-time PCR demonstrated downregulation of the ABC transporter mRNAs ABCC1 and ABCB1 but not ABCA3 in B-ALL cells transfected with miR-326 mimic, as confirmed by bioinformatic data. Western blot analysis showed a possible cross talk between miR-326 and P53 through the upregulation of Mdm2 and P53 proteins. The heightened functional activity of P53 was subsequently validated through the observed augmentation in levels of P21 and CCND1, alongside the evident disruption in the expression levels of Bcl-2, Bcl-xl, and Bax genes. Subsequently, the ceRNA network between miR-326 and LncRNAs was exhibited and the impact of exogenous miR-326 on the expression levels of its molecular sponges, H19 and SNHG1 was examined using RT-qPCR. Future studies will explore the potential impact of miR-326 on its targets, and how this may influence the development of novel therapeutic strategies for ALL.
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Introduction
Pediatric acute lymphoblastic leukemia (pALL) is a highly aggressive and life-threatening hematological cancer in children [1]. Despite notable progress in improving survival rates, multidrug resistance (MDR) remains a major challenge in pALL treatment [2]. Several mechanisms have been identified or proposed as contributory factors to cancer development, among which dysregulation of miRNAs emerges as a pivotal determinant [3]. miR-326, a tumor suppressor miRNA, plays a role in tumor induction through regulating ABC transporters and signaling molecules involved in apoptosis and cell cycle [4, 5]. Downregulation of miR-326 in pALL patients was associated with adverse outcomes, including overall survival and disease progression, particularly in cases of relapse and MDR [6].
The P53 signaling pathway plays a crucial role in the development of pALL. TP53 mutations are associated with poor treatment response and reduced overall survival rates in pALL. However, these mutations are rare in pediatric B-cell precursor pALL, occurring in only 2–4% of cases at diagnosis and 12% during relapse. Consequently, P53 inactivation is more likely due to regulatory abnormalities rather than TP53 mutations [7, 8]. Significantly, P53 plays a pivotal role in regulating cellular apoptosis through essential interactions with key effectors, such as Bcl-xl, Bcl-2, and Bax [9]. Furthermore, the induction of cell cycle arrest can be attributed to the P53-dependent activation of P21 or the suppression of CCND1 through its interference with the Hedgehog (Hh) signaling pathway, which is widely recognized as a crucial pathway in the development of cancer stem cells. Notably, our previous research effectively elucidated a remarkable correlation between the upregulation of SMO, a pivotal constituent of the Hh pathway, and the development of resistance in pALL [10].
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The objective of the present study was to provide, for the first time, in silico and empirical evidence substantiating the regulatory network of miR-326 in pALL and its possible cross talk with the tumor suppressor gene TP53. The outcomes have the potential to facilitate the identification of molecular therapeutic targets associated with pALL.
Materials and methods
Multilayer functional enrichment analysis of miR-326 interactions networks data collection
The PubMed database was searched for articles published until October 15, 2022, containing experimental data related to has-miR-326 (URS00000A939F_9606). The search utilized keywords such as "miR-326", "hsa-miR-326," and "microRNA-326," resulting in a total of 391 articles. Selection criteria were then applied, including reporter assay, western blot, and RT-qPCR evidence. The final selection process included articles that described experimentally verified interactions between miR-326 and mRNA transcripts of proteins, lncRNAs, or circRNAs, irrespective of cancer type or biological process. After screening, 207 articles were chosen for further annotation, all of which satisfied the specified selection criteria and provided sufficient experimental evidence of miR-326 interactions (Supplemental Table S1).
Functional enrichment analysis of miR-326: targets interaction network
The miR-326 target interaction network was constructed using 118 unique experimentally verified interactions from the selected 207 articles. Functional enrichment analysis was performed to gain insights into the biological processes associated with miR-326's target interactions.
For the analysis, Cytoscape plugins, BiNGO [11], and GOlorize [12] were utilized. The BiNGO settings selected for analysis included 'Cluster from Network', 'Overrepresentation', and 'No Visualization'. The Hypergeometric statistical test was employed to determine the significance of enriched categories, and the Benjamini & Hochberg false discovery rate (FDR) correction was applied to account for multiple testing. Enriched categories with an FDR-adjusted P value ≤ 0.05 were considered significant.
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The reference set for functional enrichment analysis consisted of human entities annotated to the Gene Ontology (GO) term 'biological process' or its descendants. The Gene Ontology file (go-basic.obo) (released 22nd December 2022) was obtained from the GO Consortium website (http://geneontology.org/page/download-ontology). Additionally, gene association files for human proteins (goa human.gaf) and ncRNAs (goa human rna.gaf) were downloaded from the European Bioinformatics Institute ftp site (ftp://ftp.ebi.ac.uk/pub/databases/GO/goa/HUMAN/) (released 4th May 2022) and merged into a single file named 'gene association.goa human'. No evidence codes were excluded during the analysis process to ensure comprehensive coverage.
Identifying miR-326 regulatory networks related to ALL
We conducted a kinases-TF regulatory network analysis to identify regulators of miR-326 target genes in ALL. The analysis included a transcription factor (TF) enrichment analysis using the ChEA tool from the Enrichr database and a kinase enrichment analysis using the KEA tool of Enrichr [13]. Both analyses used a statistical significance threshold of adjusted P value ≤ 0.05. Additionally, we obtained STRING network with ALL associated protein from the STRING disease query database with confidence score of 0.4 and maximum number of proteins 1500 [14]. By performing a Venn diagram analysis on the enriched TFs, kinases, and ALL-related networks, we identified a shared gene set, providing insights into potential regulatory mechanisms influenced by miR-326 in ALL. ClueGO plug-in was used to perform GO functional enrichment and KEGG pathway enrichment analysis of the differentially expressed genes (DEGs) in Cytoscape.
Visualization and analysis of the miR-326-associated ceRNA network
The miR-326-associated ceRNA network was visualized and analyzed using Cytoscape. The network integration included experimentally validated data from luciferase assays for circRNA: miR-326, lncRNA: miR-326, and miR-326: mRNA interactions. Additionally, protein–protein interactions from the STRING algorithm were incorporated to illustrate the interactions between miR-326 and circRNAs, lncRNAs, and mRNAs.
Cell culture and transfection
The RN95 cells, a TP53 wild-type precursor B-ALL cell line, were previously established and authenticated in our laboratory [15]. These cells were cultured in RPMI-1640 medium (Bio Idea, Tehran, Iran) supplemented with 10% heat-inactivated FBS (Fetal Bovine Serum) (Bio Idea, Tehran, Iran) and 1% penicillin/streptomycin, in a humidified incubator at 37 °C under 5% CO2. The electroporation system (Eppendorf Multiporator®, Germany) was used to transfect RN95 cells with 250 nM of the miR-326 mimic and scrambled miR-326 negative control (referred to as scrambled control, Exiqon, Denmark). In brief, during the exponential growth phase, cells were suspended in RPMI-1640 medium supplemented with 0.5% FBS and 2.5 × 106 cells were transfected under optimized conditions (250 V, 40 ms width and one pulse). Mock control cells were transfected in the same conditions without any oligonucleotide. Following electroporation, the transfected cells were placed in 6-well plates (2.5 × 106 cells/well) and allowed to recover in 4 ml of RPMI 1640 supplemented with 10% FBS at 37 °C and 5% CO2. The transfected cells were used for further analysis at 48 h after transfection.
RNA extraction, cDNA synthesis and quantitative real time-PCR (RT-qPCR)
Total RNA was extracted from the transfected cells, 48 h after transfection, using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. cDNA synthesis and RT-qPCR were then carried out to identify relative gene expression levels of miR-326 as previously described [6]. Primers for miR-326 and RNU6 small nuclear RNA (as endogenous control for data normalization) were purchased from Qiagen (Exiqon, Denmark) as part of their validated primer sets. The exact sequences are proprietary but have been validated by Qiagen for RT-qPCR experiments. After cDNA synthesis using PrimeScript™ RT reagent kit (Takara, Tokyo, Japan), RT-qPCR was performed to assess relative gene expression levels of ABCC1, ABCB1, ABCA3, CCND1, SMO, Bcl-xl, Bcl-2, Bax, SNHG1, and H19 using SYBR Premix Ex TaqII (Takara, Tokyo, Japan) and the specific primers as previously described [10, 16, 17]. GAPDH was used as a house keeping gene for data normalization (Supplementary Table S2).
Western blotting
Forty-eight hours after transfection, total protein was extracted using a homemade lysis buffer. The buffer was prepared by dissolving 144 g Glycine, 30 g Trisbase, and 10 g SDS in 800 ml dH2O, adjusting the volume to 1 L without pH adjustment (final pH ~ 8.3). The buffer was stored at room temperature for up to six months and diluted to 1 × with dH2O before use. The protein concentration was quantified by Bradford assay (Bio-Rad) according to the standard protocol.Equal amounts of total proteins (35 µg) were separated by SDS–PAGE (Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis) using gradient gel and transferred to nitrocellulose membranes (Amersham Biosciences). After blocking with 10% BSA (Bovine Serum Albumin) (w/v) in TBST (Tris-Buffered Saline with Tween-20) for 1 h, membranes were incubated at 4 °C overnight with the mouse anti-human primary antibodies, Mdm2 (1:300, Merck Millipore), p21 (1:100, Calbiochem), p53 (1:250, Santacruz Biotechnology, INC.), and Actin (1:250, Santacruz Biotechnology, INC). Membranes were then treated with goat anti-mouse IgG-HRP (Horseradish Peroxidase) secondary antibody (1:1000, Dako) at room temperature for 1 h. Finally, proteins were visualized on sensitive X-ray films in a dark room using ECL (Enhanced Chemiluminescence) solution (Bio-rad, Hercules, CA). The visualized bands were quantified and analyzed for intensity using Image J software (National Institute of Health, USA). Band intensities were quantified using ImageJ software. Regions of interest for each band were carefully selected to exclude extra signals, and normalization was performed against Actin as the loading control. Despite minor saturation in the Actin signal, the analysis was conducted rigorously and in line with standard practices in Western blot experiments.
Statistical analysis
The data in this study were presented as mean ± standard error of mean. To compare multiple groups, ordinary one-way analysis of variance (One-Way ANOVA) was employed. The findings represent the outcomes of two independent experiments conducted in duplicate. Student's t-test was performed for pairwise comparisons between two groups. GraphPad Prism 9.3.1 (GraphPad Software, San Diego, CA) software was utilized for data analysis. A significance level of P value < 0.05 was employed to determine statistical significance.
Results
Bioinformatic studies on the role of miR-326 in cancer biology
This research focused on investigating the regulation of signaling pathways targeted by miR-326 and analyzing gene ontology networks through a dedicated dataset of experimentally validated miR-326 targets. Using Cytoscape, we constructed an interaction network comprising 118 unique validated miR-326 target genes. By integrating this network with Gene Ontology (GO) term enrichment analysis using BiNGO and GOlorize plugins, we revealed 3035 regulated biological processes influenced by at least one miR-326 target mRNA. The comprehensive miR-326 interaction network, shown in Fig. 1, includes processes relevant to cancer behavior and associated signaling pathways (refer to Supplemental Table S3 for details). Interestingly, this regulatory network showed miR-326 multifaceted effects on drug resistance, directly regulating MDR-associated genes, and impacting signaling pathways and biological processes related to MDR.
Fig. 1
Contribution of miR-326 in cancer biology. The network created in Cytoscape and by utilizing GOlorize and BinGO plugins, enriched GO terms were identified (Supplemental Table S3). MiR-326 targets were annotated and clustered based on the cancer related processes they are in-volved in. Nodes are color-coded by annotated GO terms. Node size reflects the number of inter-actions it has with other nodes in this network, larger indicating more interactions (protein–protein interactions not shown). White nodes, named miscellaneous, correspond to the biological processes that were either not annotated or were not the intended focus of the researcher. Gray edges represent "physical association" where direct binding of miRNA to mRNA is confirmed through luciferase assay. Pink edges depict an "association" between miRNA and its target expression, which has been predicted using bioinformatics and validated through Western blotting and RT-qPCR after inducing miR-326 mimic or antagomiR into cells. EMT: epithelial-to-mesenchymal transition
Investigating the role of miR-326 in acute lymphoblastic leukemia
The regulatory network analysis identified specific transcription factors (TFs) and kinases that may govern miR-326 target genes in ALL, suggesting complex regulatory mechanisms at play in disease-specific contexts. To investigate the role of miR-326 in ALL, a protein–protein interaction (PPI) network was constructed using established databases dedicated to ALL within the STRING disease database (supplemental file S4). Considering the importance of hub genes in disease pathogenesis, we identified nodes with the highest degree, betweenness, and closeness centralities in the different layers of the ALL-regulatory networks. Through this analysis of the ALL-PPI network, 109 shared hub genes emerged among the top 10% of network genes based on their centrality scores. Notably, TP53 stood out as the highest score hub gene. These hub genes were found to be enriched in processes related to apoptosis through KEGG and GO enrichment analyses (Supplemental Table S5).
the miR-326 regulatory network was then extended through kinase and TF enrichment analyses, followed by a PPI network analysis. This comprehensive network included miR-326 validated target genes, controlling TFs, and TF-regulating kinases. Notably, certain TFs and kinases in this network could be direct miR-326 targets. Pivotal hub genes (MYC, TP53, CTNNB1, JUN, AKT1) were identified based on highest score of degree, betweenness, and closeness centralities (Supplemental Table S6). Among the 1501 DEGs in ALL, 126 genes were found to be under the direct or indirect influence of miR-326 (Fig. 2).
Fig. 2
Identification of genes regulated by miR-326 in acute lymphoblastic leukemia. A The kinases-TF-mRNA network related to Acute lymphoblastic leukemia were retrieved from STRING disease query database. TP53 emerged as the gene with the highest hub score. B The kinas-es-TF-mRNA network related to miR-326. C A Venn diagram illustrates the intersections between genes particular to ALL and genes associated with miR-326. 126 genes targeted by miR-326 have been identified in ALL
Validating the bioinformatics data regarding the impact of miR-326 in drug resistance
The bioinformatics data pertaining to miR-326 target interactions was evaluated through the transfection of RN95 cells with a miR-326 mimic, followed by the assessment of mRNA expression levels of key elements involved in multidrug resistance. The efficacy of transfection was confirmed showing 10.722-fold increase in the miR-326 expression levels in the miR-326 mimic transfected cells compared with negative controls (Fig. 3A). RN95 cells transfected with miR-326 mimic showed significantly reduced expression levels of ABCC1 and ABCB1, but not ABCA3 [0.355 (P = 0.006), 0.586 (P = 0.036) fold changes, respectively) (Fig. 2B). Interestingly, SMO expression levels, were additionally decreased in the positive control group (fold change = 0.795, P = 0.034) (Supplemental Figure S8).
Fig. 3
Exogenous expression of miR-326 and its impact on MDR gene expression. A The ex-pression of miR-326 was significantly increased in transfected RN95 cells with miR-326 mimic compared with untreated, Scrambled control, and Mock cells. B Real time analysis exhibited de-crease of ABCB1, ABCC1 at mRNA levels in the transfected RN95 cells with miR-326 mimic com-pared with scrambled control group. However, the ABCA3 expression was not significantly different between miR-326 mimic and Scrambled control groups. Untreated = RN95 cells without electroporation that were not subjected to any treatment, Mock = electroporated RN95 cells, Scramble control = RN95-electroporated cells transfected with Scrambled miR-326, miR-326 mimic = RN95-electroporated cells transfected with miR-326 mimic. Values are reported as the mean ± SEM of two separate experiments in triplicates (*, P < 0.05; **, P < 0.01; ***, P < 0.001)
The influence of miR-326 on P53 and its dependent signaling pathways
Considering the critical role of P53 in pALL pathogenesis [9], we focused on the most central TF identified in the networks of miR-326. A PPI network was constructed, including 98 proteins, consisting of miR-326 target genes, Mdm2, P53, and P21. P53 emerged as a hub protein with the highest connectivity (degree = 63) in the network (Fig. 4A). Western blot analysis confirmed that miR-326 upregulated the Mdm2/p53 feedback loop in the RN95 cells transfected with exogenic miR-326 mimic, leading to increased Mdm2 expression and modulation of p53/p21 expression (Fig. 4B). The expression profiling analysis of the anti-apoptotic Bcl-2 and Bcl-xl genes revealed a significant decrease in the transfected cells [0.428 (P < 0.0001) and 0.568 (P = 0.005) fold changes, respectively] and increased levels of the pro-apoptotic Bax gene (fold change = 0.008739, P = 0.0114). Moreover, CCND1 expression was reduced in the positive control RN95 transfected cells compared with the control cells (fold change = 0.850, P = 0.0015) (Fig. 4C). These data showed the increased functional activity of p53 followed by its miR-326 dependent upregulation.
Fig. 4
Activation of the P53 signaling pathway after induction of miR-326 mimic expression in RN95 cells. A PPI network of miR-326 target genes constructed using STRING database and Cytoscape software (v3.8.1). Black edges depict their relationship with P53, with hub genes located in the center. B Western blotting measured P53 pathway components in RN95 cells transfected with miR-326 mimic. β-actin served as a loading control. Comparisons were made between miR-326 mimic, miR-326 scramble, and Mock (electroporated RN95 cells) samples. Analysis revealed significant dysregulation in protein expression levels of MDM2, P53, and P21 in miR-326 mimic versus control groups. C The mRNA expression levels of Bax, Bcl-xl, Bcl-2 and CCND1 were significantly dysregulated in miR-326 mimic compared with scrambled control group. Two separate experiments were conducted in triplicate, and the data is presented as mean ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (D) The proposed miR-326 mechanism in improving drug resistance by inducing apoptosis, arresting the cell cycle, and suppressing the self-renewal attributes of cancer stem by activating P53 and its signaling pathway in pALL
miR326 related ceRNA network analysis and the impact of exogenous miR-326 expression on H19 and SNHG1 in RN95 cells
We constructed a competing endogenous RNA (ceRNA) network to facilitate the identification of potential biomarkers for novel cancer treatments (Fig. 5A). The network analysis highlighted key hub proteins, including Akt1, c-Myc, Egfr, Notch1, and Cyclin D1, with the highest interactions. Among non-coding regulatory RNAs, H19 and SNHG1 were selected for experimental analyses, according to their role in drug resistance across different cancers [18, 19] including ALL [17]. Results showed that elevated levels of exogenic miR-326 significantly downregulated H19 (fold change = 0.578, P = 0.0111) and SNHG1 (fold change = 0.578, P = 0.0103) in transfected cells compared to the control group (Fig. 5B).
Fig. 5
Reconstruction of experimentally validated miR-326 ceRNA network in cancers and modulation of Drug Resistance-Related lncRNAs by miR-326 in RN95 Cells. A The presented network was reconstructed using Cytoscape 3.9.1 software. The external ring of the gene network was generated by STRING on the basis of experimentally validated gene targets, with node size reflecting the number of interactions between genes in the network. B Real-time analysis showed decreased drug resistance-related lncRNA (H19 and SNHG1) in RN95 cells transfected with miR-326 mimic compared to the control group. Two separate experiments were conducted in triplicate, and the data is presented as mean ± SEM, * = P < 0.05
The present study offers a comprehensive examination of the functional implication of miR-326 in cellular processes and its regulatory networks within the context of ALL. The research is primarily focused on elucidating the gene ontology of miR-326 and its molecular targets, with two specific objectives in mind: first to illuminate the biological processes associated with drug resistance modulated by miR-326, and second, to demonstrate an overlap analysis of enriched functional gene sets (GO terms) linked to miR-326. The gene ontology analysis revealed a significant role for miR-326 in the regulation of signal transduction through the P53 class mediator. Notably, the P53 protein exerts a profound influence on various signaling pathways involved in multidrug resistance (MDR), including Hh signaling, apoptosis induction, cell cycle arrest, migration regulation, and adhesion modulation. Although there is no specific study that has directly explored the association between P53 and miR-326, the functional enrichment analysis of miR-326-target interactions suggested the involvement of miR-326 in the regulation of this signaling pathway (Figs. 1 and 2).
Gene ontology analysis of the miR-326 target interaction network also reveals involvement of ABCC1 in cell migration, a key indicator of epithelial-mesenchymal transition (EMT) and MDR (Fig. 1). As an ATP-dependent membrane transporter, ABCC1 not only enhances cancer cell progression but also contributes to MDR by facilitating cell migration [20]. Our RT-qPCR findings further support these observations, demonstrating a significant downregulation of drug resistance-related genes ABCC1 and ABCB1, followed by the upregulation of miR-326 (Fig. 3). Notably, cancer stem cells (CSCs) are recognized contributors to drug resistance and disease recurrence, primarily due to their heightened expression of drug resistance-associated genes, including ABC transporters [21‐24]. Besides, the level of cellular P53 significantly regulates the expression of ABCC1 and ABCB1 [25]. Such intricate interplay emphasizes the role of miR-326 in modulating key pathways associated with both cell migration and drug resistance, providing valuable insights into potential therapeutic interventions.
On the other hand, RT-qPCR demonstrated a downregulation of SMO, the pivotal member of the Hh pathway, in RN95 cells transfected with exogenous miR-326 mimic (Supplemental Figure S8). These results aligned with our prior investigations, which established a positive correlation between miR-326 and SMO expression levels in patients diagnosed with B-ALL [10]. Previous studies implicated increased Hh pathway activity and elevated ABCC1 expression in the development of drug resistance in multiple myeloma cell lines. Furthermore, it was highlighted that components of the Hh signaling pathway play a critical role in promoting the survival of cancer stem cells [26]. Collectively, it is hypothesized that the miR-326-mediated inhibition of ABC transporters could conceivably hinder disease recurrence and the retrieval of leukemia stem cells in B-ALL.
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The Western blot analysis was utilized to investigate the interplay between miR-326 and P53 in the treatment-resistant pALL cell line, RN95. The results demonstrated that the introduction of exogenous miR-326-mimic led to an elevation in the expression levels of P53 (Fig. 4). Consequently, the heightened activity of P53 on the cell cycle was validated through increased expression levels of Mdm2 and P21 proteins, as well as the downregulation of CCND1 gene (Fig. 4B, C). To substantiate the functional impact of P53 on apoptosis, the upregulation of apoptotic Bax and the reduced expression levels of antiapoptotic Bcl-2 and Bcl-xl genes were observed (Fig. 4C).
In silico studies revealed that miR-326 indirectly affects P53 by inhibiting the mRNA translation of AKT1/2 (Figs. 2 and 4D). The association between PI3K/AKT upregulation and unfavorable prognosis in pre-B-ALL, which includes drug resistance and decreased apoptosis, provides support for these findings [27]. Additionally, prior research has demonstrated a decrease in phosphorylated AKT following transfection with the miR-326 mimic in pancreatic cancer [28]. Consequently, we proposed that in the cytarabine-resistant RN95 cells, miR-326 may impede Mdm2 phosphorylation by inhibiting AKT1/2. This may prevent the nuclear translocation of Mdm2, thereby triggering P53 activation (Figs. 4C and 6). This hypothesis was substantiated by the analogous protective effect observed in drug-resistant ALL cells, where PTEN played a crucial role in safeguarding P53 activity through the suppression of PI3K, thus effectively inhibiting MDM2 [29] (Also shown in Fig. 4D). Further investigation is imperative to elucidate the underlying mechanisms by which miR-326 exerts its influence on P53, a pivotal regulator of apoptosis.
Finally, the results from RT-qPCR and in silico assays demonstrated that miR-326 exerts a downregulatory effect on the stability of H19 and SNHG1 in B-ALL cells (Fig. 5). It is noteworthy that H19 has been implicated in leukemia cell proliferation and apoptosis, while SNHG1 has been associated with drug resistance in liver cancer [30, 31]. In the context of CD133 + liver cancer stem cells, elevated H19 expression has been shown to correlate with increased levels of MDR1 and Gst-π proteins. Importantly, inhibition of H19 through the induction of ROS production has been observed to enhance apoptosis and impede the MAPK/ERK pathway, consequently reducing drug resistance in CD133 + CSCs [32]. Further investigation is required to elucidate the underlying mechanisms responsible for the reduced expression of H19 and SNHG1.
Although we acknowledge that additional experiments, such as the use of miRNA inhibitors, would have strengthened our findings, the lack of sufficient funding and resources in the current phase prevented us from performing these experiments. We aim to address this limitation in future studies to better validate the role of ABC transporters. Additionally, although the MDM2-p53-p21 pathway was explored, due to constraints related to experiment repetition and antibody availability, the data presented here is limited. We plan to investigate this pathway further in subsequent experiments, where we can fully optimize the methodology. Additionally, it is necessary to assess the in vivo efficacy of miR-326.
Conclusion
The current study represents the inaugural comprehensive investigation into the mutual interactions between miR-326, lncRNAs, and proteins involved in pediatric ALL, with a special focus on P53, utilizing bioinformatics design in conjunction with experimental analyses. Based on our novel findings, it is evident that the introduction of miR-326 mimic may exhibit a tumor suppressor role in ALL. This may be achieved through the inhibition of ABC transporters, facilitating apoptosis via intricate communication with p53, arresting cell proliferation, and suppressing the expression of genes associated with self-renewal (Fig. 6). Further experiments are required to determine the therapeutic significance of these findings in ALL patients.
Fig. 6
Suggested view of the multifaceted positive role of miR-326 in overcoming multidrug resistance by reducing the expression of genes and lncRNAs affecting drug resistance in B-ALL cell line which is established during disease relapse. → is the sign of activation or expression, ⊣ is the sign of inhibition or suppression, and
represents chemotherapy drugs, Gli A refers to activated Gli
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Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis
SMO
Smoothened, Frizzled Class Receptor
SNHG1
Small Nucleolar RNA Host Gene 1
TF
Transcription factor
TP53
Tumor Protein 53
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Die Zahl der diagnostizierten Depressionen steigt – gleichzeitig fehlen flächendeckend Therapieplätze. Für Betroffene ist die Hausarztpraxis eine erste Anlaufstelle. Doch wie kann dort der passende Raum entstehen? Allgemeinmedizinerin Prof. Dr. med. Anne Simmenroth und Psychologin Maike Krauthausen berichten, wie niederschwellig Hilfe angeboten werden kann.
Ergebnisse der Phase-II-Studie QUIWI nach längerem Follow-up sprechen weiter dafür, dass auch Personen mit neu diagnostizierter FLT3-ITD-negativer AML von einer Erweiterung der Standardchemotherapie um den zielgerichteten Wirkstoff Quizartinib profitieren.
Wiederholte Insulininjektionen an derselben Stelle können in eine Lipohypertrophie münden. Eine andere, ähnlich aussehende Folgekomplikation, die nicht von selbst verschwindet, ist seltener und weniger bekannt. Eine Kasuistik.
Fünf Jahre nach Diagnose eines frühen Brusttumors leidet jede siebte Langzeitüberlebende unter ausgeprägter Rezidivangst. Besonders betroffen sind Frauen mit Nebenwirkungen der endokrinen Therapie oder ohne eine solche Behandlung.
represents chemotherapy drugs, Gli A refers to activated Gli
