MicroRNA-26-5p functions as a new inhibitor of hepatoblastoma by repressing lin-28 homolog B and aurora kinase a expression.
ABSTRACT: Hepatoblastoma (HB) is the most common liver tumor in children. Despite recent improvements in treatment strategies, the survival of children with hepatoblastoma remains poor. In this study, we identified a novel role of microRNA-26a-5p (miR-26a-5p), lin-28 homolog B (LIN28B), Ras-related nuclear protein (RAN), and aurora kinase A (AURKA) in HB. The expression of LIN28B, RAN, and AURKA was significantly up-regulated in human HB livers and cell lines. Knockdown of LIN28B and RAN by small interfering RNAs inhibited HB tumor cell proliferation and foci formation. We also elucidated miR-26a-5p-mediated translational inhibition of LIN28B and AURKA in HB. Overexpression of miR-26a-5p markedly decreased LIN28B and AURKA 3'-untranslated region activities and protein expression and repressed HB cell proliferation and colony formation. In contrast, re-expression of LIN28B and AURKA rescued miR-26a-5p-mediated suppression of HB cell growth and clonality. Importantly, a decreased miR-26a-5p expression correlated with the poor outcome of patients with HB. Conclusion: miR-26a-5p is a newly identified repressor of HB growth through its inhibition of the oncogenic LIN28B-RAN-AURKA pathway. (Hepatology Communications 2018;2:481-491).
Project description:Hepatoblastoma (HB) is the most common hepatic neoplasm in childhood and the therapeutic outcomes remain undesirable due to its recurrence and metastasis. Increasing evidence shows that dipeptidase 1 (DPEP1) has pivotal function in tumorigenesis in multiple tumors. However, the expression pattern, biological function, and underlying mechanism of DPEP1 in HB have not been reported. Here we showed that DPEP1 was significantly upregulated and was associated with poor prognosis in HB patients. In vitro and in vivo assays indicated that silencing DPEP1 significantly suppressed HB cell proliferation, migration, and invasion, while DPEP1 overexpression exhibited the opposite effect. In addition, we identified that DPEP1 was a direct target of microRNA-193a-5p (miR-193a-5p). Functional experiments demonstrated that overexpression of miR-193a-5p significantly inhibited cell proliferation and invasion of HB cells, while the inhibitory effect could be reversed by DPEP1 overexpression. Moreover, miR-193a-5p was decreased in HB tumor tissues and associated with a poor clinical prognosis. Mechanistically, our results indicated that the miR-193a-5p/DPEP1 axis participated to the progression of HB via regulating the PI3K/Akt/mTOR (phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin) signaling. In conclusion, our findings suggest that the miR-193a-5p /DPEP1 axis might be a good prognostic predictor and therapeutic target in HB.
Project description:LIN28B has been identified as an oncogene in various tumor entities, including neuroblastoma, a childhood cancer that originates from neural crest-derived cells, and is characterized by amplification of the MYCN oncogene. Recently, elevated LIN28B expression levels were shown to contribute to neuroblastoma tumorigenesis via let-7 dependent de-repression of MYCN. However, additional insight in the regulation of LIN28B in neuroblastoma is lacking. Therefore, we have performed a comprehensive analysis of the regulation of LIN28B in neuroblastoma, with a specific focus on the contribution of miRNAs. We show that MYCN regulates LIN28B expression in neuroblastoma tumors via two distinct parallel mechanisms. First, through an unbiased LIN28B-3'UTR reporter screen, we found that miR-26a-5p and miR-26b-5p regulate LIN28B expression. Next, we demonstrated that MYCN indirectly affects the expression of miR-26a-5p, and hence regulates LIN28B, therefore establishing an MYCN-miR-26a-5p-LIN28B regulatory axis. Second, we provide evidence that MYCN regulates LIN28B expression via interaction with the LIN28B promoter, establishing a direct MYCN-LIN28B regulatory axis. We believe that these findings mark LIN28B as an important effector of the MYCN oncogenic phenotype and underline the importance of MYCN-regulated miRNAs in establishing the MYCN-driven oncogenic process.
Project description:Purpose:Aurora kinase A (AURKA), which belongs to the serine/threonine protein kinase family, has been identified as a key driver of the genesis and progression of diverse tumors. The aim of this study was to determine the clinical significance of AURKA in patients with hepatoblastoma (HB) and the effect of inhibiting AURKA in the HB cell line HuH-6. Methods:The expression of AURKA in HB tissue and adjacent normal liver tissue was detected by immunohistochemistry. Then, statistical analysis was performed to evaluate the association between AURKA expression and the clinicopathological characteristics of HB. The effect of AURKA knockdown on cell viability was assessed by CCK-8 assay. EdU and CCK-8 assays, Western blotting, flow cytometry, and transmission electron microscopy (TEM) were used to examine the effect of alisertib (ALS), a selective AURKA small-molecule inhibitor, on the cell cycle, proliferation, apoptosis, and autophagy in HuH-6 human hepatoblastoma cells. Results:The expression of AURKA was significantly higher in HB tissue than in adjacent normal tissue. Furthermore, high AURKA expression was associated with advanced Children's Oncology Group (COG) stage and tumor metastasis of HB. In vitro, AURKA knockdown significantly reduced the viability of HuH-6 cells, while ALS treatment significantly suppressed HuH-6 cell proliferation and induced G1-phase cell cycle arrest by reducing cyclin-D1 expression. Moreover, ALS promoted apoptosis and autophagy by decreasing the activity of p38 MAPK in HuH-6 cells. Conclusion:High expression of AURKA is a potential predictor of poor prognosis in HB patients. AURKA knockdown reduced the viability of HuH-6 cells, and ALS treatment inhibited cell proliferation and induced apoptosis and autophagy via the p38 MAPK signaling pathway. Our results suggest that AURKA may be a novel therapeutic target and ALS a potential therapeutic drug for the treatment of HB.
Project description:Circular RNAs (circRNAs), a novel class of endogenous RNAs, have been recently shown to participate in cellular development and several pathophysiological processes. The identification of dysregulated circRNAs and their function in cancer have attracted considerable attention. Nevertheless, the expression profile and role of circRNAs in human hepatoblastoma (HB) remain to be studied. In this report, we analyzed the expression prolife of circRNAs in HB tissues and identified circHMGCS1 (3-hydroxy-3-methylglutaryl-CoA synthase 1; hsa_circ_0072391) as a remarkably upregulated circRNA. <b>Methods:</b> The expression prolife of circRNAs in HB tissues were investigated through circRNA sequencing analyses. ISH and qRT-PCR assays were performed to measure the expression level of circHMGCS1. The effect of knocking down circHMGCS1 in HB cells <i>in vitro</i> and <i>in vivo</i> were evaluated by colony formation assay, flow cytometry, xenograft tumors assay and untargeted metabolomics assay. MRE analysis and dual luciferase assay were performed to explore the underlying molecular mechanisms. <b>Results:</b> HB patients with high circHMGCS1 expression have shorted overall survival. Knockdown of circHMGCS1 inhibits HB cells proliferation and induces apoptosis. CircHMGCS1 regulates IGF2 and IGF1R expression via sponging miR-503-5p, and affects the downstream PI3K-Akt signaling pathway to regulate HB cell proliferation and glutaminolysis. <b>Conclusions:</b> The circHMGCS1/miR-503-5p/IGF-PI3K-Akt axis regulates the proliferation, apoptosis and glutaminolysis of HB cells, implying that circHMGCS1 is a promising therapeutic target and prognostic marker for HB patients.
Project description:Hepatoblastoma is one of the malignant liver tumors in children. However, genetic mechanisms underpinning the initiation of hepatoblastoma remain largely unclear. The previous study showed that lin-28 homolog B (LIN28B) might play a role in the development of hepatoblastoma. To detect the association between LIN28B gene polymorphisms and hepatoblastoma risk in Chinese children, we conducted a five-center case-control study of 275 hepatoblastoma patients and 1018 cancer-free controls. Four potentially functional polymorphisms were genotyped using the Taqman method. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to evaluate the strength of the associations. We found that the rs314276 C>A polymorphism (AA vs. CC: adjusted OR=2.05, 95% CI=1.36-3.10, P=0.0006; AA vs. CA/CC: adjusted OR=2.11, 95% CI=1.43-3.12, P=0.0002) and rs9404590 T>G (GG vs. TT: adjusted OR=1.89, 95% CI=1.20-3.00, P=0.007; GG vs. TT/TG: adjusted OR=1.87, 95% CI=1.20-2.92, P=0.006) were associated with increased hepatoblastoma risk. Combination analysis of risk genotypes showed that patients with four risk genotypes had a higher chance of developing hepatoblastoma than carriers of 1 to 3 risk genotypes. Stratification analysis showed the significant association between the rs314276 AA genotype and hepatoblastoma risk in both age and sex groups, as well as clinical stages III+IV cases. The rs9404590 GG genotype was associated with hepatoblastoma risk in participants' ?17 months, in females, and for those with clinical stages III+IV disease. Furthermore, four risk genotypes confer higher hepatoblastoma susceptibility in both age and sex groups, as well as groups with clinical stages III+IV disease. Genotype-based gene expression analysis confirmed that the rs9404590 T>G polymorphism was significantly associated with altered LIN28B gene expression. We further validated our findings using false-positive probability analysis. This finding suggested that LIN28B gene polymorphisms may be associated with an increased predisposition to hepatoblastoma.
Project description:A more complete understanding of aberrant oncogenic signaling in neuroblastoma, a malignancy of the developing sympathetic nervous system, is paramount to improving patient outcomes. Recently, we identified LIN28B as an oncogenic driver in high-risk neuroblastoma. Here, we identify the oncogene RAN as a LIN28B target and show regional gain of chromosome 12q24 as an additional somatic alteration resulting in increased RAN expression. We show that LIN28B influences RAN expression by promoting RAN Binding Protein 2 expression and by directly binding RAN mRNA. Further, we demonstrate a convergence of LIN28B and RAN signaling on Aurora kinase A activity. Collectively, these findings demonstrate that LIN28B-RAN-AURKA signaling drives neuroblastoma oncogenesis, suggesting that this pathway may be amenable to therapeutic targeting.
Project description:Hepatoblastoma is the most common liver tumor of early childhood, which is usually characterized by unusual hypervascularity. Recently, long non-coding RNAs (lncRNA) have emerged as gene regulators and prognostic markers in several cancers, including hepatoblastoma. We previously reveal that lnRNA-TUG1 is upregulated in hepatoblastoma specimens by microarray analysis. In this study, we aim to elucidate the biological and clinical significance of TUG1 upregulation in hepatoblastoma. We show that TUG1 is significantly upregulated in human hepatoblastoma specimens and metastatic hepatoblastoma cell lines. TUG1 knockdown inhibits tumor growth and angiogenesis in vivo, and decreases hepatoblastoma cell viability, proliferation, migration, and invasion in vitro. TUG1, miR-34a-5p, and VEGFA constitutes to a regulatory network, and participates in regulating hepatoblastoma cell function, tumor progression, and tumor angiogenesis. Overall, our findings indicate that TUG1 upregulation contributes to unusual hypervascularity of hepatoblastoma. TUG1 is a promising therapeutic target for aggressive, recurrent, or metastatic hepatoblastoma.
Project description:Hepatoblastoma (HB) is the most common liver tumor of childhood and occurs predominantly within the first 3 years of life. In accordance to its early manifestation, HB has been described to display an extremely low mutation rate. As substitute, epigenetic modifiers seem to play an exceptional role in its tumorigenesis, which holds promise to develop targeted therapies and establish biomarkers for patient risk stratification.We examined the role of a newly described protein complex consisting of three epigenetic regulators, namely E3 ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), ubiquitin-specific-processing protease 7 (USP7), and DNA methyltransferase 1 (DNMT1), in HB. We found the complex to be located on the promoter regions of the pivotal HB-associated tumor suppressor genes (TSGs) HHIP, IGFBP3, and SFRP1 in HB cells, thereby leading to strong repression through DNA methylation and histone modifications. Consequently, knockdown of UHRF1 led to DNA demethylation and loss of the repressive H3K9me2 histone mark at the TSG loci with their subsequent transcriptional reactivation. The observed growth impairment of HB cells upon UHRF1 knockdown could be attributed to reduced expression of genes involved in cell cycle progression, negative regulation of cell death, LIN28B signaling, and the adverse 16-gene signature, as revealed by global RNA sequencing. Clinically, overexpression of UHRF1 in primary tumor tissues was significantly associated with poor survival and the prognostic high-risk 16-gene signature.These findings suggest that UHRF1 is critical for aberrant TSG silencing and sustained growth signaling in HB and that UHRF1 overexpression levels might serve as a prognostic biomarker and potential molecular target for HB patients.
Project description:BACKGROUND:N6-Methyladenosine (m6A) modification has been implicated in many biological processes. It is important for the regulation of messenger RNA (mRNA) stability, splicing, and translation. However, its role in cancer has not been studied in detail. Here we investigated the biological role and underlying mechanism of m6A modification in hepatoblastoma (HB). METHODS:We used Reverse transcription quantitative real-time PCR (RT-qPCR) and Western blotting to determine the expression of m6A related factors. And we clarified the effects of these factors on HB cells using cell proliferation assay, colony formation, apoptotic assay. Then we investigated of methyltransferase-like 13 (METTL3) and its correlation with clinicopathological features and used xenograft experiment to check METTL3 effect in vivo. m6A-Seq was used to profiled m6A transcriptome-wide in hepatoblastoma tumor tissue and normal tissue. Finally, methylated RNA immunoprecipitation (MeRIP) assay, RNA remaining assay to perform the regulator mechanism of MEETL3 on the target CTNNB1 in HB. RESULTS:In this research, we discovered that m6A modifications are increased in hepatoblastoma, and METTL3 is the main factor involved with aberrant m6A modification. We also profiled m6A across the whole transcriptome in hepatoblastoma tumor tissues and normal tissues. Our findings suggest that m6A is highly expressed in hepatoblastoma tumors. Also, m6A is enriched not only around the stop codon, but also around the coding sequence (CDS) region. Gene ontology analysis indicates that m6A mRNA methylation contributes significantly to regulate the Wnt/?-catenin pathway. Reduced m6A methylation can lead to a decrease in expression and stability of the CTNNB1. CONCLUSION:Overall our findings suggest enhanced m6A mRNA methylation as an oncogenic mechanism in hepatoblastoma, METTL3 is significantly up-regulated in HB and promotes HB development. And identify CTNNB1 as a regulator of METTL3 guided m6A modification in HB.
Project description:Hepatoblastoma (HB) is one of the most common hepatic malignancies in the pediatric population. HB are composed of a variety of tumors, which derived from different origins and had varying clinical outcomes. However, the unclear underlying mechanisms of HB limited exploring novel biomarkers and effective therapeutic targets. We searched microarray datasets on Gene Expression Omnibus (GEO) database and selected GSE75271 and GSE75283 datasets for comprehensive analysis. Weighted gene correlation network analysis (WGCNA) was employed to identify genes which were associated with tumor malignant phenotypes, including HB subtypes, Cairo classification and tumor stage. Coexpression analysis of identified genes was also performed and lncRNA-miRNA-mRNA network was finally conducted. Our results showed that a total of 22 lncRNAs, 13 miRNAs and 66 mRNAs were identified to be associated with tumor malignant phenotypes. Mechanistically, these molecules might promote the malignant phenotypes via regulating metabolic pathways. Among of them, 6 miRNAs (hsa-miR-106b, hsa-miR-130b, hsa-miR-19a, hsa-miR-19b, hsa-miR-20a and hsa-miR-301a), 8 lncRNAs (NR_102317, XR_245338, XR_428373, XR_924945, XR_929728, XR_931611, XR_935074 and XR_946696), and 6 mRNAs (EGFR, GAREM, INSIG1, KRT81, SAR1B and SDC1) were selected to conduct a lncRNA-miRNA-mRNA network. Taken together, our findings provide evidence for exploring molecular mechanisms of HB. Those identified malignant phenotype-associated molecules might be potential biomarkers and anti-cancer therapeutic targets in future.