Inducible Expression of Amphinase in Neuroblastoma Cells using Cre/LoxP System.

PURPOSE: To induce the expression of Amphinase, an antitumor ribonuclease from Rana pipiens oocytes, in neuroblastoma cell lines and build a foundation for mechanism study. METHODS: A loxP-cassette vector was constructed comprising a sequence of loxP -Puro-3∗polyA-loxP, followed by amphinase cDNA. The vector was transfected into neuroblastoma cell lines, SK-N-BE(2)-C, by Lipofectamine LTX. The transfected cells were selected by puromycin for two weeks. Polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) were conducted to verify that the loxP-cassette vector was stably transfected. The expression of amphinase was activated by the addition of Cre recombinase delivered by a lentiviral vector and identified by qPCR and Western blotting (WB). CCK8 assay and colony formation assay were conducted to check the effect of amphinase on cell proliferation. RNA sequencing (RNA-seq) was conducted to explore the targeted pathway of Cre/loxP-mediated amphinase and recombinant amphinase. RESULTS: Stably transfected cell clones were achieved through puromycin selection. After Cre recombinase was delivered to the cells, the loxP-flanked fragment was deleted and the expression of amphinase was induced, which were tested by PCR and qPCR. It was shown that cell proliferation was significantly inhibited by the amphinase mediated by the Cre/loxP system. KEGG enrichment and GSEA analysis indicated that amphinase had an impact on the ER function of neuroblastoma cells, which was identical to the effect of the recombinant amphinase. CONCLUSION: We successfully induce the expression of amphinase in neuroblastoma cell lines via Cre/loxP system. The Cre/loxP-mediated amphinase had a similar antitumor mechanism to the recombinant amphinase, providing a powerful tool for the mechanism study of amphinase.

Discovery, evaluation and mechanism study of WDR5-targeted small molecular inhibitors for neuroblastoma.

Neuroblastoma is the most common and deadliest tumor in infancy. WDR5 (WD Repeat Domain 5), a critical factor supporting an N-myc transcriptional complex via its WBM site and interacting with chromosome via its WIN site, promotes the progression of neuroblastoma, thus making it a potential anti-neuroblastoma drug target. So far, a few WIN site inhibitors have been reported, and the WBM site disruptors are rare to see. In this study we conducted virtual screening to identify candidate hit compounds targeting the WBM site of WDR5. As a result, 60 compounds were selected as candidate WBM site inhibitors. Cell proliferation assay demonstrated 6 structurally distinct WBM site inhibitors, numbering as compounds 4, 7, 11, 13, 19 and 22, which potently suppressed 3 neuroblastoma cell lines (MYCN-amplified IMR32 and LAN5 cell lines, and MYCN-unamplified SK-N-AS cell line). Among them, compound 19 suppressed the proliferation of IMR32 and LAN5 cells with EC50 values of 12.34 and 14.89 µM, respectively, and exerted a moderate inhibition on SK-N-AS cells, without affecting HEK293T cells at 20 µM. Analysis of high-resolution crystal complex structure of compound 19 against WDR5 revealed that it competitively occupied the hydrophobic pocket where V264 was located, which might disrupt the interaction of MYC with WDR5 and further MYC-medicated gene transcription. By performing RNA-seq analysis we demonstrated the differences in molecular action mechanisms of the compound 19 and a WIN site inhibitor OICR-9429. Most interestingly, we established the particularly high synergy rate by combining WBM site inhibitor 19 and the WIN site inhibitor OICR-9429, providing a novel therapeutic avenue for neuroblastoma.

Rapamycin induces autophagy and apoptosis in Kaposiform hemangioendothelioma primary cells in vitro.

BACKGROUND: Rapamycin has been recommended to treat Kaposiform hemangioendothelioma (KHE) with Kasabach-Merritt phenomenon (KMP), but the underlying mechanism of the clinical effect has not been established. Therefore, we determined rapamycin cytotoxicity on KHE cells in vitro and the underlying mechanism. METHODS: KHE primary cells were derived from a tumor specimen and treated with rapamycin. Immunofluorescence was applied to identify the cells. Cell viability was measured using the Cell Counting Kit-8 (CCK-8) assay. Cell cycle and apoptosis were assessed using flow cytometry (FCM). Western blots (WB) were performed to determine phosphorylation of mammalian target of rapamycin (mTOR), p70 S6 kinase (S6K1), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), as well light chain 3 (LC3) expression. RESULTS: Rapamycin inhibited the growth of KHE primary cells in a dose- and time-dependent manner. Cell cycle progression was arrested in the G0/G1 phase and apoptosis was induced. WB results showed that LC3-II/I expression was significantly elevated in KHE primary cells treated with rapamycin, while the level of p-mTOR, p-S6K1, and p-4E-BP1 expression was reduced. LC3 fluorescent spots were increased in the rapamycin treatment group. CONCLUSIONS: Rapamycin inhibited KHE primary cell proliferation, induced apoptosis and autophagy, and blocked the mTOR signaling pathway.

LINC01296 promotes neuroblastoma tumorigenesis via the NCL-SOX11 regulatory complex.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Long non-coding RNA LINC01296 has been shown to predict the invasiveness and poor outcomes of patients with NB. Our study validated its prognostic value and investigated the biological function and potential mechanism of LINC01296 regulating NB. Results illuminated that LINC01296 expression was significantly correlated with unfavorable prognosis and malignant clinical features according to the public NB database. We identified that silencing LINC01296 repressed NB cell proliferation and migration and promoted apoptosis. Moreover, LINC01296 knockdown inhibited tumor growth in vivo. The opposite results were observed through the dCas9 Synergistic Activation Mediator System (dCas9/SAM) activating LINC01296. Mechanistically, we revealed that LINC01296 could directly bind to nucleolin (NCL), forming a complex that activated SRY-box transcription factor 11 (SOX11) gene transcription and accelerated tumor progression. In conclusion, our findings uncover a crucial role of the LINC01296-NCL-SOX11 complex in NB tumorigenesis and may serve as a prognostic biomarker and effective therapeutic target for NB.

circRNA-TBC1D4, circRNA-NAALAD2 and circRNA-TGFBR3: Selected Key circRNAs in Neuroblastoma and Their Associations with Clinical Features.

OBJECTIVE: The roles of circRNAs in neuroblastoma (NB) are unclear. We used next-generation sequencing to detect the circRNA expression profiles in NB to identify the key circRNAs and analyzed the relationships between the circRNAs and clinical features. METHODS: Five paired neuroblastoma tumor and adjacent normal fetal adrenal medulla samples were collected for high-throughput RNA sequencing. Bioinformatics analysis was performed for functional annotation of the host genes of differentially expressed circRNAs. Validation of dysregulated circRNAs was performed by real-time quantitative reverse transcription polymerase chain reaction. The relationships between the key circRNAs and clinical features were analyzed. In addition, overexpression of key circRNAs in an NB cell line, as well as cell proliferation assays, colony formation assays and cell migration assays, was conducted to investigate the biological functions of key circRNAs. RESULTS: A total of 4704 differentially expressed circRNAs were found, including 2462 up-regulated and 2242 down-regulated circRNAs. According to our previous studies, the predicted target circRNAs of miR-21 involved in tumorigenic signaling pathways were selected, including circRNA-TBC1D4, circRNA-NAALAD2 and circRNA-TGFBR3. These circRNAs were associated with clinical features, and the circRNA expression was significantly lower (P < 0.05) in the NB tissues than in normal adrenal tissues. Overexpression of circRNA-TBC1D4 promotes NB cell migration, but not proliferation and colony-formation in vitro. CONCLUSION: We suggest that circRNA-TBC1D4, circRNA-NAALAD2 and circRNA-TGFBR3 may be cancer suppressor genes, which act by sponging miR-21 in NB. Further investigations are needed to elucidate the underlying mechanism.

Overexpression Prox1 in HemECs resembles Kaposiform hemangioendothelioma and cytotoxicity of sirolimus in vitro.

BACKGROUND: Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor that occurs in children. Prox1 is a specific lymphatic marker for KHE. We intended to establish a Prox1 transgenic cell line resembling KHE and investigate the mechanism of sirolimus in treating KHE. METHODS: Prox1 was stably expressed in infantile hemangioma cell HemECs. RT-qPCR and Western blot were conducted to measure the expression of target genes. CCK-8, EdU assay, and cell cycle analysis were conducted to detect cell proliferation. Wound healing and transwell assay were used to evaluate cell migration and invasion. RESULTS: Both mRNA and protein levels of Prox1, LYVE-1, Podoplanin were upregulated in Prox1+ HemECs. An acceleration of cell growth and a rise in migration and invasion were observed with Prox1 overexpression. Sirolimus inhibited cell proliferation, promoted apoptosis and led to G1 phase arrest in Prox1+ HemECs. The expression of p-mTOR, p-4EBP1, and p-P70S6K decreased and the ratio of LC-3 II/LC-3 I elevated after treatment of sirolimus. CONCLUSIONS: Stable overexpression of Prox1 in HemECs induced a lymphatic endothelial reprogramming, and enhanced aggressive biological effects, partly resembled the invasion of KHE, and could serve as a novel model for KHE. Sirolimus may block mTOR-mediated pathways and induced autophagy in KHE.

Clinical features, treatment, and outcomes of bilateral Wilms' tumor: A systematic review and meta-analysis.

BACKGROUND: Wilms' tumor(WT) is the most common malignant renal tumor of childhood. Despite the good prognosis of WT, bilateral Wilms' tumor (BWT) still has a poor outcome. We systematically reviewed the literature on BWT, aiming to define its clinical features, treatment, and outcomes. METHODS: PubMed, OVID EMbase, Web of Science, and Cochrane Library were systematically searched for studies published from 1980 to 2017. Case series and comparative studies reported clinical data of BWT patients were included. RESULTS: A total of 32 studies comprising 1457 patients were retained for primary outcome. Hemihypertrophy, cryptorchidism, and Beckwith-Wiedemann syndrome(BWS) are the most common congenital anomalies and syndrome. 86% of patients had favorable histology (FH). Patients with local stage I or II accounted for 64%, and 12.6% had metastasis at diagnosis. Bilateral nephron-sparing surgery (NSS) was achieved in 33.8%. Recurrence and renal failure occurred in 20% and 8%. The overall survival (OS) was 73%. In comparative studies, OS of patients undergoing bilateral NSS was similar to that of other operation types. CONCLUSION: Prognosis of BWT has been improved but is significantly poorer than WT. Bilateral NSS was recommended by most centers to preserve more renal volume. However, finding a balance between retaining renal function and avoiding recurrence remains a question. TYPE OF STUDY: Systematic review. LEVEL OF EVIDENCE: Level IV.