Immunohistochemical Analysis of mTOR Pathway-Related Proteins in Kaposiform Hemangioendothelioma.

BACKGROUND: Mammalian target of rapamycin (mTOR) inhibitors have been shown to have excellent effects in the management of kaposiform hemangioendothelioma (KHE); however, the mechanism of action is unclear. This study identified the expressions of mTOR pathway-related proteins in different vascular tumors to provide insight into the pathogenesis of KHE. METHODS: We retrospectively reviewed the pathologic specimens of 30 patients (KHE, 15; tufted angioma [TA], 5; infantile hemangioma [IH], 5; and lymphatic malformation [LM], 5). The immunohistochemical expression of mTOR-related proteins tuberous sclerosis complex 2 (TSC2), phosphatase and tensin homologue (PTEN), phosphorylated eukaryotic translation initiation factor 4E binding protein 1 (p-4EBP1), phosphorylated mTOR (p-mTOR), and phosphorylated ribosomal protein S6 kinase B1 (p-P70S6K) were analyzed using Image-Pro Plus software. KHE had the following pattern of expression in the spindle vascular endothelial cells: TSC2 (-); PTEN (-); p-4EBP1 (+); p-mTOR (+); and p-P70S6K (+). RESULTS: All 3 patients treated with sirolimus had good responses. The TA results were similar to KHE with no significant differences (p-4EBP1: p = 0.0687; p-mTOR: p = 0.0832). The expressions of TSC2, PTEN, p-4EBP1, p-mTOR, and p-P70S6K were negative or weakly positive in IH with a statistically significant difference compared to KHE (p-4EBP1: p < 0.001; p-mTOR: p < 0.001; p-P70S6K: p < 0.001). LM had no significant differences when compared to KHE. CONCLUSIONS: The absence of TSC2 and PTEN caused abnormal activation of the mTOR signaling pathway and may be involved in the pathogenesis of KHE. The expression of mTOR-related proteins in TA and LM was similar to KHE, unlike IH. The KHE pattern of expression [PTEN (-), TSC2 (-), p-mTOR (+), p-P70S6K (+), and p-4EBP1 (+)] suggested that sirolimus may be a good therapeutic choice.

Retroperitoneal kaposiform hemangioendothelioma complicated by Kasabach-Merritt phenomenon and obstructive jaundice: A retrospective series of 3 patients treated with sirolimus.

We present a retrospective case series of 3 patients with retroperitoneal kaposiform hemangioendothelioma (KHE) complicated by Kasabach-Merritt phenomenon (KMP) and biliary obstruction. We found sirolimus to be a safe and effective treatment for these patients who were refractory to other treatment modalities. However, our patients were slow to respond in comparison to published reports of sirolimus use for KHE without biliary obstruction. We postulate that therapeutic serum levels of sirolimus may be affected by biliary obstruction and improved with surgical alleviation of the obstruction.

Clinical characteristics and prognosis of adrenocortical tumors in children.

PURPOSE: The purpose of this study was to review the clinical characteristics and prognosis of children with adrenocortical tumors (ACT). METHODS: We retrospectively reviewed the medical records of 28 patients with ACT at our hospital between March 2010 and March 2017. RESULTS: The main clinical presentations were sexual prematurity (n = 17) and Cushing's syndrome (n = 15). All patients without metastasis underwent complete resection by laparotomy (n = 19) or laparoscopic surgery (n = 9). Pathological diagnosis confirmed adrenocortical carcinomas (ACC, n = 12) and adrenocortical adenomas (ACA, n = 16). Dehydroepiandrosterone (939.4 ± 148.2 µg/dl vs 630.9 ± 376.3 µg/dl; p = 0.031) and testosterone (235.7 ± 89.1 ng/dl vs 164.6 ± 47.5 ng/dl; p = 0.012) were significantly increased in ACC compared with ACA. The ACC tumor volumes were larger than those in ACA (107.5 ± 69 vs 25.5 ± 23.1 cm3; average diameter 6 cm vs 4 cm p = 0.001) and the immunochemical expression of Ki-67 was higher in ACC than in ACA (30.2 ± 22.7 vs 9.9 ± 4.9 p = 0.013). The mean follow-up of patients with ACA was 40 ± 23 months without recurrence. Seven patients with ACC had postoperative distant metastases and five patients died within 2 years. Five patients with ACC survived with a median follow-up of 27 months. The 2-year overall survival was 44.6%. CONCLUSIONS: Patients with ACC had significantly larger tumor volumes than those with ACA. The discordantly elevated serum levels of sexual corticosteroid hormones and lactate dehydrogenase may predict the malignant nature of these tumors. The prognosis of patients with ACA was good, while those with ACC had high postoperative metastasis and mortality rates.

Epidermal growth factor receptor is overexpressed in neuroblastoma tissues and cells.

Neuroblastoma is the most common abdominal malignant tumor in childhood. Immunotoxin (IT) that targets the tumor cell surface receptor is a new supplementary therapeutic treatment approach. The purpose of this study is to detect the expression of epidermal growth factor receptor (EGFR) in neuroblastoma cell lines and tissues, and to explore if IT therapy can be used to treat refractory neuroblastoma. The EGFR expression in human neuroblastoma tissue samples was detected by immunohistochemistry staining. The positive rate of EGFR expression was 81.0% in neuroblastoma tissue and 50.0% in gangliocytoma, respectively, but without statistical significance between them (P > 0.05). The positive rate of EGFR expression in favorable type and unfavorable type was 62.5% and 92.3%, respectively, but they were not statistically different (P > 0.05). Results from pre-chemotherapy and post-chemotherapy samples showed that there was no significant statistical difference (P > 0.05) between them in the EGFR expression. Furthermore, the EGFR expression levels in five neuroblastoma cell lines were measured using cell-based ELISA assay and western blot analysis. The results showed that the expression of EGFR was higher in KP-N-NS and BE(2)-C than those in other cell lines. Our results revealed that there are consistent and widespread expressions of EGFR in neuroblastoma tissues as well as in neuroblastoma cell lines, suggesting that it is possible to develop future treatment strategies of neuroblastoma by targeting at the EGFR.

Analysis of clinical features and outcomes for inflammatory myofibroblastic tumors in China: 11 years of experience at a single center.

PURPOSE: Inflammatory myofibroblastic tumor (IMT) is a rare benign neoplasm. The purpose of this study was to review the clinical characteristics, imaging and pathological features, and outcomes of children with IMTs from a single center in China. METHODS: A retrospective file review was conducted involving 23 cases of pathologically confirmed IMTs treated at the Children's Hospital between April 2003 and April 2014. RESULTS: The tumor locations included multiple anatomic sites, as follows: abdomen or pelvis (n = 17); lungs (n = 2); head and neck (n = 1); trunk (n = 1); and extremities (n = 2). The tumors were associated with various clinical presentations. The predominant symptoms included an anemic appearance, fevers, and an asymptomatic mass. Computed tomography scanning showed solid, heterogeneous, well-demarcated masses; the appearance of enhancement was variable. MRI appeared hypointense on T1-weighted images and hypointense or hyperintense on T2-weighted images. Immunohistochemical staining revealed anaplastic lymphoma kinase was negative in 11 of 13 cases tested. One patient quit treatment for the unresectable mass after biopsy and died 2 years later, and another patient with incompletely resection is alive at 30 months following chemotherapy. The remaining 21 cases had complete resections; one patient died due to a recurrence, and the other 20 patients survived and were tumor free. The follow-up ranged from 7 to 141 months, with a mean of 56 months. The 3-year OS was 88 % (95 % CI, 57-97 %). CONCLUSIONS: IMT is a benign neoplasm that rarely presents with malignant features. Complete resection is curative in most patients. ALK+ is variable for diagnosis. Close follow-up is necessary for patients who undergo surgical resection.

Successful treatment of kaposiform lymphangiomatosis with sirolimus.

Generalized lymphatic anomaly (GLA) is a rare and often fatal congenital lymphatic disorder that also commonly affects bone. Kaposiform lymphangiomatosis (KLA) is a novel subtype of GLA with poor prognosis and no proper treatment guidelines. A 9-year-old male with recurrent pleural effusion was clinically diagnosed as KLA. Following sirolimus therapy at a dose of 0.8 mg/m(2) twice daily, pleural effusion was significantly decreased and the general status of the patient markedly improved. The clinical course indicates that sirolimus may present an effective therapeutic option in KLA. Moreover, KLA should be considered in differential diagnosis for cases of GLA with coagulopathy.

Steroid-resistant kaposiform hemangioendothelioma: a retrospective study of 37 patients treated with vincristine and long-term follow-up.

BACKGROUND: Kaposiform hemangioendothelioma (KHE) with Kasabach-Merritt phenomenon (KMP) still remains a particular and life-threatening disease. The purpose of this study was to evaluate the efficacy of vincristine (VCR) and the possibility of replacement with steroids in the treatment of steroid-resistant KHE with KMP. PROCEDURE: We retrospectively reviewed the medical records of 37 patients with steroid-resistant KHE who were treated at the Children's Hospital of Fudan University between March 2003 and March 2013. RESULTS: The age of initial diagnosis with KHE was between 1 day and 10 months. Eight and 29 cases were located in the superficial and deep soft tissues, respectively. Thirty-seven KHE lesions did not respond well to steroids before starting VCR treatment. Twenty-six KHE lesions achieved complete remission, with platelet counts reaching normal levels within7.6 ± 5.2 weeks after VCR treatment. The vascular tumor began to decrease in size or soften at an average of 4.9 ± 2.7 weeks. Two KHE lesions had partial responses and one remains in treatment. Eight KHE lesions had no apparent response to VCR and thus received other therapies. Twenty-eight patients have ended treatment with VCR; the average length of treatment was 31.2 ± 5.9 weeks. Side effects occurred in 48.6% of patients who received steroids, and in 11.4% of patients who received VCR treatment. The mean follow-up time was 3.5 years. No recurrences have been reported. CONCLUSIONS: VCR appears to be a safe and effective treatment option in the management of steroid-resistant KHE with KMP, and recommended as first-choice treatment.

Recreational ball games are effective in improving social communication impairments among preschoolers diagnosed with autism spectrum disorder: a multi-arm controlled study.

BACKGROUND: This study aimed to compare the effects of two 12-week training intervention experimental ball games combined with standard behavioral rehabilitation against a control group solely utilizing standard behavioral rehabilitation on social communication impairments (SCI) in preschool children with Autism Spectrum Disorder (ASD). METHODS: A multi-arm controlled study design was implemented, involving 41 children diagnosed with ASD (mean age: 4.99 ± 0.76 years). 41 participants were randomized assigned to two experimental groups and a control group, The experimental group carried out ball combination training program group (BCTP) and mini-basketball training program group (MBTP) on the basis of routine behavioral rehabilitation, which underwent 12-week training interventions 5 times a week. The control group (n = 14) received only standard behavioral rehabilitation. Evaluations were conducted before and after interventions using the Social Responsiveness Scale, Second Edition (SRS-2). RESULTS: The results suggest that both 12-week interventions, BCTP, and MBTP, led to significant improvements in social communication impairment among children with ASD (p < 0.05). Despite enhancing the overall scores on the SRS-2, these interventions displayed varying impacts across different sub-dimensions. BCTP primarily exhibited significant enhancements in social awareness and behavior pattern (p < 0.05), whereas MBTP significantly improved social cognition and social communication (p < 0.05). Both interventions showed slight improvements in social motivation. CONCLUSIONS: The utilization of recreational ball games has showed to be effective in decreasing the impairment levels of children with ASD, while the control group experienced a worsening of outcomes. This suggests that irrespective of the specific ball game strategy employed, both can be employed on a weekly basis to complement standard behavioral rehabilitation and enhance the ability to improve the quality of life for children diagnosed with ASD. TRIAL REGISTRATION: The trial is retrospectively registered on the Chinese Clinical Trial Registry (ChiCTR1900024973;August 5, 2019).

CAMK2G Promotes Neuronal Differentiation and Inhibits Migration in Neuroblastoma.

PURPOSE: Neuroblastoma (NB) originates from differentiation arrest of sympathoadrenal progenitors in the neural crest. It is necessary to reveal the differentiation mechanism of NB. Previously, we reported that Purkinje cell protein 4 (PCP4) is a well-differentiated marker of NB tissues. Herein, we explored the underlying mechanism of PCP4 induced differentiation in order to find better treatment options for patients. METHODS: We screened the interacting proteins of PCP4 by co-immunoprecipitation (Co-IP) and liquid chromatography-mass spectrometry (LC-MS/MS). Then we investigated the relevance between expression of calmodulin-dependent protein kinase II gamma (CAMK2G) and clinical features using R2 platform. We also explored the function of CAMK2G in NB cells by knockdown and RNA sequencing. RESULTS: Here, we verified the binding of PCP4 and calmodulin (CaM) by Co-IP and identified a target kinase of CaM, CAMK2G, by LC-MS/MS. PCP4 overexpression activates the autophosphorylation of CAMK2G. Patients with high CAMK2G expression had better survival while low CAMK2G was associated with unfavorable clinical features including MYCN-amplification, unfavorable histology, progression and high INSS stage. CAMK2G knockdown inhibited neurite outgrowth and down-regulated neuronal differentiation markers (NF-H, MAP2), yet promoted migration, invasion and proliferation. Gene Ontology (GO) analysis showed that knockdown of CAMK2G downregulated the expression of neuronal differentiation-related genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that knockdown of CAMK2G upregulated the expression of migration-related genes. CONCLUSION: These findings indicate that CAMK2G activated by PCP4/CaM complex promotes differentiation and inhibits migration in NB cells. LEVEL OF EVIDENCE: Not applicable.

Case Report: Kaposiform hemangioendothelioma with PIK3CA mutation successfully treated with sirolimus.

Kaposiform hemangioendothelioma (KHE) is an extremely rare, locally aggressive vascular neoplasm. The etiopathogenesis of KHE is still poorly understood. In the present study, we found a new mutation in KHE (c.685delA, p.Thr229fs). The KHE patient with the PIK3CA mutation showed complete regression after sirolimus treatment. We propose that the presence of the PIK3CA mutation in KHE may correlate with good response to sirolimus.

Successful Treatment of Fibro-Adipose Vascular Anomaly with Sirolimus.

BACKGROUND: The purpose of this study was to present our initial experience in using sirolimus therapy to treat fibro-adipose vascular anomaly (FAVA). METHODS: We retrospectively reviewed the medical records of eight patients with FAVA who were treated with sirolimus at our hospital between July 2017 and October 2020. RESULTS: Six girls (75%) and two boys (25%) were included in the cohort; the average age was 8 years (range, 1-13 years). Vascular tumors developed mainly on the extremities, including the forearm (n = 2; 25.0%), calf (n = 4; 50.0%), and thigh (n = 2; 25.0%). The predominant symptoms included swelling of the lesion (n = 8; 100%), pain (n = 7; 87.5%), contracture (n = 3; 37.5%), and phlebectasia (n = 3; 37.5%). Magnetic resonance imaging was the primary method used for FAVA diagnosis, and all patients underwent enhanced MRI. All lesions were heterogeneous with hyperintense T1 signals. The fat-suppressed T2-weighted images also revealed heterogeneous hyperintense masses, thus indicating fibrofatty infiltration. All eight patients received a sirolimus treatment regimen after FAVA diagnosis. One patient underwent tumor resection but experienced recurrence, whereas the other six patients underwent biopsy. Histological examination revealed that the lesions consisted of fibrofatty tissue with abnormal venous channels and anomalous lymphatic vascular components. Sirolimus softened the masses and caused tumor shrinkage within 5.25 ± 2.6 weeks (range, 2-10 weeks) after treatment initiation. The tumors also involuted rapidly and became stable within 7.75 ± 2.25 months after treatment initiation (range, 6-12 months). All seven patients experiencing pain reported relief within 3.8 ± 1.8 weeks (range, 2-7 weeks) after initiation of sirolimus therapy. Sirolimus alleviated but did not fully resolve the contracture in three patients. Remarkably, five patients exhibited a complete response, and three patients exhibited a partial response. At the time of the last follow-up, three patients had begun to gradually taper off sirolimus after 24 months of treatment and maintained a low blood sirolimus concentration. No serious adverse effects were observed during treatment. CONCLUSION: FAVA is a complex vascular malformation that appears to respond well to sirolimus treatment. Thus, sirolimus may be an effective and safe treatment for FAVA. LEVEL OF EVIDENCE: LEVEL IV.

Removal of cadmium and polychlorinated biphenyls by clover and the associated microbial community in a long-term co-contaminated soil.

Legumes such as clover are cost-effective and environmentally friendly components of strategies for remediating soils contaminated with heavy metals or organic pollutants. However, the mechanisms by which clover remediates co-contaminated soils are unclear. The present study explored the effects of phytoremediation by clover on pollutant removal and the microbial community in soil co-contaminated with cadmium (Cd) and polychlorinated biphenyls (PCBs). After 18 months of phytoremediation, Cd removal increased from 20.25 % in the control to 40.65 % in soil planted with clover, while PCB removal increased from 29.81 % to 60.02 %. High-throughput sequencing analysis showed that the relative abundances of the bacterial phylum Proteobacteria and the diazotrophic genus Rhizobium increased significantly after phytoremediation. Random forest analysis showed that bacterial and diazotrophic diversity significantly influenced Cd and PCB removal. Furthermore, co-occurrence network and correlation analyses revealed that Rhizobiales and Micromonosporales were the main bacteria associated with Cd removal, while Rhizobiales, Burkholderiales, and Xanthomonadales were identified as the main degraders of PCBs. PICRUSt functional prediction demonstrated that the gene bphC, which is related to PCB degradation, was significantly increased in the rhizosphere soil in the presence of clover. These results provide a better understanding for further studies of remediation efficiency by clover, rhizosphere microbial response and remediation mechanisms of co-contaminated soils under in situ conditions in the field.

Chemodiversity of soil organic matters determines biodegradation of polychlorinated biphenyls by a graphene oxide-assisted bacterial agent.

A promising strategy for degrading persistent organic pollutants (POPs) in soil is amendment with nanomaterial-assisted functional bacteria. However, the influence of soil organic matter chemodiversity on the performance of nanomaterial-assisted bacterial agents remains unclear. Herein, different types of soil (Mollisol soil, MS; Ultisol soil, US; and Inceptisol soil, IS) were inoculated with a graphene oxide (GO)-assisted bacterial agent (Bradyrhizobium diazoefficiens USDA 110, B. diazoefficiens USDA 110) to investigate the association between soil organic matter chemodiversity and stimulation of polychlorinated biphenyl (PCB) degradation. Results indicated that the high-aromatic solid organic matter (SOM) inhibited PCB bioavailability, and lignin-dominant dissolved organic matter (DOM) with high biotransformation potential was a favored substrate for all PCB degraders, which led to no stimulation of PCB degradation in MS. Differently, high-aliphatic SOM in US and IS promoted PCB bioavailability. The high/low biotransformation potential of multiple DOM components (e.g., lignin, condensed hydrocarbon, unsaturated hydrocarbon, etc.) in US/IS further resulted to the enhanced PCB degradation by B. diazoefficiens USDA 110 (up to 30.34%) /all PCB degraders (up to 17.65%), respectively. Overall, the category and biotransformation potential of DOM components and the aromaticity of SOM collaboratively determine the stimulation of GO-assisted bacterial agent on PCB degradation.

MRI-based radiomics in distinguishing Kaposiform hemangioendothelioma (KHE) and fibro-adipose vascular anomaly (FAVA) in extremities: A preliminary retrospective study.

OBJECTIVE: To investigate the pretreatment differentiation between Kaposiform hemangioendothelioma (KHE) and fibro-adipose vascular anomaly (FAVA) in extremities of pediatric patients. To build and validate an MRI-based radiomic model. METHOD: In this retrospective study, we obtained imaging data from 43 patients. We collected and compared clinical information, sketched region of interest (ROI), and extracted radiomic features from fat-suppressed T2-weighted (T2FS) images of the two cohorts of 30 and 13 patients respectively (training versus testing cohort 7:3). To select features, we used two sample t-test and the least absolute shrinkage and selection operator (LASSO) regression. The support vector machine (SVM) classification was constructed and evaluated by receiver operating characteristic (ROC) analysis. RESULTS: Thirty patients with KHE and 13 patients with FAVA in the extremities were included. Most lesions demonstrated low to intermediate signal intensity on T1-weighted images and hyperintense signals on T2-weighted ones. They also showed similar traits pathologically. Initially, 107 radiomic features were acquired and then three were finally selected. The support vector machine (SVM) model was able to differentiate the two anomalies from each other with an area under the curve (AUC) of 0.807 (95%CI 0.602-1.000) and 0.846 (95%CI 0.659-1.000) in training and testing cohort, respectively. CONCLUSION: The derived radiomic features were helpful in differentiating KHE from FAVA. A model which contained these features might further improve the performance and hopefully could serve as a potential tool for identification.

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.

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.

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.