Objective:To investigate differentially expressed long noncoding RNAs (lncRNAs) and mRNAs between proliferating and involuting infantile hemangioma (IH) .Methods:Eight IH specimens were surgically resected from 4 children with proliferating IH and 4 with involuting IH in Department of Pediatric Surgery, West China Hospital, Sichuan University from January to March in 2019. Differentially expressed lncRNAs and mRNAs between proliferating and involuting IH (a P value < 0.05 and a fold change ≥ 2.0) were screened by microarray analysis, and verified by real-time fluorescence-based quantitative PCR (qRT-PCR) . Bioinformatics analysis was further performed, including GO and KEGG pathway analyses of differentially expressed mRNAs, construction of a lncRNA-mRNA co-expression network, and prediction of cis-acting target genes of differentially expressed lncRNAs. Results:A total of 405 differentially expressed lncRNAs and 772 differentially expressed mRNAs were identified between the proliferating and involuting IH specimens by using microarray technology. Of them, 108 lncRNAs and 107 mRNAs were downregulated, 297 lncRNAs and 665 mRNAs were upregulated in the proliferating IH specimens. Four lncRNAs (n335248, ENST00000450864, n333319, and n335185) and 4 mRNAs (EDNRA, IFI6, HK2, and ITGA1) were verified by qRT-PCR, and the results were consistent with those of microarray analysis. GO and KEGG enrichment analyses showed that differentially expressed mRNAs were mainly involved in the biological processes blood coagulation, axon guidance, angiogenesis and cell adhesion. Besides, differentially expressed mRNAs were mostly enriched in the metabolic pathways focal cell adhesion, regulation of actin cytoskeleton, leukocyte transendothelial migration and phosphatidylinositol 3-kinase/serine-threonine protein kinase and other signaling pathways. In addition, a lncRNA-mRNA co-expression network was constructed with 23 mRNAs and 58 lncRNAs with the degree ≥ 15.Conclusion:Lots of differentially expressed lncRNAs and mRNAs were identified between proliferating and involuting IH tissues, and these lncRNAs may play important roles in the development of IH by regulating corresponding target mRNAs.

Objective:To investigate the effect of the key glycolysis enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) on the biological activity of hemangioma-derived endothelial cells (HemECs) .Methods:Totally, 4 proliferating infantile hemangioma (IH) tissues and 4 involuting IH tissues were collected. Primary HemECs were isolated from the proliferating IH tissues, and human umbilical vein endothelial cells (HUVECs) served as controls. Immunohistochemical study and Western blot analysis were performed to determine the expression of PFKFB3 in the IH tissues and HemECs, respectively. Cell counting kit-8 (CCK8) assay was conducted to evaluate the effect of PFK15 (a specific inhibitor of PFKFB3) at concentrations of 0 - 10 μmol/L on the proliferation of HemECs, and HemECs treated without PFKFB3 served as the control group. Some in vitro cultured HemECs were treated with 5 μmol/L PFK15, and served as a PFK15 intervention group, while HemECs treated without PFK15 served as a control group; then, the migratory ability of HemECs was assessed by Transwell assay, and the apoptosis level of HemECs was detected by flow cytometry. Comparisons between groups were performed by using t test or analysis of variance. Results:Immunohistochemical study showed that the positive rate of PFKFB3 was significantly higher in the proliferating IH tissues (74.34% ± 5.26%) than in the involuting IH tissues (41.46% ± 2.99%, t = 9.40, P < 0.001). Western blot analysis showed that the relative expression level of PFKFB3 was also significantly higher in HemECs (0.73 ± 0.05) than in HUVECs (0.45 ± 0.04, t = 8.50, P < 0.001). CCK8 assay revealed significantly decreased proliferative activity of HemECs in the 0.625-, 1.25-, 2.5-, 5-, and 10-μmol/L PFK15 groups compared with the control group (all P < 0.01). Compared with the control group, the PFK15 intervention group showed significantly decreased number of migratory HemECs (297 ± 15 vs. 422 ± 8, t = 12.59, P < 0.001), but significantly increased apoptosis rates of HemECs (6.69% ± 0.64% vs. 0.34% ± 0.07%, t = 17.07, P < 0.001) . Conclusion:The key glycolytic enzyme PFKFB3 was highly expressed in the proliferating IH tissues and HemECs, and the PFKFB3 inhibitor PFK15 could suppress the proliferation, migration, and increase the apoptosis of HemECs.

Objective:To preliminarily assess the clinical value of sirolimus combined with prednisone in the treatment of Kaposiform hemangioendothelioma (KHE) complicated by Kasabach-Merritt phenomenon (KMP) .Methods:A retrospective study was conducted. General clinical data and relevant data on efficacy and adverse reactions were collected from 36 patients with KHE complicated by KMP, who received oral sirolimus combined with prednisone in Department of Pediatric Surgery, West China Hospital, Sichuan University from January 2011 to January 2018.Results:Among the 36 patients with KHE complicated by KMP, the male-to-female ratio was 1∶0.8; their average age was 15.0 months (range, 4.0 - 60.0 months) , and the average age of onset was 6.3 months (range, 0.8 - 48.0 months) ; 32 (88.9%) patients were diagnosed with mixed KHE, and 4 (11.1%) with deep KHE; the tumor size ranged from 2.5 cm × 4.0 cm to 20.0 cm × 24.0 cm. During the combined treatment, the average duration of prednisone administration was 6.4 weeks (range, 5.0 - 9.0 weeks) , and that of sirolimus administration was 19.3 months (range, 13.0 - 27.0 months) . After 1-5 weeks of combined treatment, platelet counts and fibrinogen levels of the 36 patients gradually returned to normal. After short-term prednisone combined with long-term sirolimus treatment, the average disease severity score decreased from 4.0 before treatment to 2.4 at 6 months and 1.6 at 12 months. After 12 months of the combined treatment, tumors mostly regressed in 32 (88.9%) patients, partially regressed in 3 (8.3%) , showed no obvious change in 1 (2.8%) . During the treatment, common adverse reactions included gastrointestinal discomfort and oral ulcers, no patient discontinued the treatment due to severe adverse reactions, and no drug toxicity-related death occurred.Conclusion:Sirolimus combined with prednisone is effective and safe for the treatment of KHE complicated by KMP.

Objective:To analyze demographic and clinical characteristics of infantile hemangioma (IH) , and to explore related risk factors for IH.Methods:A multicenter case-control study was conducted. IH patients (case group) and healthy children (control group) were collected from West China Hospital of Sichuan University, West China Second University Hospital of Sichuan University and Yulin Community Central Hospital of Chengdu from October 2018 to December 2020. The data on patients′ demographic characteristics, and risk factors during their mothers′ pre-pregnancy, pregnancy and perinatal period were collected and retrospectively analyzed. Univariate and multivariate analyses were performed using binary logistic regression.Results:A total of 1 479 patients with IH and 1 086 healthy children were included in this study. There were 456 males and 1 023 females in the case group, with the age being 3.74 ± 2.82 months, and there were 359 males and 727 females in the control group, with the age being 3.95 ± 2.77 months. There was no significant difference in the gender ratio, age, ethnic composition, birth weight or birth height between the case group and control group (all P > 0.05) . IH lesions mostly affected the head and face (564 cases, 38.1%) , followed by the trunk (449 cases, 30.6%) and limbs (356 cases, 24.1%) . At the visit, 1 109 (75.0%) patients presented with proliferating IH, 1 059 (71.6%) with superficial IH, and 1 306 (88.3%) with focal IH. The IH lesion area ranged from 0.01 to 168.00 (6.24 ± 12.91) cm 2, and the segmental IH area ranged from 7.50 to 168.00 (32.17 ± 26.94) cm 2. Univariate logistic regression analysis showed some factors influencing the occurrence of IH (all P < 0.05) , including pre-pregnancy factors (delivery history and miscarriage history) , pregnancy factors (fetal distress, cord entanglement, history of threatened abortion, placenta previa, oligohydramnios, gestational hypothyroidism, gestational anemia, history of progesterone supplementation, history of thyroxine drug use, history of uterus myomas) , and perinatal factors (including fetal position, gestational weeks, premature rupture of membranes and preterm premature rupture of membranes) . Multivariate binary logistic regression adjusted analysis showed that fetal breech presentation, preterm birth, cord entanglement and history of thyroxine drug use during pregnancy did not influence the occurrence of IH (all P > 0.05) ; the delivery history was the strongest independent risk factor for IH (adjusted OR = 5.624, 95% CI: 4.275 to 7.398, P < 0.001) , and gestational hypothyroidism and history of uterus myomas were protective factors for IH. Conclusions:In this study, the average age of IH patients at visit was 4 months, skin lesions mostly occurred on the head and face, and most were superficial and focal in the proliferative stage. The occurrence and development of IH may be associated with placental diseases, hypoxia, maternal hormone levels during pregnancy, etc.

Objective:To investigate the effect of the glucose transporter 1 (Glut-1) inhibitor resveratrol on the activity of infantile hemangioma (IH) -derived endothelial cells (HemEC) .Methods:IH tissues were collected from 4 cases of proliferating IH and 4 cases of involuting IH, and immunohistochemical study was performed to determine the Glut-1 expression. Primary HemEC were extracted from 4 proliferating IH tissues, real-time fluorescence-based quantitative PCR (qPCR) and Western blot analysis were performed to determine the mRNA and protein expression of Glut-1 in HemEC and human umbilical vein endothelial cells (HUVEC) , respectively. HemEC were cultured in vitro and treated with 0 (control group) , 50, 100, 200, 400 and 800 μmol/L resveratrol for 24 hours, respectively. Cell counting kit-8 (CCK8) assay was performed to evaluate the proliferative ability of HemEC in the above groups, and the 50% inhibitory concentration (IC50) was calculated. The migratory ability and apoptosis level of HemEC were assessed by Transwell assay and flow cytometry, respectively. Intergroup comparisons were performed using t test or analysis of variance, and multiple comparisons were performed using least significant difference- t test. Results:Immunohistochemical study showed that Glut-1 was expressed in vascular endothelial cells derived from both proliferating and involuting IH tissues, and the Glut-1 expression was abundant in the proliferating IH but markedly decreased in the involuting IH tissues. The mRNA and protein expression levels of Glut-1 were significantly higher in HemEC (1.793 ± 0.041, 1.959 ± 0.144, respectively) than in HUVEC (0.820 ± 0.073, 0.648 ± 0.046, t = 16.35, 12.28, respectively, both P < 0.001) . After the treatment with Glut-1 inhibitor resveratrol at different concentrations, the proliferative ability of HemEC significantly differed among the control group, 50-, 100-, 200-, 400- and 800-μmol/L resveratrol groups ( F = 1 043.00, P < 0.001) , and was significantly lower in all the resveratrol groups than in the control group (all P < 0.05) . The IC50 of resveratrol was calculated to be 150 μmol/L by using GraphPad Prism 8 software. Transwell assay and flow cytometry showed significantly decreased number of migratory HemEC but significantly increased apoptosis rate respectively in the 150 μmol/L resveratrol group (61 ± 5, 13.01% ± 0.45%, respectively) compared with the control group (150 ± 6, 3.93% ± 0.68%, t = 15.11, 19.34, respectively, both P < 0.001) . Conclusion:The key glycolytic enzyme Glut-1 was highly expressed in proliferating IH tissues and HemEC, and resveratrol could inhibit the proliferation and migration of HemEC, but promote their apoptosis.

OBJECTIVE: To investigate the effect of danusertib (Danu), an inhibitor of Aurora kinase, on the proliferation, cell cycle, apoptosis, and autophagy of hepatocellular carcinoma HepG2 cells and explore the underlying mechanisms. METHODS: MTT assay was used to examine the effect of Danu on the viability of HepG2 cells to determine the IC50 of Danu. The effect of Danu on cell cycle distribution, apoptosis and autophagy were determined using flow cytometry. Western blotting was used to detect the expressions of the proteins related to cell cycle, apoptosis and autophagy. Chloroquine was used to suppress Danuinduced autophagy to test the apoptosis-inducing effect of Danu. RESULTS: Danu significantly inhibited the proliferation of HepG2 cells with IC of 39.4 μmol and 14.4 μmol at 24 h and 48 h, respectively. Danu caused cell cycle arrest in G/M phase in HepG2 cells and led to polyploidy accumulation via up-regulating the expressions of p53 and p21 and down-regulating the expressions of cyclin B1 and DC2. Danu also caused apoptosis of HepG2 cells through up-regulating the expressions of Bax, Puma, cleaved caspase-3, cleaved caspase-9, cleaved PARP and cytochrome C and down-regulating the expressions of Bcl-xl and Bcl-2. Danu induced autophagy via activating AMPK signaling and inhibiting PI3K/PTEN/AKT/mTOR axis, and inhibition of Danu-induced autophagy with chloroquine enhanced the pro-apoptotic effect of Danu. CONCLUSIONS Danu inhibits cell proliferation and induces cell cycle arrest in G/M phase, apoptosis and cytoprotective autophagy in HepG2 cells.
Apoptosis ; drug effects ; Autophagy ; drug effects ; Benzamides ; pharmacology ; Carcinoma, Hepatocellular ; pathology ; Cell Cycle ; drug effects ; Cell Division ; drug effects ; Cell Proliferation ; drug effects ; Hep G2 Cells ; Humans ; Liver Neoplasms ; pathology ; Neoplasm Proteins ; metabolism ; Protein Kinase Inhibitors ; pharmacology ; Pyrazoles ; pharmacology