Hsa_circ_0009096/miR-370-3p modulates hepatic stellate cell proliferation and fibrosis during biliary atresia pathogenesis.

Background: Hepatic stellate cell (HSC) activation and hepatic fibrosis mediated biliary atresia (BA) development, but the underlying molecular mechanisms are poorly understood. This study aimed to investigate the roles of circRNA hsa_circ_0009096 in the regulation of HSC proliferation and hepatic fibrosis. Methods: A cellular hepatic fibrosis model was established by treating LX-2 cells with transforming growth factor ß (TGF-ß1). RNaseR and actinomycin D assays were performed to detect hsa_circ_0009096 stability. Expression of hsa_circ_0009096, miR-370-3p, and target genes was detected using reverse transcription-qPCR. Direct binding of hsa_circ_0009096 to miR-370-3p was validated using dual luciferase reporter assay. Cell cycle progression and apoptosis of LX-2 cells were assessed using flow cytometry. The alpha-smooth muscle actin (α-SMA), collagen 1A1 (COL1A1), and TGF beta receptor 2 (TGFBR2) protein levels in LX-2 cells were analyzed using immunocytochemistry and western blotting. Results: Hsa_circ_0009096 exhibited more resistance to RNase R and actinomycinD digestion than UTRN mRNA. Hsa_circ_0009096 expression increased significantly in LX-2 cells treated with TGF-ß1, accompanied by elevated α-SMA and COL1A1 expression. Hsa_circ_0009096 siRNAs effectively promoted miR-370-3p and suppressed TGFBR2 expression in LX-2 cells, mediated by direct association of hsa_circ_0009096 with miR-370-3p. Hsa_circ_0009096 siRNA interfered with the cell cycle progression, promoted apoptosis, and reduced α-SMA and COL1A1 expression in LX-2 cells treated with TGF-ß1. MiR-370-3p inhibitors mitigated the alterations in cell cycle progression, apoptosis, and α-SMA, COL1A1, and TGFBR2 expression in LX-2 cells caused by hsa_circ_0009096 siRNA. In conclusion, hsa_circ_0009096 promoted HSC proliferation and hepatic fibrosis during BA pathogenesis by accelerating TGFBR2 expression by sponging miR-370-3p.

Role of the circular RNA regulatory network in the pathogenesis of biliary atresia.

Circular RNAs (circRNAs) serve an essential role in the occurrence and development of cholangiocarcinoma, but the expression and function of circRNA in biliary atresia (BA) is not clear. In the present study, circRNA expression profiles were investigated in the liver tissues of patients with BA as well as in the choledochal cyst (CC) tissues of control patients using RNA sequencing. A total of 78 differentially expressed circRNAs (DECs) were identified between the BA and CC tissues. The expression levels of eight circRNAs (hsa_circ_0006137, hsa_circ_0079422, hsa_circ_0007375, hsa_circ_0005597, hsa_circ_0006961, hsa_circ_0081171, hsa_circ_0084665 and hsa_circ_0075828) in the liver tissues of the BA group and control group were measured using reverse transcription-quantitative polymerase chain reaction. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis demonstrated that the identified DECs are involved in a variety of biological processes, including apoptosis and metabolism. In addition, based on the GO and KEGG pathway enrichment analyses, it was revealed that target genes that can be affected by circRNAs regulatory network were enriched in the TGF-ß signaling pathway, EGFR tyrosine kinase inhibitor resistance pathway and transcription factor regulation pathway as well as other pathways that may be associated with the pathogenesis of BA. The present study revealed that circRNAs are potentially implicated in the pathogenesis of BA and could help to find promising targets and biomarkers for BA.

CircUTRN24/miR-483-3p/IGF-1 Regulates Autophagy Mediated Liver Fibrosis in Biliary Atresia.

Biliary atresia (BA) is a rare neonatal cholestatic disease that presents with a marked bile duct reaction and rapid fibrotic development. Our earlier research has shown that circUTRN24 is highly elevated in BA, but the exact molecular mechanism is still unknown. This study attempted to investigate whether circUTRN24 induces BA liver fibrosis through regulation of autophagy and to elucidate its molecular mechanism. Using TGF-ß-treated hepatic stellate cells (HSC) LX-2, we created a liver fibrosis model. qRT-PCR was used to analyze the expression of circUTRN24, miR-483-3p, and IGF-1. Western blot analysis was used to assess the expression of IGF-1, HSC activation-related proteins, and autophagy-related proteins. The TGF-ß-induced LX-2 cell fibrosis model was then supplemented with circUTRN24 siRNA, miR-483-3p mimics, and the autophagy activator Rapamycin, and functional rescue tests were carried out to investigate the role of circUTRN24, miR-483-3p, and autophagy in BA liver fibrosis. Using a luciferase reporter assay, a direct interaction between miR-483-3p and circUTRN24 or IGF-1 was discovered. With the increase of TGF-ß treatment concentration, circUTRN24 expression also gradually increased, as did HSC activation and autophagy-related protein. si-circUTRN24 significantly decreased circUTRN24 expression and inhibited HSC activation and autophagy, which was reversed by Rapamycin. Through bioinformatics prediction and validation, we found circUTRN24 might act through miR-483-3p targeting IGF-1 in the autophagy-related mTOR pathway. Furthermore, miR-483-3p mimics significantly increased miR-483-3p expression and inhibited HSC activation and autophagy, which were reversed by Rapamycin. Functional rescue experiments showed that si-circUTRN24 inhibited circUTRN24 and IGF-1 expressions and promoted miR-483-3p expression, while the miR-483-3p inhibitor abolished these effects. These findings imply that circUTRN24/miR-483-3p/IGF-1 axis mediated LX-2 cell fibrosis by regulating autophagy.

miR-139-3p/Wnt5A Axis Inhibits Metastasis in Hepatoblastoma.

In order to examine new potential treatment options for the treatment of hepatoblastoma (HB), we identified the differential expression of five-candidate tumor suppressor miRNAs in HB and explored possible regulatory mechanisms of target miRNA molecule. By using real-time quantitative polymerase chain reaction (qPCR), we examined the expression of miRNAs in HB tissues and cells. The effect of has-miR-139-3p mimics on the invasion and migration ability was assessed by transwell assay and scratch-wound assay in HepG2 cells. Subsequently, we analyzed the target genes of miR-139-3p and their enrichment signaling pathways through bioinformatics. qPCR, Western-blot and dual-luciferase assays were further used to assess whether has-miR-139-3p targets Wnt5A. The results showed that hsa-miR-139-3p was significantly decreased in HB cells. Upregulation of hsa-miR-139-3p inhibited the invasive and migratory ability of HepG2. Bioinformatics analysis showed that hsa-miR-139-3p may target Wnt5A to regulate the WNT pathway, which was further confirmed by Western-blot and dual-luciferase assays. Overexpression of Wnt5A can reverse the miR-139-3p mimic-induced declines in the expression of WNT pathway-related proteins and restore the invasion and migration of HepG2. These data indicated that the hsa-miR-139-3p/Wnt5A axis inhibited HB metastasis, suggesting that miR-139-3p and Wnt5A may be potential targets for the treatment of HB.

Aminoacylase 1 (ACY-1) Mediates the Proliferation and Migration of Neuroblastoma Cells in Humans Through the ERK/Transforming Growth Factor ß (TGF-ß) Signaling Pathways.

BACKGROUND Aminoacylase 1 (ACY-1) is a cytosolic enzyme that catalyzes amino acid deacylation and has been reported to participate in various human diseases. However, the role and mechanism of ACY-1 in neuroblastoma (NB) are not completely understood. The aim of this study was to elucidate the role of ACY-1 in NB. MATERIAL AND METHODS Overexpression and knockdown of ACY-1 in human NB cells were performed, and the transfection efficiency was assessed through fluorescence microscopy, real-time PCR, and western blotting. The effect of ACY-1 on tumorigenesis and metastasis was determined by cell counting, colony formation, wound healing, flow cytometry, and transwell invasion assays in vitro, and the signaling pathway was examined using western blotting. RESULTS ACY-1 overexpression inhibited proliferation and induced apoptosis in human NB cells. ACY-1 inhibited the colony formation ability, migration, and invasion of SH-SY5Y cell lines. Moreover, the ERK1/2 and TGF-ß1 signaling pathways were more active when ACY-1 was overexpressed in NB cells. However, the knockdown of ACY-1 in SH-SY5Y cell lines showed the opposite effects. CONCLUSIONS ACY-1 regulates the proliferation, migration, and invasion of human NB cells through the ERK1/2 and TGF-ß1 signaling pathways, implying that ACY-1 may serve as a therapeutic target for patients with NB.

Suppressing microRNA-29c promotes biliary atresia-related fibrosis by targeting DNMT3A and DNMT3B.

This study was designed to investigate the potential role of microRNA-29c (miR-29c) in biliary atresia-related fibrosis. The expression of miR-29c was determined in 15 pairs of peripheral blood samples from infants with biliary atresia (BA) and infants with non-BA neonatal cholestasis using quantitative real-time PCR. EMT was established by induction with TGF-ß1 in HIBEpiC cells. MiR-29c was inhibited by lipofectamine transfection. The expressions of proteins related to epithelial-mesenchymal transition (EMT), i.e., E-cadherin, N-cadherin and vimentin, were determined using quantitative real-time PCR and western blotting. Direct interaction between miR-29c and DNMT3A and DNMT3B was identified using a luciferase reporter assay. The expressions of DNMT3A and DNMT3B were suppressed by treatment with SGI-1027. Patients with BA showed significantly lower miR-29c levels in peripheral blood samples than the control subjects. In vitro, TGF-ß1-induced EMT significantly decreased the expression of miR-29c. Downregulation of miR-29c had a promotional effect on BA-related fibrosis in HIBEpiC cells, as confirmed by the decrease in E-cadherin and increase in N-cadherin and vimentin levels. MiR-29c was found to target the 3'UTR of DNMT3A and DNMT3B and inhibit their expression. Suppression of DNMT3A and DNMT3B reversed the effects of miR-29c downregulation on BA-related fibrosis in HIBEpiC cells. These data suggest that BA-related fibrosis is closely associated with the occurrence of EMT in HIBEpiC cells. MiR-29c might be a candidate for alleviating BA-related fibrosis by targeting DNMT3A and DNMT3B.

Downregulation of microRNA-145 may contribute to liver fibrosis in biliary atresia by targeting ADD3.

BACKGROUND AND OBJECTIVES: Biliary atresia (BA) is a pediatric liver disease characterized by fibro-obliteration and obstruction of the extrahepatic biliary system, that invariably leads to cirrhosis and even death, if left untreated for extended time. However, its pathology and etiology still remained unknown. In this study, we tested the expression of adducin 3 (ADD3), the gene identified as a susceptibility gene in BA by GWAS, and uncovered its upstream regulatory microRNA in the pathogenesis of BA. METHODS: In this study, 14 infants with BA and 14 infants with choledochal cyst (CC) were enrolled as experimental group and control group, respectively. ADD3 and microRNA-145 (miR-145) expression profiles in liver tissues of BA and CC were determined using qPCR. Luciferase reporter assay was performed to verify the direct interaction between miR-145-5p and ADD3 3' Untranslated Regions (3'UTR). The Lentiviral vectors containing miR-145, miR-145-3p inhibitor, miR-145-5p inhibitor, empty vector were transfected into human hepatic stellate cell line (LX-2) to determine the functional effect of miR-145 on ADD3 expression at both mRNA and protein level. RESULTS: MiR-145 was shown to be down-regulated in liver tissues of infants with BA compared to CC (p = 0.0267). ADD3, verified as a target of miR-145-5p, was shown to be overexpressed in infants with BA at the mRNA level (p = 0.0118). Transfection of lentiviruses containing miR-145 into LX-2 cells decreased the expression of ADD3 at both mRNA and protein level compared to negative control group, and suppressed the expression of p-Akt at protein level. CONCLUSIONS: Our study has shown that overexpressed ADD3 and downregulated miR-145 were detected in BA liver tissues. MiR-145-5p was confirmed to target ADD3 by luciferase reporter assay. The downregulation of miR-145 may contribute to liver fibrosis in BA by upregulating the expression of ADD3.

Learning Curve Analysis of Laparoscopic Kasai Portoenterostomy.

OBJECTIVE: To compare the surgical outcomes of Kasai Portoenterostomy (PE) and investigate the learning curve of laparoscopic Kasai Portoenterostomy (Lap-PE). MATERIALS AND METHODS: Retrospective chart review of 80 cases of biliary atresia (type III) undergoing Lap-PE at Shenzhen Children's Hospital from January 2011 to June 2015, all of which were performed by the same surgical team. According to the operative sequences, the cases were equally divided into four phases (Phase I, II, III, and IV), which contain 20 cases in each. The age, weight, and gender, the volume of intraoperative blood loss, the postoperative clearance rate of bilirubin, the incidence of postoperative cholangitis, and the first and second year native liver survival rate were all reviewed. RESULTS: There was no significant difference among the four phases regarding age, weight at operation, and gender (P > .05). Statistical difference was observed with respect to operative time, the volume of intraoperative blood loss, the postoperative jaundice clearance, and the first and second year native liver survival rate among Phase III and IV compared to Phase I and II (P < .05), but showed no significant difference in neither the first two phases nor the latter two phases (P > .05). The incidence of postoperative cholangitis showed no significant variation among the four phases. CONCLUSION: A surgeon is able to become more experienced after performing approximately 40 laparoscopic Kasai Portoenterostomys.