STS â ¡A inhibited angiogenesis of lung adenocarcinoma by activating FOXO3 to inhibit CXCL1/STAT3/VEGF pathway.

BACKGROUND: Lung adenocarcinoma (LUAD) is the most popular type of lung cancer. Sulfotanshinone IIA sodium (STS IIA) has been proven to have an anticancer effect. However, its role in LUAD and its underlying mechanism remain unclear. OBJECTIVE: To investigate the role and mechanism of STS IIA in LUAD angiogenesis. METHODS: The mRNA levels of genes, including forkhead box O3 (FOXO3) and chemokine C-X-C motif ligand 1 (CXCL1), were detected by qRT-PCR. The levels of proteins, including FOXO3, CXCL1, and vascular endothelial growth factor (VEGF), were measured by Western blot. The proliferation and angiogenesis of human umbilical vein endothelial cells (HUVECs) were detected by the EdU assay and the tubule formation assay, respectively. The binding relationship between FOXO3 and CXCL1 was detected by dual-luciferase reporter assay. RESULTS: Our results illustrated that different concentrations of STS IIA inhibited the proliferation and angiogenesis of HUVECs. FOXO3 regulated the proliferation and angiogenesis of HUVECs inhibited by STS ⅡA via targeting CXCL1. Subsequently, we proved that exogenous CXCL1 alleviated the inhibition of proliferation and angiogenesis of HUVECs regulated by STS IIA via activating the STAT3/VEGF pathway. Finally, we found that STS IIA inhibited the angiogenesis of lung adenocarcinoma though FOXO3 to inhibit the CXCL1/STAT3/VEGF pathway. CONCLUSION: Our study finally elucidated the underlying molecular mechanism by which STS ⅡA inhibits LUAD angiogenesis.

High-quality haplotype-resolved genome assembly for ring-cup oak (Quercus glauca) provides insight into oaks demographic dynamics.

Quercus section Cyclobalanopsis represents a dominant woody lineage in East Asian evergreen broadleaved forests. Regardless of its ecological and economic importance, little is known about the genomes of species in this unique oak lineage. Quercus glauca is one of the most widespread tree species in the section Cyclobalanopsis. In this study, a high-quality haplotype-resolved reference genome was assembled for Q. glauca from PacBio HiFi and Hi-C reads. The genome size, contig N50, and scaffold N50 measured 902.88, 7.60, and 69.28 Mb, respectively, for haplotype1, and 913.28, 7.20, and 71.53 Mb, respectively, for haplotype2. A total of 37,457 and 38,311 protein-coding genes were predicted in haplotype1 and haplotype2, respectively. Homologous chromosomes in the Q. glauca genome had excellent gene pair collinearity. The number of R-genes in Q. glauca was similar to most East Asian oaks but less than oak species from Europe and America. Abundant structural variation in the Q. glauca genome could contribute to environmental stress tolerance in Q. glauca. Sections Cyclobalanopsis and Cerris diverged in the Oligocene, in agreement with fossil records for section Cyclobalanopsis, which document its presence in East Asia since the early Miocene. The demographic dynamics of closely related oak species were largely similar. The high-quality reference genome provided here for the most widespread species in section Cyclobalanopsis will serve as an essential genomic resource for evolutionary studies of key oak lineages while also supporting studies of interspecific introgression, local adaptation, and speciation in oaks.

Sodium tanshinone IIA sulphate inhibits angiogenesis in lung adenocarcinoma via mediation of miR-874/eEF-2K/TG2 axis.

CONTEXT: Sodium tanshinone IIA sulphate (STS) is a product originated from Salvia miltiorrhiza Bunge [Lamiaceae], which exerts an antitumour effect. However, the role of STS on lung adenocarcinoma (LUAD) remains unexplored. OBJECTIVE: Our study explores the effect and mechanism of STS against LUAD. MATERIALS AND METHODS: LUAD cells were treated with 100 µM STS for 24 h and control group cells were cultured under normal medium conditions. Functionally, the viability, migration, invasion and angiogenesis of LUAD cells were examined by MTT, wound healing, transwell and tube formation assay, respectively. Moreover, cells were transvected with different transfection plasmids. Dual luciferase reporter and RNA immunoprecipitation (RIP) assays were used to verify the relationship between miR-874 and eEF-2K. RESULTS: STS significantly decreased the viability (40-50% reduction), migration (migration rate of A549 cells from 0.67 to 0.28, H1299 cells from 0.71 to 0.41), invasion (invasion numbers of A549 cells from 172 to 55, H1299 cells from 188 to 35) and angiogenesis (80-90% reduction) of LUAD cells. Downregulation of miR-874 partially abolished the antitumour effect of STS. EEF-2K was identified to be the target of miR-874, and its downregulation markedly abolished the effects of miR-874 downregulation on tumourigenesis of LUAD. Moreover, silencing of TG2 abrogated eEF-2K-induced progression of LUAD. DISCUSSION AND CONCLUSIONS: STS attenuated the tumourigenesis of LUAD through the mediation of the miR-874/eEF-2K/TG2 axis. STS is a promising drug to fight against lung cancer, which might effectively reverse drug resistance when combined with classical anticancer drugs.

eEF-2K knockdown synergizes with STS treatment to inhibit cell proliferation, migration, and invasion via the TG2/ERK pathway in A549 cells.

Emerging research has suggested the anticancer potential of tanshinone IIA, the bioactive ingredient isolated from the traditional Chinese herb Salvia miltiorrhiza. However, the molecular mechanism of sodium tanshinone IIA sulfonate (STS) antilung cancer effect is not very clear. In this study, our purpose is to investigate the roles of STS and elongation factor-2 kinase (eEF-2K) in regulating the proliferation, migration, and invasion of A549 cells and explore the implicated pathways. We found that STS suppressed A549 cell survival and proliferation in a time- and xdose-dependent manner. Knockdown of eEF-2K and treatment with STS synergistically exerted antiproliferative, -migratory, and -invasive effects on A549 cells. These effects were caused by attenuation of the extracellular signal-regulated kinase (ERK) pathway via inhibition of tissue transglutaminase (TG2). In summary, the inhibition of eEF-2K synergizes with STS treatment, exerting anticancer effects on lung adenocarcinoma cells through the TG2/ERK signaling pathway, which provides a potential therapeutic target for treating lung adenocarcinoma.

Cropland redistribution to marginal lands undermines environmental sustainability.

Cropland redistribution to marginal land has been reported worldwide; however, the resulting impacts on environmental sustainability have not been investigated sufficiently. Here we investigated the environmental impacts of cropland redistribution in China. As a result of urbanization-induced loss of high-quality croplands in south China (∼8.5 t ha-1), croplands expanded to marginal lands in northeast (∼4.5 t ha-1) and northwest China (∼2.9 t ha-1) during 1990-2015 to pursue food security. However, the reclamation in these low-yield and ecologically vulnerable zones considerably undermined local environmental sustainability, for example increasing wind erosion (+3.47%), irrigation water consumption (+34.42%), fertilizer use (+20.02%) and decreasing natural habitats (-3.11%). Forecasts show that further reclamation in marginal lands per current policies would exacerbate environmental costs by 2050. The future cropland security risk will be remarkably intensified because of the conflict between food production and environmental sustainability. Our research suggests that globally emerging reclamation of marginal lands should be restricted and crop yield boost should be encouraged for both food security and environmental benefits.

Folium Ginkgo extract and tetramethylpyrazine sodium chloride injection (Xingxiong injection) protects against focal cerebral ischaemia/reperfusion injury via activating the Akt/Nrf2 pathway and inhibiting NLRP3 inflammasome activation.

CONTEXT: Folium Ginkgo extract and tetramethylpyrazine sodium chloride injection (Xingxiong injection) is a compound preparation commonly used for treating cerebral ischaemia/reperfusion injury in ischaemic stroke in China. However, its potential mechanisms on ischaemic stroke remain unknown. OBJECTIVE: This study explores the potential mechanisms of Xingxiong injection in vivo or in vitro. MATERIALS AND METHODS: Sprague-Dawley (SD) rats were randomly assigned to five groups: the sham (normal saline), the model (normal saline) and the Xingxiong injection groups (12.5, 25 or 50 mL/kg). The rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) followed by reperfusion for 14 d. Xingxiong injection was administered via intraperitoneal (i.p.) injection immediately after ischaemia induction for 14 d. Afterwards, rats were sacrificed at 14 d induced by administration of Xingxiong injection. RESULTS: Xingxiong injection significantly reduces infarct volume (23%) and neurological deficit scores (93%) compared with the MCAO/R group. Additionally, Xingxiong injection inhibits the loss in mitochondrial membrane potential (43%) and reduces caspase-3 level (44%), decreases NOX (41%), protein carbonyl (29%), 4-HNE (40%) and 8-OhdG (41%) levels, inhibits the expression of inflammatory factors, such as TNF-α (26%), IL-1ß (34%), IL-6 (39%), MCP-1 (36%), CD11a (41%) and ICAM-1 (43%). Moreover, Xingxiong injection can increase p-Akt/Akt (35%) and Nrf2 (47%) protein expression and inhibit NLRP3 (42%) protein expression. CONCLUSIONS: Xingxiong injection prevents cerebral ischaemia/reperfusion injury via activating the Akt/Nrf2 pathway and inhibiting NLRP3 inflammasome. These findings provide experimental evidence for clinical use of drugs in the treatment of ischaemic stroke.

[Circ_0001666 promotes the proliferation and inhibits apoptosis of non-small cell lung cancer cells by targeting the miR-330-5p/HMGA2 axis].

Many studies have shown that circular RNAs (circRNAs) play a key regulatory role in the whole biological process of tumors. The purpose of this study was to explore the biological function and molecular mechanism of circ_0001666 in non-small cell lung cancer (NSCLC), so as to provide new targets for the diagnosis and treatment of NSCLC. Gene expression profiles were downloaded from Gene Expression Omnibus (GEO, GSE101586) and the differential genes were obtained by using GEO2R analysis. The quantitative real time polymerase chain reaction (qRT-PCR) was used to detect the expression level of circ_0001666 in NSCLC cells. Cell counting kit-8 (CCK-8) and Annexin V-FITC apoptosis detection kit were respectively used to assess the cell proliferation and apoptosis, where circ_0001666 was knockdown in NSCLC cells. The targeted relationship among mircoRNA 330-5p (miR-330-5p), circ_0001666, and high mobility group A2 protein (HMGA2) was verified by bioinformatics prediction, dual-luciferase reporter gene, RNA immunoprecipitation (RIP) and RNA pull down assay. The results showed that the expression of circ_0001666 in NSCLC cells was significantly up-regulated than that in normal lung epithelial cells. Circ_0001666 knockdown reduced the cell viability and promoted the apoptosis of NSCLC cells, which could be reversed by miR-330-5p inhibitors. MiR-330-5p is the downstream target of circ_0001666 and can be adsorbed by circ_0001666. HMGA2 is a target gene of miR-330-5p, which can be indirectly regulated by circ_0001666. The results suggest that circ_0001666 promotes the proliferation and inhibits apoptosis of NSCLC cells via miR-330-5p/HMGA2 axis.

Notoginsenoside R1 activates the NAMPT-NAD+-SIRT1 cascade to promote postischemic angiogenesis by modulating Notch signaling.

Nicotinamide phosphoribosyltransferase (NAMPT) maintains mitochondrial function and protects against cerebral ischemic injury by improving energy metabolism. Notoginsenoside R1 (R1), a unique constituent of Panax notoginseng, has been shown to promote the proliferation and tube formation of human umbilical vein endothelial cells. Whether R1 has proangiogenesis on the activation of NAMPT in ischemic stroke remains unclear. The purpose of this study was to investigate the pharmacodynamic effect and mechanism of R1 on angiogenesis after ischemic stroke. We used male Sprague-Dawley (SD) rats subjected to middle cerebral artery occlusion/reperfusion (MCAO/R). R1 was administered via intraperitoneal (i.p.) injection immediately after ischemia induction. The promotion of R1 on angiogenesis were detected by immunofluorescence staining, 3D stereoscopic imaging and transmission electron microscopy detection. HBMEC cells were pretreated with different concentrations of R1 for 12 h before oxygen-glucose deprivation/reoxygenation (OGD/R) exposure. Afterward, scratch assay, EdU staining and tube formation were determined. Western blot analyses of proteins, including those involved in angiogenesis, NAMPT-SIRT1 cascade, VEGFR-2, and Notch signaling, were conducted. We showed that R1 significantly restored cerebral blood flow, improved mitochondrial energy metabolism and promoted angiogenesis. More importantly, incubation with 12.5-50 µM R1 significantly increased the migration, proliferation and tube formation of HBMECs in vitro. The promotion of R1 on angiogenesis were associated with the NAMPT-NAD+-SIRT1 cascade and Notch/VEGFR-2 signaling pathway, which was partially eliminated by inhibitors of NAMPT and SIRT1. We demonstrated that R1 promotes post-stroke angiogenesis via activating NAMPT-NAD+-SIRT1 cascade. The modulation of Notch signaling and VEGFR-2 contribute to the post-stroke angiogenesis. These findings offer insight for exploring new therapeutic strategies for neurorestoration via R1 treatment after ischemic stroke.

Cassane diterpenoid derivative induces apoptosis in IDH1 mutant glioma cells through the inhibition of glutaminase in vitro and in vivo.

BACKGROUND: Glioblastoma multiforme (GBM) is the most frequent, lethal and aggressive tumour of the central nervous system in adults. The discovery of novel anti-GBM agents based on the isocitrate dehydrogenase (IDH) mutant phenotypes and classifications have attracted comprehensive attention. PURPOSE: Diterpenoids are a class of naturally occurring 20-carbon isoprenoid compounds, and have previously been shown to possess high cytotoxicity for a variety of human tumours in many scientific reports. In the present study, 31 cassane diterpenoids of four types, namely, butanolide lactone cassane diterpenoids (I) (1-10), tricyclic cassane diterpenoids (II) (11-15), polyoxybutanolide lactone cassane diterpenoids (III) (16-23), and fused furan ring cassane diterpenoids (IV) (24-31), were tested for their anti-glioblastoma activity and mechanism underlying based on IDH1 mutant phenotypes of primary GBM cell cultures and human oligodendroglioma (HOG) cell lines. RESULTS: We confirmed that tricyclic-type (II) and compound 13 (Caesalpin A, CSA) showed the best anti-neoplastic potencies in IDH1 mutant glioma cells compared with the other types and compounds. Furthermore, the structure-relationship analysis indicated that the carbonyl group at C-12 and an α, ß-unsaturated ketone unit fundamentally contributed to enhancing the anti-glioma activity. Studies investigating the mechanism demonstrated that CSA induced oxidative stress via causing glutathione reduction and NOS activation by negatively regulating glutaminase (GLS), which proved to be highly dependent on IDH mutant type glioblastoma. Finally, GLS overexpression reversed the CSA-induced anti-glioma effects in vitro and in vivo, which indicated that the reduction of GLS contributed to the CSA-induced proliferation inhibition and apoptosis in HOG-IDH1-mu cells. CONCLUSION: Therefore, the present results demonstrated that compared with other diterpenoids, tricyclic-type diterpenoids could be a targeted drug candidate for the treatment of secondary IDH1 mutant type glioblastoma through negatively regulating GLS.

Long noncoding RNA LEF1-AS1 acts as a microRNA-10a-5p regulator to enhance MSI1 expression and promote chemoresistance in hepatocellular carcinoma cells through activating AKT signaling pathway.

Long noncoding RNAs (lncRNAs) contribute to the development of hepatocellular carcinoma (HCC), which could regulate various HCC biological characteristics. Here, the study seeks to investigate the role of lncRNA LEF1-AS1 in HCC cell chemoresistance by regulating microRNA (miR)-10a-5p and Musashi1 (MSI1). The microarray-based analysis was employed to identify the HCC-related lncRNA-miRNA-gene regulatory network. Expression patterns of LEF1-AS1, miR-10a-5p, and MSI1 in the HCC cell lines, tissues were accessed by means of reverse transcription-quantitative polymerase chain reaction. Next, the interaction among LEF1-AS1, miR-10a-5p, and MSI1 in HCC was accessed by bioinformatics and dual-luciferase reporter gene assay. Then, the cell line resistant to cisplatin was established, which was then treated with sh/oe-lncRNA LEF1-AS1, miR-10a-5p-mimic, and oe/sh-MSI1 vectors alone or in combination. Afterward, the effect of LEF1-AS1, miR-10a-5p, and MSI1 on HCC cell chemoresistance, proliferation, and apoptosis was assessed. At last, in vivo experiments confirmed the role of MSI1 in tumor growth and chemoresistance in HCC. LEF1-AS1 might potentially affect the growth and chemoresistance of HCC cells by regulating miR-10a-5p and MSI1. LEF1-AS1 and MSI1 expression patterns were elevated, while miR-10a-5p was repressed in HCC tissues and cell lines. LEF1-AS1 combined to miR-10a-5p and regulated MSI1, thereby activating the protein kinase B (AKT) signaling pathway. Knockdown of LEF1-AS1 and MSI1 or elevation of miR-10a-5p compromised the proliferation of Huh7 cell line resistant to DDP and promoted its chemosensitivity and apoptosis. At last, these in vitro findings were also confirmed in vivo. Our results unraveled LEF1-AS1 acts as a miR-10a-5p modulator to promote chemoresistance of HCC cells by stimulating MSI1 and activating the AKT signaling pathway, which might provide a novel therapeutic target for HCC.

Silencing of long non-coding RNA FOXD2-AS1 inhibits the progression of gallbladder cancer by mediating methylation of MLH1.

Evidence has documented the tumor-promoting properties of long non-coding RNA (lncRNA) FOXD2 adjacent opposite strand RNA 1 (FOXD2-AS1) in many cancers. However, little is known about its role in gallbladder cancer. Here, we aimed to characterize the functional relevance of lncRNA FOXD2-AS1 in gallbladder cancer and the possible mechanisms associated with methylation of MutL homolog-1 (MLH1). Initially, microarray-based gene expression profiling of gallbladder cancer was employed to identify differentially expressed lncRNAs. Next, the expression of lncRNA FOXD2-AS1 was examined, and the cell line presenting with the highest lncRNA FOXD2-AS1 expression was selected for subsequent experimentation. Then, the interaction between lncRNA FOXD2-AS1 and MLH1 was identified. The effect of lncRNA FOXD2-AS1 on proliferation, migration, invasion, and apoptosis as well as tumorigenicity of transfected GBC-SD cells was examined with gain- and loss-of-function experiments. We found that lncRNA FOXD2-AS1 was highly expressed, while MLH1 was poorly expressed in gallbladder cancer cells. Besides, lncRNA FOXD2-AS1 could promote MLH1 methylation by recruiting DNMT1 to the MLH1 promoter, and consequently inhibit MLH1 transcription. Silencing of lncRNA FOXD2-AS1 or overexpression of MLH1 inhibited gallbladder cancer cell proliferation, invasion, and migration, while facilitating cell apoptosis in vitro as well as retarding tumor growth in vivo. Thus, silencing of lncRNA FOXD2-AS1 suppressed the progression of gallbladder cancer via upregulation of MLH1 by inhibiting MLH1 promoter methylation. These findings present lncRNA FOXD2-AS1 knockdown as a potential candidate for the treatment of gallbladder cancer.

Xuesaitong injection (lyophilized) combined with aspirin and clopidogrel protect against focal cerebral ischemic/reperfusion injury in rats by suppressing oxidative stress and inflammation and regulating the NOX2/IL-6/STAT3 pathway.

BACKGROUND: Combination of aspirin (ASA) and clopidogrel (CLP) [dual antiplatelet therapy (DAPT)] has been limited in reducing early recurrent stroke events. Xuesaitong injection (lyophilized) (XST) made of total saponins from P. notoginseng, which significantly improves cerebral circulation and has been widely used in clinical applications for decades to treat and prevent ischemic stroke. Here, we confirmed the protective effect and mechanism of XST combined with DAPT (XST+ASA+CLP) on cerebral ischemia/reperfusion injury, exploring their better pharmacological action for clinical patients. METHODS: Sprague-Dawley rats (SD rats) (n=9 in each group) were randomly assigned to three groups and pretreated with XST, ASA+CLP, or XST+ASA+CLP for 7 days. Then rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) followed by reperfusion for 24 h. Therapeutic effect of XST+ASA+CLP was measured by infarct volume, neurological behavior and regional cerebral blood flow (rCBF). Inhibition of neuronal apoptosis and glial cells was determined by immunofluorescent staining. We studied the protein levels of neurotrophic factors, neuroplasticity-related factors, oxidative stress indicators and inflammatory factors by ELISA assay. RESULTS: XST+ASA+CLP group showed significant reduction in infarct volumes and neurological deficit scores. XST+ASA+CLP group also had higher levels in rCBF and synaptic growth, and showed remarkable inhibition of microglia and astrocytes activation and the neuronal apoptosis. In addition, XST+ASA+CLP group had lower levels of NADPH, protein carbonyl, 4-hydroxynonenal (4-HNE), 8-hydroxydeoxyguanosine (8-OHdG) and several inflammatory cytokines. Moreover, XST+ASA+CLP group also had lower levels of NOX2, inducible nitric oxide synthase (iNOS), interleukin (IL)-6, and p-STAT3/STAT3. CONCLUSIONS: These results demonstrate that a combination of XST, ASA, and CLP effectively protected rats against middle cerebral artery occlusion/reperfusion (MCAO/R) injury by suppressing the NOX2/IL-6/ STAT3 pathway. These novel findings provide theoretical basis and experimental evidence for the rationality of clinical combined use of drugs in the treatment of ischemic stroke.

Long noncoding RNA LINC00324 exerts protumorigenic effects on liver cancer stem cells by upregulating fas ligand via PU box binding protein.

Hepatocellular carcinoma (HCC) represents a major cause of cancer death, but the molecular mechanism for its development has not yet been well characterized. Long noncoding RNAs (lncRNAs) are involved in a wide range of biological processes via their roles as oncogenes or tumor suppressor genes. The present study aimed to elucidate the role of LINC00324 in HCC through its interaction with Fas ligand (FasL). Initially, microarray-based gene expression profiling of HCC was employed to identify differentially expressed genes. Next, the expression of LINC00324 in HCC tissues and liver cancer stem cell (LCSC) lines was examined using RT-qPCR. Then, the interaction among LINC00324, PU box binding protein (PU.1) and FasL was identified with RIP, ChIP and dual-luciferase reporter gene assays. The effect of LINC00324 on viability, proliferation, migration, invasion, and apoptosis as well as the tumorigenesis of transfected cells was examined with gain- and loss-of-function experiments. LINC00324 and FasL were highly expressed in HCC. LINC00324 regulated FasL expression via interaction with PU.1. Silencing of LINC00324 or FasL suppressed expression of stemness-related genes, cell viability, proliferation, migration, invasion, self-renewal, and tumorigenesis, but enhanced cell apoptosis. Taken together, LINC00324 promotes the expression of FasL through the recruitment of PU.1, which ultimately maintains the biological properties of LCSCs, thus, highlighting LINC00324 as a promising therapeutic candidate for HCC.

LncRNA LEF1-AS1 silencing diminishes EZH2 expression to delay hepatocellular carcinoma development by impairing CEBPB-interaction with CDCA7.

Hepatocellular carcinoma (HCC) is recognized for its high mortality rate worldwide. Based on intensive studies, long non-coding RNA (lncRNA) expression exerts significant effects on tumor suppression. Herein, we investigated the molecular mechanism of lymphoid enhancer-binding factor-1 antisense RNA 1 (LEF1-AS1) in HCC cells. Microarray-based gene expression analysis was adopted to predict and verify the differentially expressed genes in HCC, which predicted cell division cycle-associated 7 (CDCA7) and LEF1-AS1 to be highly expressed in HCC. The expression of LEF1-AS1, CDCA7, CCAAT/enhancer-binding protein beta (CEBPB) and enhancer of zeste homolog 2 (EZH2) was determined by means of reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. LncMap was used to predict the lncRNA-transcription factor-gene interaction in HCC. ChIP, RIP assay and dual luciferase reporter gene assay were employed to verify the relationship between the transcription factor and gene. Silencing of LEF1-AS1 could downregulate CDCA7 expression through CEBPB. Overexpression of LEF1-AS1, EZH2 and CDCA7 promoted proliferation and invasion in HCC cells. LEF1-AS1 promoted CDCA7 expression to further upregulate EZH2. Tumor formation in nude mice was assessed to verify the experimental results. Silencing of LEF1-AS1 inhibited the growth of tumors in vivo. Collectively, silencing LEF1-AS1 inhibited the proliferation and invasion of HCC cells by down-regulating EZH2 through the CEBPB-CDCA7 signaling pathway, which provides scientific evidence for the treatment of HCC.Abbreviations: HCC: Hepatocellular carcinoma; lncRNA: long non-coding RNA; LEF1-AS1: lymphoid enhancer-binding factor-1 antisense RNA 1; EZH2: enhancer of zeste homolog 2; CDCA7: cell division cycle-associated 7; GEO: Gene Expression Omnibus; NC: negative control; oe: overexpressed; RT-qPCR: reverse transcription quantitative polymerase chain reaction; PBS: phosphate buffered saline; HRP: horseradish peroxidase; OD: optical density; RIP: Radioimmunoprecipitation; ChIP: Chromatin immunoprecipitation; WT: wild type.

Upregulated microRNA-194 impairs stemness of cholangiocarcinoma cells through the Rho pathway via inhibition of ECT2.

Cholangiocarcinoma (CCA) is devastating for its delayed presence, difficulty in diagnosis, and high mortality. Other studies have supported the important role of microRNAs (miRNAs) in the pathogenesis of CCA, and the role of miR-194 was investigated in several human cancers, though, the molecular mechanism of miR-194 in CCA stem cells remains largely unknown. We aimed to identify the functional significance of miR-194 in CCA. The microarray-based analysis was applied to detect the epithelial cell transforming sequence 2 (ECT2) expression and predict the miRNA-regulated ECT2, followed by the identification of relationship between ECT2 and obtained miRNA by dual-luciferase reporter gene assay. The effects of depletion or ectopic expression of miR-194 on Rho pathway and the biological characteristics of CCA were assessed by reverse transcription quantitative polymerase chain reaction, immunoblotting, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, scratch test, Transwell, and flow cytometry. Lastly, tumor growth was assessed by xenograft tumor in nude mice. ECT2 was highly expressed while miR-194 was poorly expressed in CCA stem cells, and the targeting relation between ECT2 and miR-194 was proved. More important, the elevated expression of miR-194 or ECT2 silencing inhibited the Rho pathway, and further promoted the apoptosis and suppressed the stem cell proliferation, migration, and invasion of CCA in vitro. miR-194 inhibited the tumor growth in vivo. In a word, miR-194 inhibits ECT2 and blocks the activation of Rho signaling pathway, thus promoting apoptosis, inhibiting proliferation and migration of CCA stem cells, and suppressing tumor growth. The mechanism can be regarded as a target for treating CCA.

Circ-TCF4.85 silencing inhibits cancer progression through microRNA-486-5p-targeted inhibition of ABCF2 in hepatocellular carcinoma.

The current study aimed to explore the role of the circular RNA circ-TCF4.85 and its downstream target microRNA-486-5p (miR-486-5p) in hepatocellular carcinoma (HCC) development. Circ-TCF4.85 was detected to be highly expressed in HCC tissues. Next, we found that silencing of circ-TCF4.85 repressed HCC cell proliferation, invasion, and migration, while enhancing apoptosis. In addition, biotin-coupled probe pull-down and miRNA capture assays, as well as fluorescence in situ hybridization, confirmed that circ-TCF4.85 could bind to miR-486-5p. In rescue experiments, miR-486-5p had the potential to eliminate the tumor-suppressive effects of circ-TCF4.85 knockdown in HCC. Moreover, miR-486-5p was shown to target ABCF2 gene, which was positively regulated by circ-TCF4.85. Finally, nude mice subcutaneously injected with si-circ-TCF4.85-transfected HCC cells presented with inhibited xenograft tumor formation in vivo. Taken together, our results reveal that silencing of circ-TCF4.85 suppresses HCC progression via miR-486-5p-targeted inhibition of ABCF2.

microRNA-485-5p inhibits the progression of hepatocellular carcinoma through blocking the WBP2/Wnt signaling pathway.

microRNA-485-5p (miR-485-5p) has been shown to act as a tumor-suppressor gene in some cancers, such as ovarian epithelial tumors and oral tongue squamous cell carcinoma. However, with regard to the anti-tumor role of miR-485-5p in hepatocellular carcinoma (HCC), evidence is unexpectedly limited. In the present study, we investigated the expression and the role of miR-485-5p in the progression of HCC. Microarray analysis revealed that miR-485-5p was downregulated and WBP2 was upregulated in HCC, which was consistent with RT-qPCR and immunohistochemistry assays in the HCC tissues we collected. A negative correlation between the expression of miR-485-5p and WBP2 was also found in HCC tissues. It was predicted and confirmed that miR-485-5p could bind to WW domain binding protein 2 (WBP2) through in silico analysis of genetic sequences and an in vitro dual-luciferase reporter gene assay. Next, gain- or loss-of-function studies were applied in the HCC cell line (Huh7) to examine the effects of miR-485-5p and WBP2 on HCC cell behavior. The effects of miR-485-5p and WBP2 on the Wnt/ß-catenin signaling pathway were determined by TOP/FOP flash luciferase assays. miR-485-5p was shown to downregulate WBP2 and block the Wnt/ß-catenin signaling pathway. As expected, elevated miR-485-5p levels and inhibition of WBP2 protein expression exerted inhibitory effects on HCC cell proliferation, migration and invasion and, induced apoptosis. In vivo experiments were finally conducted, which confirmed that upregulation of miR-485-5p or depletion of WBP2 attenuated tumor growth. Collectively, our results suggest miR-485-5p can downregulate WBP2 to inhibit the development of HCC by the blockade of the Wnt/ß-catenin signaling, providing a novel molecular target for HCC treatment.

Xuezhitong capsule, an extract of Allium macrostemon Bunge, exhibits reverse cholesterol transport and accompanies high-density lipoprotein levels to protect against hyperlipidemia in ApoE-/- mice.

BACKGROUND: Xuezhitong capsules (XZT) are derived from Xie Bai and used for abnormal lipid homeostasis treatment through maintained metabolic balance. However, their mechanisms are largely unknown. Here, we mainly assessed the contribution of reverse cholesterol transport (RCT) and the accompanying increase in the high-density lipoprotein (HDL) effects of XZT to cholesterol dysfunction amelioration in mice. METHODS: We assessed serum lipids by using enzymatic kits. We observed atherosclerotic plaque formation by hematoxylin-eosin (HE) and Oil Red O staining. We studied the lipid metabolism, fatty acid synthase (FAS), HDL, low-density lipoprotein receptor (LDLR), triglyceride (TG) metabolic enzyme expression levels, and RCT function in various tissues upon stimulation with high-fat diet, XZT, and some positive drugs by ELISA. RESULTS: After 34 weeks of high-fat diet administration, blood lipids levels increased because attenuated by XZT treatment (800 and 1,600 mg/kg, i.g.). XZT improved the lipid metabolism instability, induced RCT activation, and subsequently increased the HDL levels in hyperlipidemic mice (P<0.05). FAS (P<0.05) and LDLR (P<0.01) levels also remarkably improved. The effects of XZT were closely associated with RCT activation and the accompanying increase in the HDL levels, as characterized by XZT-induced preservation in ATP-binding cassette transporter member 1 (ABCA1), scavenger receptor class B type 1 (SRB1), acyl coenzyme A: cholesterol acyltransferase (ACAT), lecithin cholesterol acyltransferase (LCAT), apolipoprotein A I (ApoA1) and apolipoprotein B (ApoB). However, XZT showed no effect on high fat diet-activated TG metabolic enzyme expression levels (P>0.05). CONCLUSIONS: XZT are promising drugs in balancing the cholesterol dysfunction from hyperlipidemia through RCT activation and accompanying increase in HDL levels.

Intrahepatic bile duct exploration lithotomy is a useful adjunctive hepatectomy method for bilateral primary hepatolithiasis: an eight-year experience at a single centre.

BACKGROUND: To evaluate the perioperative and long-term results of intrahepatic bile duct exploration lithotomy (IHBDIL) combined with hepatectomy for patients with complicated bilateral primary hepatolithiasis. METHODS: A study was conducted involving 56 patients with complicated bilateral primary hepatolithiasis who underwent IHBDIL combined with hepatectomy at our hospital from January 2006 to December 2014. The perioperative and long-term outcomes that were retrospectively analysed included the stone clearance rate, operative morbidity and mortality, and stone recurrence rate. Patients with a preoperative diagnosis of cholangiocarcinoma were excluded. RESULTS: In all 56 patients, hepatic duct stones were located in the bilateral IHBD. The surgical method was IHBDIL combined with hepatectomy. Postoperative complications occurred in 15 patients (26.8%), 14 patients responded to conservative management, and there was 1 case of postoperative mortality because of hepatic failure. The overall initial success rate of stone clearance was 85.7%, and the final clearance rate was 92.9% following postoperative choledochoscopic lithotripsy. The stone recurrence rate was 13.5%, and the occurrence of postoperative cholangitis was 10.9% during the follow-up period. CONCLUSION: IHBDIL combined with hepatectomy is a safe, effective, and promising treatment for patients with complicated bilateral primary hepatolithiasis. The perioperative and long-term outcomes are satisfactory for complicated bilateral primary hepatolithiasis.