miRNA326-5p Targets DKC1 Gene to Regulate Apoptosis-Related Proteins and Intervene in the Development of Neuroblastoma.

Objective: To study the effect of congenital dyskeratosis 1 (DKC1) on neuroblastoma and its regulation mechanism. Methods: The expression of DKC1 in neuroblastoma was analyzed by TCGA database and molecular assay. NB cells were transfected with siDKC1 to observe the effects of DKC1 on proliferation, cloning, metastasis, and invasion, and apoptosis and apoptosis-related proteins. The tumor-bearing mouse model was constructed, shDKC1 was transfected to observe the tumor growth and tumor tissue changes, and the expression of DKC1 and Ki-67 was detected. Screening and identification of miRNA326-5p targeting DKC1. NB cells were treated with miRNA326-5p mimic or inhibitors to detect the expression of DKC1. NB cells were transfected with miRNA326-5p and DKC1 mimics to detect cell proliferation, apoptosis, and apoptotic protein expression. Results: DKC1 was highly expressed in NB cells and tissues. The activity, proliferation, invasion, and migration of NB cells were significantly decreased by DKC1 gene knockout, while apoptosis was significantly increased. The expression level of B-cell lymphoma-2 in shDKC1 group was significantly lower than that of the control group, while the expression level of BAK, BAX, and caspase-3 was significantly higher than that of the control group. The results of experiments on tumor-bearing mice were consistent with the above results. The results of miRNA assay showed that miRNA326-5p could bind DKC1 mRNA to inhibit the protein expression, thereby inhibiting the proliferation of NB cells, promoting their apoptosis, and regulating the expression of apoptotic proteins. Conclusion: miRNA326-5p targeting DKC1 mRNA regulates apoptosis-related proteins to inhibit neuroblastoma proliferation and promote the apoptotic process.

The mechanism and effects of remdesivir-induced developmental toxicity in zebrafish: Blood flow dysfunction and behavioral alterations.

The antiviral drug remdesivir has been used to treat the growing number of coronavirus disease 2019 (COVID-19) patients. However, the drug is mainly excreted through urine and feces and introduced into the environment to affect non-target organisms, including fish, which has raised concerns about potential ecotoxicological effects on aquatic organisms. Moreover, studies on the ecological impacts of remdesivir on aquatic environments have not been reported. Here, we aimed to explore the toxicological impacts of microinjection of remdesivir on zebrafish early embryonic development and larvae and the associated mechanism. We found that 100 µM remdesivir delayed epiboly and impaired convergent movement of embryos during gastrulation, and dose-dependent increases in mortality and malformation were observed in remdesivir-treated embryos. Moreover, 10-100 µM remdesivir decreased blood flow and swimming velocity and altered the behavior of larvae. In terms of molecular mechanisms, 80 differentially expressed genes (DEGs) were identified by transcriptome analysis in the remdesivir-treated group. Some of these DEGs, such as manf, kif3a, hnf1ba, rgn, prkcz, egr1, fosab, nr4a1, and ptgs2b, were mainly involved in early embryonic development, neuronal developmental disorders, vascular disease and the blood flow pathway. These data reveal that remdesivir can impair early embryonic development, blood flow and behavior of zebrafish embryos/larvae, probably due to alterations at the transcriptome level. This study suggests that it is important to avoid the discharge of remdesivir to aquatic ecosystems and provides a theoretical foundation to hinder remdesivir-induced ecotoxicity to aquatic environments.

SOX17 inhibits proliferation and invasion of neuroblastoma through CXCL12/CXCR4 signaling axis.

SOX17 has been shown to be involved in the transcriptional regulation of CXCR4, and CXCL12 functions by binding to its receptor CXCR4. Here, we explored the expression of SOX17 in neuroblastoma (NB), its mutual regulation with CXCL12, and its effects on cancer cell proliferation, migration and invasion. Five human NB cell lines and 15 pairs of NB and adjacent tissue specimens were used, to conduct RT-qPCR, immunohistochemistry, western blot, ELISA, CCK-8, colony formation, Edu, transwell, chromatin immunoprecipitation (ChIP), and dual-luciferase assays, to study the role of SOX17 in NB. SOX17 levels were reduced in both NB tissues and cell lines. SOX17 inhibited NB tumor growth, migration and invasion in vivo and suppressed NB cell proliferation, migration, and invasion in vitro. SOX17 knockdown or overexpression revealed a negative correlation between SOX17 and CXCL12/CXCR4 pathway activation. ChIP and dual-luciferase assays in NB cells demonstrated that SOX17 significantly inhibited CXCL12 gene and protein levels by binding to CXCL12 promoter regions. In vivo and in vitro experiments using the CXCR4 antagonist, AMD3100, demonstrated that cell proliferation, migration and invasion were significantly abrogated by AMD3100 in NB cells with SOX17 knocked down. Further, AMD3100 impaired growth of NB tumors with SOX17 knocked down in mice. Importantly, SOX17 bound to the CXCL12 promoter, which then activated downstream targets to regulate cell viability, proliferation, and migration. In conclusion, our data demonstrate that SOX17 expression is repressed in NB tissues and cells, and that SOX17 suppresses NB tumor formation and proliferation through inhibition of CXCL12/CXCR4 signaling.

Effect of FibroScan test in antiviral therapy for HBV-infected patients with ALT <2 upper limit of normal.

OBJECTIVE: The objective of this study is to detect the liver stiffness of hepatitis B virus (HBV)-infected patients with an alanine aminotransferase (ALT) level of <2 upper limit of normal (2ULN) by FibroScan and compare histological changes to assess the progression of liver lesions and its test results. METHODS: There were 36 patients who had a liver FibroScan degree of >7.3 KD (F1), and a liver biopsy was conducted. Along with serology of liver fibrosis, indexes and hierarchical processing were used for evaluation. The correlation between these factors was analyzed. RESULTS: The histopathological results of the liver were closely correlated with liver hardness. In the pathological diagnosis of chronic hepatitis, G represents the grade of inflammation and S represents the stage of hepatic fibrosis. Pathological examination results of H&E staining of liver tissue sections revealed that the area under the work characteristic curve of the subjects in G2S1, G2S2, G3S2, and G3S3 stages was 0.923, 0.916, 0.955, and 0.971, respectively, with diagnostic cut-off values of 9.03, 9.85, 15.14, and 30.67, respectively. Furthermore, hydroxyapatite, type III procollagen, laminin, and type IV collagen of serum fibrosis indexes are associated with liver stiffness values (P < 0.05). CONCLUSION: FibroScan can be used as an alternative to liver biopsy. It is meaningful in determining whether HBV infected patients with an ALT level of <2 ULN should receive antiviral therapy.

IGF1R facilitates epithelial-mesenchymal transition and cancer stem cell properties in neuroblastoma via the STAT3/AKT axis.

BACKGROUND: Neuroblastoma (NB) displays the most heterogeneity in clinical manifestation. The insulin-like growth factor 1 receptor (IGF1R) has long been recognized for its role in tumourigenesis and growth. The IGF/IGF1R pathway is important in maintaining cell survival. It is reported that IGF1R participates in the occurrence of NB, but the mechanism is still unclear. METHODS: Human NB cell lines IMR-32 and SH-SY5Y were recruited in this study. IGF1R was knocked down by transfection with short hairpin RNA. Signal transducer and activator of transcription 3 (STAT3) expression was inhibited by Cryptotanshinone treatment. Cell proliferation, migration, and invasion were determined by MTT assay, wound healing assay, and cell invasion assay, respectively. The cancer stem cell properties were characterized by tumour sphere formation assay and colony formation assay. The mRNA and protein expression levels of related proteins were detected by RT-PCR and Western blot, respectively. RESULTS: The knockdown of IGF1R inhibits NB cell tumourigenesis and the epithelial-mesenchymal transition (EMT) of NB cells. Additionally, IGF1R was found to stimulate cancer stem cell-like properties in NPC cells. The knockdown of IGF1R significantly reduced the phosphorylation of AKT, and STAT3, indicating that the activation of the AKT and STAT3 pathways was inhibited by IGF1R knockdown. Furthermore, IGF1R was demonstrated to stimulate cancer stem cell-like properties in NB cells via the regulation of the STAT3/AKT axis. CONCLUSION: IGF1R promotes cancer stem cell properties to facilitate EMT in neuroblastoma via the STAT3/AKT axis.

FGF represses metastasis of neuroblastoma regulated by MYCN and TGF-ß1 induced LMO1 via control of let-7 expression.

BACKGROUND: MYCN and LMO1 amplification are commonly observed in neuroblastoma (NB), which was often accompanied by genetic loss of let-7 microRNA (miRNA). Fibroblast growth factor (FGF) was found to regulate let-7 miRNA expression via FGF receptor substrate 2 (FRS2), which then activates transforming growth factor beta (TGF-ß) signaling. METHODS: Expression of MYCN, LMO1, FRS2, let-7, and TGF-ß receptor I (TGFßRI) was selectively knocked-down or enhanced in NB cells. Proliferation, invasion, migration, metastasis and tumorigenesis of NB, expression of downstream signaling factors and metastasis-associated protein were evaluated. RESULTS: Knock-down on either MYCN or LMO1 has led to inhibition on proliferation, invasion, migration, and metastasis of NB cells, and knock-down of FRS2 resulted in increases in MYCN and LMO1 expression and enhanced invasion, migration and metastasis of NB cells. Decreased expression of TGF-ß1 or TGFßRI led to decrease expression in LMO1 and proliferation, invasion, migration and metastasis markers, except MYCN expression which appeared not to be regulated by TGF-ß1 or TGFßRI. Furthermore, let-7 miRNA was shown to decrease the expression levels of TGF-ßRI, LMO1 and MYCN. CONCLUSIONS: FGF regulates MYCN and TGF-ß1-induced LMO1 and metastasis of NB cells via let-7 miRNA.

Oridonin synergizes with Nutlin-3 in osteosarcoma cells by modulating the levels of multiple Bcl-2 family proteins.

The small-molecule inhibitors of p53-murine double minute 2 interaction, such as Nutlin-3, are effective against cancers bearing wild-type p53. However, murine double minute 2 inhibitors often are unable to completely eliminate solid tumor cells. To address this issue, we investigated the anticancer effects of Nutlin-3 in combination with Oridonin in osteosarcoma cells. We found that Oridonin at sub-toxic concentrations synergistically enhanced Nutlin-3-mediated cell viability inhibition in wild-type p53 U2OS and SJSA-1, but not in p53-mutant MNNG/HOS and in null-p53 Saos-2 osteosarcoma cell lines. Importantly, in the presence of Oridonin, Nutlin-3 could completely abolish cell viability in the wild-type p53 osteosarcoma cell lines. Western blotting analysis showed that Oridonin treatment rapidly and distinctly increased the levels of all three forms of Bim and also markedly reduced the levels of Bcl-2 and Bcl-xl in osteosarcoma cells. Western blotting analysis further showed that Oridonin considerably enhanced Nutlin-3-triggered activation of caspases-9 and -3 and poly(ADP-ribose) polymerase cleavage. Flow cytometry assay showed that Oridonin significantly enhanced Nutlin-3-mediated apoptosis in wild-type p53 osteosarcoma cells. Overall, our results suggest that the combined treatment of Nutlin-3 plus Oridonin may offer a novel therapeutic strategy for osteosarcoma.

EGF Protects Cells Against Dox-Induced Growth Arrest Through Activating Cyclin D1 Expression.

It has been reported that the antitumor drug doxorubicin (Dox) exerts its toxic effects via GATA-4 depletion and that over-expression of GATA-4 reverses Dox-induced toxicity and apoptosis; however, the precise mechanisms remain unclear. In this study, we observed, for the first time, that EGF protects cells against Dox-mediated growth arrest, G2/M-phase arrest, and apoptosis. Additionally, EGF expression was down-regulated in Dox-treated cells and up-regulated in GATA-4 over-expressing cells. Utilizing real-time PCR and western blotting analysis, we found that the expression of the cell cycle-associated protein cyclin D1 was inhibited in GATA-4-silenced cells and Dox-treated cells and was enhanced in GATA-4 over-expressing cells and EGF-treated cells. Furthermore, EGF treatment reversed the inhibited expression of cyclin D1 that was mediated by GATA-4 RNAi or Dox. Our results indicate that EGF, as a downstream target of Dox, may be involved in Dox-induced toxicity as well as in the protective role of GATA-4 against toxicity induced by Dox via regulating cyclin D1 expression, which elucidates a new molecular mechanism of Dox toxicity with important clinical implications.

Insulin induces proliferation and cardiac differentiation of P19CL6 cells in a dose-dependent manner.

Insulin is a peptide hormone produced by beta cells of the pancreas. The roles of insulin in energy metabolism have been well studied, with most of the attention focused on glucose utilization, but the roles of insulin in cell proliferation and differentiation remain unclear. In this study, we observed for the first time that 10 nmol/L insulin treatment induces cell proliferation and cardiac differentiation of P19CL6 cells, whereas 50 and 100 nmol/L insulin treatment induces P19CL6 cell apoptosis and blocks cardiac differentiation of P19CL6 cells. By using real-time polymerase chain reaction (PCR) and Western blotting analysis, we found that the mRNA levels of cyclin D1 and α myosin heavy chain (α-MHC) are induced upon 10 nmol/L insulin stimulation and inhibited upon 50/100 nmol/L insulin treatment, whereas the mRNA levels of BCL-2-antagonist of cell death (BAD) exists a reverse trend. The similar results were observed in P19CL6 cells expressing GATA-6 or peroxisome proliferator-activated receptor α (PPARα). Our results identified the downstream targets of insulin, cyclin D1, BAD, α-MHC, and GATA-4, elucidate a novel molecular mechanism of insulin in promoting cell proliferation and differentiation.

Insulin induces C2C12 cell proliferation and apoptosis through regulation of cyclin D1 and BAD expression.

Insulin is a secreted peptide hormone identified in human pancreas to promote glucose utilization. Insulin has been observed to induce cell proliferation and myogenesis in C2C12 cells. The precise mechanisms underlying the proliferation of C2C12 cells induced by insulin remain unclear. In this study, we observed for the first time that 10 nM insulin treatment promotes C2C12 cell proliferation. Additionally, 50 and 100 nM insulin treatment induces C2C12 cell apoptosis. By utilizing real-time PCR and Western blotting analysis, we found that the mRNA levels of cyclinD1 and BAD are induced upon 10 and 50 nM/100 nM insulin treatment, respectively. The similar results were observed in C2C12 cells expressing GATA-6 or PPARα. Our results identify for the first time the downstream targets of insulin, cyclin D1, and BAD, elucidate a new molecular mechanism of insulin in promoting cell proliferation and apoptosis.

miR-200b targets GATA-4 during cell growth and differentiation.

GATA-4 is an important transcription factor involved in several developmental processes of the heart, such as cardiac myocyte proliferation, differentiation and survival. The precise mechanisms underlying the regulation of GATA-4 remain unclear, this is especially true for the mechanisms that mediate the post-transcriptional regulation of GATA-4. Here, we demonstrate that miR-200b, a member of the miR-200 family, is a critical regulator of GATA-4. Overexpression of miR-200b leads to the downregulation of GATA-4 mRNA and a decrease in GATA-4 protein levels. Moreover, miR-200b not only inhibits cell growth and differentiation but also reverses the growth response mediated by GATA-4, whereas depletion of miR-200b leads to a slight reversal of the anti-growth response achieved by knocking down endogenous GATA-4. More importantly, the cell cycle-associated gene cyclin D1, which is a downstream target of GATA-4, is also regulated by miR-200b. Thus, miR-200b targets GATA-4 to downregulate the expression of cyclin D1 and myosin heavy chain (MHC), thereby regulating cell growth and differentiation.

Involvement of heat shock protein 27 in the susceptibility of KT human breast cancer cells to UVC and interferon lethality.

Revealing the key molecules regulating the stress-response pathways in human cells is an intriguing problem. Chaperones, such as glucose-regulated protein 78 (GRP78) and heat shock protein 27 (HSP27), are important molecules for protecting the viability of human cells; however, it remains to be further clarified whether the molecules differentially modulate cellular responses to various types of stressors, such as DNA-damaging ultraviolet ray C (principally 254-nm wavelength, UVC) and cytocidal cytokine interferons. In the present study, the human breast cancer cell lines KT and MCF-7 were examined for GRP78 and HSP27 expression following exposure to UVC and human interferon-ß (HuIFN-ß). The KT cells demonstrated a higher sensitivity to both UVC and HuIFN-ß lethality than MCF-7 cells. The cellular expression levels of GRP78 in KT cells, assessed by western blot analysis, were approximately 2-fold higher than that in MCF-7 cells, while the expression of HSP27 in the KT cells was 20% of the expression in the MCF-7 cells. Decreased resistance to UVC lethality was observed in GRP78 siRNA-transfected KT cells. In addition, HSP27 cDNA transfection of KT cells resulted in an increased resistance to UVC lethality. The cDNA-transfected KT cells showed an increased viability against HuIFN-ß, compared with that of empty vector-transfected cells. By contrast, KT cells pretreated with HuIFN-ß and irradiated with UVC demonstrated an increased resistance to UVC lethality, in association with increased levels of HSP27 expression. Thus, HSP27 may control the survival response pathways to both UVC and HuIFN-ß in the human cells examined.

Erythropoietin attenuates cardiac dysfunction by increasing myocardial angiogenesis and inhibiting interstitial fibrosis in diabetic rats.

BACKGROUND: Recent studies revealed that erythropoietin (EPO) has tissue-protective effects in the heart by increasing vascular endothelial growth factor (VEGF) expression and attenuating myocardial fibrosis in ischemia models. In this study, we investigated the effect of EPO on ventricular remodeling and blood vessel growth in diabetic rats. METHODS: Male SD rats were randomly divided into 3 groups: control rats, streptozotocin (STZ)-induced diabetic rats, and diabetic rats treated with 1000 U/kg EPO by subcutaneous injection once per week. Twelve weeks later, echocardiography was conducted, and blood samples were collected for counting of peripheral blood endothelial progenitor cells (EPCs). Myocardial tissues were collected, quantitative real-time PCR (RT-PCR) was used to detect the mRNA expression of VEGF and EPO-receptor (EPOR), and Western blotting was used to detect the protein expression of VEGF and EPOR. VEGF, EPOR, transforming growth factor beta (TGF-ß), and CD31 levels in the myocardium were determined by immunohistochemistry. To detect cardiac hypertrophy, immunohistochemistry of collagen type I, collagen type III, and Picrosirius Red staining were performed, and cardiomyocyte cross-sectional area was measured. RESULTS: After 12 weeks STZ injection, blood glucose increased significantly and remained consistently elevated. EPO treatment significantly improved cardiac contractility and reduced diastolic dysfunction. Rats receiving the EPO injection showed a significant increase in circulating EPCs (27.85 ± 3.43%, P < 0.01) compared with diabetic untreated animals. EPO injection significantly increased capillary density as well as EPOR and VEGF expression in left ventricular myocardial tissue from diabetic rats. Moreover, EPO inhibited interstitial collagen deposition and reduced TGF-ß expression. CONCLUSIONS: Treatment with EPO protects cardiac tissue in diabetic animals by increasing VEGF and EPOR expression levels, leading to improved revascularization and the inhibition of cardiac fibrosis.

Effects of Doxorubicin and Fenofibrate on the activities of NADH oxidase and citrate synthase in mice.

Doxorubicin (Dox) has widely been used as an anticancer drug, but its use is limited by serious toxicity to the heart, kidney and liver. Mitochondrial dysfunction is one of the potential mechanisms of toxicity but not fully understood. Fenofibrate, one of the peroxisome proliferator-activated receptor-alpha (PPARα) ligands, is involved in lipid metabolism which takes place primarily in the mitochondria, so mitochondrial function may be affected by fenofibrate. Therefore, we investigated the effects of DOX and fenofibrate on activities of both mitochondrial citrate synthase and NADH oxidase, which are marker enzymes in the tricarboxylic acid (TCA) cycle and a measure of the complex I-III-IV activity in electron transport chain, respectively. Dox (15 mg/kg) and/or fenofibrate (100 mg/kg/day) were administered to mice for 3 or 14 days, and the activities of citrate synthase and NADH oxidase were measured. Our study showed that Dox significantly inhibits the activity of citrate synthase while fenofibrate induces the activity. Similar to citrate synthase, NADH oxidase activity was also induced by fenofibrate except in spleen but inhibited by Dox except in the heart and liver. Furthermore, fenofibrate not only protects citrate synthase activity from Dox-induced toxicity in the ventricle but also significantly rescues NADH oxidase activity in the kidney. These results reveal the actions of fenofibrate and Dox on the mitochondria, and the underlying mechanism may be related to the toxicity of Dox, which has clinical implications in the side effects of Dox treatment by modulation of mitochondrial function.

Synthesis and antitumor activity of amino acid ester derivatives containing 5-fluorouracil.

A series of amino acid ester derivatives containing 5-fluorouracil were synthesized using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC*HCl) and N-hydroxybenzotriazole (HOBt) as a coupling agent. The structures of the products were assigned by NMR, MS, IR etc. The in vitro antitumor activity tests against leukaemia HL-60 and liver cancer BEL-7402 indicated that (R)-ethyl 2-(2-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)-3-(4-hydroxyphenyl) propanoate showed more inhibitory effect against BEL-7402 than 5-FU.

Capillary electrophoretic determination of glucosamine in osteoarthritis tablets via microwave-accelerated dansylation.

A robust capillary electrophoretic method has been established to separate aminosaccharides including glucosamine, galactosamine, N-methyl-glucamine, N-acetyl-glucosamine and amino glucuronic acid. All the aminosaccharides were dansylated fast under microwave irradiation at 385 W for 6 min (about 50-fold faster than common methods) and detected via on-line UV adsorption at 214 nm. Baseline separation of the dansylatied products was achieved in 20 min using a running buffer of 320 mM borate at pH 9.50. Quantitation of glucosamine in osteoarthritis tablets was then conducted. A linear working range was found in between 2.00 microg/mL and 1.80 mg/mL with linear regression coefficient of 0.9964. The limit of detection reached 1.00 microg/mL glucosamine (signal-to-noise, S/N=3). Recoveries were determined by spiking a known amount of glucosamine in tablet-extracted solutions, giving a range of 88.0-99.7%. The run-to-run relative standard deviation was 0.24% (n=5) for migration time and 2.72% for peak area.