miR-373 inhibits autophagy and further promotes apoptosis of cholangiocarcinoma cells by targeting ULK1.

Intrahepatic cholangiocarcinoma is a malignant tumor originating from intrahepatic bile ducts. Surgical therapy, radiotherapy, and chemotherapy are taken to treat this disease, but it is prone to recurrence and metastasis, with poor prognosis. Therefore, it is of great significance to explore new targets and molecular mechanisms for the development of cholangiocarcinoma cells. Clinical cholangiocarcinoma tissues from patients and four human cholangiocarcinoma cell lines were analyzed for microRNA-373 (miR-373) expression. For investigating whether miR-373 directly modulated unc-51 like autophagy activating kinase 1 (ULK1), dual-luciferase reporter assay was performed. In addition, CCK-8 assay, flow cytometry, western blot, and immunofluorescence were applied to evaluate the proliferation, apoptosis, and autophagy of cholangiocytic hepatocellular carcinoma cells. miR-373 downregulation was observed in clinical tissues and cell lines of cholangiocarcinoma. Overexpression of miR-373 reduced proliferation, enhanced apoptosis, and raised expression levels of pro-apoptosis proteins including BCL2 associated X (Bax), Caspase-3, and Caspase-9. Moreover, overexpression of miR-373 downregulated expression levels of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II, Beclin-1, and promoted P62 expression on mRNA and protein levels. After miR-373 knockdown, all indexes of apoptosis and autophagy mentioned above were reversed. Luciferase activity was decreased after cotransfection of miR-373 mimic and wild-type ULK1 vector. Also, miR-373 overexpression inhibited ULK1 expression. Importantly, overexpression of miR-373 weakened expressions of ULK1, LC3, Beclin-1, and Bcl-2, and enhanced expressions of P62, Bax, Caspase-3, and Caspase-9. miR-373 mimic treatment and subsequent ULK1 overexpression, induced reverse regulation in expressions of these proteins, compared with overexpression of miR-373 only. miR-373 targeted ULK1 to initiate inhibition of autophagy and subsequent promotion of apoptosis in cholangiocarcinoma cells.

Highly informative single-copy nuclear microsatellite DNA markers developed using an AFLP-SSR approach in black spruce (Picea mariana) and red spruce (P. rubens).

BACKGROUND: Microsatellites or simple sequence repeats (SSRs) are highly informative molecular markers for various biological studies in plants. In spruce (Picea) and other conifers, the development of single-copy polymorphic genomic microsatellite markers is quite difficult, owing primarily to the large genome size and predominance of repetitive DNA sequences throughout the genome. We have developed highly informative single-locus genomic microsatellite markers in black spruce (Picea mariana) and red spruce (Picea rubens) using a simple but efficient method based on a combination of AFLP and microsatellite technologies. PRINCIPAL FINDINGS: A microsatellite-enriched library was constructed from genomic AFLP DNA fragments of black spruce. Sequencing of the 108 putative SSR-containing clones provided 94 unique sequences with microsatellites. Twenty-two of the designed 34 primer pairs yielded scorable amplicons, with single-locus patterns. Fourteen of these microsatellite markers were characterized in 30 black spruce and 30 red spruce individuals drawn from many populations. The number of alleles at a polymorphic locus ranged from 2 to 18, with a mean of 9.3 in black spruce, and from 3 to 15, with a mean of 6.2 alleles in red spruce. The polymorphic information content or expected heterozygosity ranged from 0.340 to 0.909 (mean = 0.67) in black spruce and from 0.161 to 0.851 (mean = 0.62) in red spruce. Ten SSR markers showing inter-parental polymorphism inherited in a single-locus Mendelian mode, with two cases of distorted segregation. Primer pairs for almost all polymorphic SSR loci resolved microsatellites of comparable size in Picea glauca, P. engelmannii, P. sitchensis, and P. abies. SIGNIFICANCE: The AFLP-based microsatellite-enriched library appears to be a rapid, cost-effective approach for isolating and developing single-locus informative genomic microsatellite markers in black spruce. The markers developed should be useful in black spruce, red spruce and other Picea species for various genetics, genomics, breeding, forensics, conservation studies and applications.

Primary human hepatocyte transplantation in the therapy of hepatic failure: 2 cases report.

Liver failure is the end stage of hepatopathy with unfavorable prognosis. In two patients with liver failure, viable primary human hepatocytes, obtained from resected liver tissue of patients with hepatolithiasis, were transplanted into the spleen by interventional therapy through femoral arterial cannula. After transplantation, the patients' clinical symptoms and liver function were significantly improved. However, their bilirubin increased within six days following transplantation. One suffered from hepatic coma and give up treatment and the other patient died fourteen days after transplantation. It is technically safe to treat liver failure by intrasplenic transplantation of adult hepatocytes and the clinical efficacy has been confirmed. How to make transplanted hepatic cells proliferate and functionally survive is the key point to maintain continuous improvement of the recipient's hepatic function.

Down-regulation of c-Myc expression inhibits the invasion of bile duct carcinoma cells.

Cholangiocarcinoma is the second most common primary hepatic tumour originating from biliary tract epithelial cells with poor prognosis. Enhanced c-Myc protein expression contributes to many aspects of tumour cell biology. Although the ability of c-Myc to drive unrestricted cell proliferation and to inhibit cell differentiation had been well recognized, whether down-regulated c-Myc expression can inhibit tumour cell invasion still remains to be explored. The c-Myc ASODN (antisense oligodeoxyribonucleotide) and NSODN (nonsense oligodeoxyribonucleotide) were designed, synthesized and transfected into human QBC939 bile duct carcinoma cells using the Lipofectamine 2000 reagent. The protein expression of c-Myc was detected by Western blot. A transwell experiment was applied to evaluate the invasive capacity of the QBC939 cells. c-Myc ASODN could significantly suppress the c-Myc protein expression (P<0.05) and the invasion (P<0.01) of QBC939 cells transfected with c-Myc ASODN compared with that in the control and c-Myc NSODN-transfected group. Thus in the present study we show that down-regulation of c-Myc expression can inhibit the invasion of QBC939 cells in vitro.

[Expression of Toll like receptor 4 in peripheral-blood mononuclear cells of patients with acute cerebral infarction and its correlation with clinical subtypes and the severity of the disease].

AIM: To investigate the expression of Toll like receptor 4 (TLR4) in peripheral-blood mononuclear cells (PBMC) of patients with acute cerebral infarction and its correlation with clinical subtypes and the severity of the disease. METHODS: The levels of mRNA and protein of TLR4 in PBMC of 66 patients with acute cerebral infraction were examined by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry (FCM), respectively. Clinical neurological deficit score and OCSP (Oxford Community Stroke Project) clinical subtype were performed in the patients within 3 days after admission. RESULTS: The expression of TLR4 in PBMC of patients with acute cerebral infraction (positive rate: 66.87+/-16.27%, fluorescence intensity: 11.62+/-4.39) was significantly higher than normal controls (P<0.01, respectively). 66 patients were divided into 4 groups based on OCSP. There were 9 patients with TACI, 39 patients with PACI, 7 patients with POCI, and 11 patients with LACI. The differences in the expression levels of TLR4 protein and mRNA in these groups have no statistic significance. Meanwhile, there were 27 mild cases, 30 median cases, and 9 severe cases according to the neurological deficit score. The differences in the expression levels of TLR4 protein and mRNA in these groups have statistic significance (P<0.05). The expression of TLR4 protein and mRNA were correlated positively with the severity of the disease (r=0.85, 0.87; P=0.01, 0.01). CONCLUSION: The increased expression of TLR4 may be associated with the progression of acute cerebral infarction. It may be a risk factor of acute cerebral infarction.

The POM monoclonals: a comprehensive set of antibodies to non-overlapping prion protein epitopes.

PrP(Sc), a misfolded and aggregated form of the cellular prion protein PrP(C), is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrP(C) in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrP(C) and PrP(Sc). Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrP(C). Three epitopes are located within the N-terminal octarepeat region, one is situated within the central unstructured region, and four epitopes are discontinuous within the globular C-proximal domain of PrP(C). Some of these antibodies recognize epitopes that are resilient to protease digestion in PrP(Sc). Other antibodies immunoprecipitate PrP(C), but not PrP(Sc). A third group was found to immunoprecipitate both PrP isoforms. Some of the latter antibodies could be blocked with epitope-mimicking peptides, and incubation with an excess of these peptides allowed for immunochromatography of PrP(C) and PrP(Sc). Amino-proximal antibodies were found to react with repetitive PrP(C) epitopes, thereby vastly increasing their avidity. We have also created functional single-chain miniantibodies from selected POMs, which retained the binding characteristics despite their low molecular mass. The POM collection, thus, represents a unique set of reagents allowing for studies with a variety of techniques, including western blotting, ELISA, immunoprecipitation, conformation-dependent immunoassays, and plasmon surface plasmon resonance-based assays.

[Screen of inflammatory genes regulated by heat shock factor 1 and corroboration with SOCS3 gene].

OBJECTIVE: To screen the inflammatory mediators genes regulated by HSF1, and explore the mechanism of downstream genes regulated by HSF1. METHODS: HSF-/- and HSF1+/+ mice were injected with 15 mg/kg LPS intraperitoneally (ip), respectively, and were treated as previous after HSR. The total RNA of lung tissues were extracted and filtrated by SuperArray gene Microarry. The promoter of candidate genes were analyzed by transcription element search software to search for heat shock element (HSE). Select the suppressor of cytokine signaling 3 (SOCS3) with HSE. Macrophage cells were stimulated with 400 ng/mL LPS, and were treated as previous after HSR, then the total RNA was extracted respectively. RT-PCR and northern blot assay were performed to detect the expression levels of SOCS3 mRNA. RESULTS: Fifteen genes were repressed by HSF1, including 9 genes with complete HSE. Eleven genes were accelerated by HSF1 possibly, including 8 genes with complete HSE. The promoter of SOCS3 gene contained one complete HSE. LPS stimulation obviously increased the levels of SOCS3 mRNA in macrophages of RAW264.7 mice, which was inhibited by HSR and over-expression of HSF1. CONCLUSION: HSR or HSF1 inhibits LPS induced expression of SOCS3 mRNA; HSF1 might inhibit LPS-induced expression of SOCS3 mRNA by binding to HSE in the promoter of SOCS3 gene.

[HSP70 inhibits smac release from the mitochondria and protects against H2O2-induced apoptosis in C2C12 myogenic cells].

OBJECTIVE: To observe whether HSP70 could protect against H2O2-induced apoptosis in C2C12 myogenic cells by inhibiting Smac release from the mitochondria. METHODS: HSP70 gene and full length Smac gene was transiently transfected in C2C12 myogenic cells by lipofectamine and the protein levels of HSP70 and Smac were analysed by Western blotting. Hoechst 33 258 staining was used to examine cell morphological changes and to calculate percentage of apoptotic nuclei. DNA ladder pattern on agarose gel electrophoresis was used to observe the DNA fragmentation. Activities of caspase-3 and caspase-9 were assayed with Western blotting. The release of Smac from the mitochondria to the cytoplasm was observed by immunofluorescence. RESULTS: H2O2 ( 0.5 mmol/L ) activated caspase-3, caspase-9 8 h after the treatment and specific morphological changes of apoptosis 12 h after the treatment, and overexpression of Smac significantly promoted H2O2-induced activation of caspase-3, caspase-9 and apoptosis in C2C12 myogenic cells. HSP70 overexpression significantly inhibited H2O2-induced and Smac-promoted apoptosis, as shown by no specific DNA ladder pattern in agarose gel electrophoresis, decrease of percentage of apoptotic nuclei, and marked inactivation of caspase-3 and caspase-9. HSP70 could inhibit the release of Smac from the mitochondria to the cytoplasm 2 h after the treatment by H2O2. CONCLUSION: HSP70 inhibits Smac release from the mitochondria and protects against H2O2-induced apoptosis in C2C12 myogenic cells.

[Mechanisms of heat shock proteins inhibiting C2C12 cell apoptosis induced by hydrogen peroxide].

OBJECTIVE: To explore the mechanisms of C2C12 cell apoptosis induced by hydrogen peroxide (H2O2) which is inhibited by heat shock proteins. METHODS: The expression of heat shock proteins in mouse embryonic myogenic cell line, C2C12 cell,was induced by heat shock response. C2C12 cell apoptosis induced by 0.5 mmol/L hydrogen peroxide (H2O2) was determined by Hoechst 33258 staining. The activities of caspase -3,8,9 were assayed by caspase colorimetric assay kit and Western-blotting. The release of cytochrome C from mitochondria was observed by Western-blotting of cell mitochondria and cytosol fractions. RESULTS: The expression of HSP70 and alphaB-crystallin in C2C12 cell significantly increased at 24 hour after the heat shock response. Heat shock response could inhibit the release of cytochrome c from mitochondria to cytoplasm,the activation of caspase -3,8,9 and the subsequent apoptosis induced by H2O2 in C2C12 cell. CONCLUSION: HSPs can inhibit the C2C12 cell apoptosis through interference with the activation of both mitochondrial and death receptor pathways, which can provide new clues for the prevention of cardiovascular diseases.

[Effect of heat shock response on the cleavage of nucleolin induced by oxidative stress].

OBJECTIVE: To observe the cleavage of nucleolin (C23) during apoptosis induced by oxidative stress and to clarify the effect of heat shock response (HSR) on the cleavage of nucleolin and its possible molecular mechanism. METHODS: We added 0.5 mmol/L peroxide hydrogen (H2O2 ) into cultured cells to mimic oxidative stress. Apoptosis and cleavage of C23 were detected using caspase-3 colorimetric assay and Western blotting respectively. HSR was performed to observe the effect of HSR on cleavage of C23 induced by oxidative stress, and over-expressions of HSP70 and HSP25 were detected by Western blotting. RESULTS: Activity of caspase-3 increased significantly after 2 hours of 0.5 mmol/L H2O2 treatment, and reached the peak after 12 hours. The cleavage of C23 appeared 30 minutes to 1 hour after the treatment of H2O2 as indicated by a cleaved fragmentation of 80 kD, which was significantly inhibited by HSR. Moreover, HSR could induce HSP70 and HSP25 over-expressions. CONCLUSION: Oxidative stress can induce the activation of caspase-3, cleavage of C23, and apoptosis. HSR can significantly inhibit the cleavage of C23 induced by oxidative stress, which is related to the over-expressions of HSP70, HSP25, and other stress proteins.

[Heat shock pretreatment in inhibiting myocardical apoptosis induced by ischemia-reperfusion and its mechanism].

OBJECTIVE: To explore the mechanisms of myocardial apoptosis during myocardial ischemia-reperfusion injury and to further clarify the molecular mechanisms by which heat shock pretreatment in inhibiting myocardial apoptosis induced by ischemia-reperfusion injury. METHODS: Myocardial ischemia-reperfusion injury was induced by the occlusion of left anterior descending branch of the coronary artery. Apoptosis was evaluated by DNA laddering assay and the activities of caspase 3, 8, or 9 was measured with Caspase Colorimetric Assay Kit. Expression of heat shock proteins was detected by Western blotting analysis. To explore the effect of heat shock pretreatment on myocardium against apoptosis, mice were pretreated with whole body hyperthermia before the myocardial ischemia-reperfusion injury. RESULTS: Ischemia-reperfusion injury induced myocardial apoptosis and activation of caspase-3,8,9. Heat shock pretreatment induced the expression of several family members of heat shock proteins and inhibited myocardial apoptosis and activation of the above caspases. CONCLUSION: Mitochondria and death receptor signaling pathways play important roles in myocardial apoptosis induced by ischemia-reperfusion injury. Heat shock pretreatment may increase the expression of several HSP, and inhibit the activation of both mitochondria and death receptor signaling pathways and apoptosis in cardiomyocytes induced by myocardial ischemia-reperfusion injury.

[Heat shock pretreatment inhibits the release of Smac from mitochondria and decreases H2O2-induced cardiomyocyte apoptosis].

OBJECTIVE: To explore the effect of heat shock pretreatment on H2O2-induced apoptosis of neonatal rat cardiomyocytes and the release of Smac from mitochondria. METHODS: After heat shock pretreatment (42 degrees C for 1 h), neonatal cardiomyocytes were exposed to H2O2 (0.5 mmol/L) for 6, 12, 24, and 36 h, respectively. The apoptotic morphological changes and percentage of apoptotic nuclei were analyzed. Activities of caspase-3, 9 were assayed with caspase colorimetric assay kit and Western-blotting. Inducible heat shock proteins were detected by Western-blotting analysis. The release of Smac from mitochondria to cytoplasm was observed by Western-blotting and immunofluorescence. RESULTS: (1) H2O2 (0.5 mmol/L) resulted in a marked release of Smac from mitochondria to cytoplasm at 2 h after the treatment, the activation of caspase-9 and caspase-3 at 4 h after the treatment and specific morphological changes of apoptosis at 24 h after the treatment. (2) Heat shock pretreatment (42 degrees C, 1 h) could increase the expression of hsp90, hsp70 and betaB-crystallin, and inhibit the H2O2-induced release of Smac from mitochondria, the activity of caspase-9, caspase-3 and apoptosis of cardiomyocytes. CONCLUSION: (1) Mitochondrial signal transduction pathway is involved in the apoptosis of cardiomyocytes induced by H2O2; (2) Heat shock pretreatment can inhibit the release of Smac from mitochondria and the activities of caspase-9, 3 and protect cardiomyocytes against H2O2-induced apoptosis.