Long non-coding RNA nuclear enriched abundant transcript 1 (NEAT1) sponges microRNA-124-3p to up-regulate phosphodiesterase 4B (PDE4B) to accelerate the progression of Parkinson's disease.

Reportedly, long non-coding RNA (lncRNA) are crucial modulators in neurodegenerative diseases. Herein, we investigated the role of lncRNA nuclear enriched abundant transcript 1 (NEAT1) in Parkinson's disease (PD). In-vitro PD model was established based on SH-SY5Y cells treated with 1-methyl-4-phenylpyridinium (MPP+). NEAT1, microRNA (miR) -124-3p and phosphodiesterase 4B (PDE4B) expression levels were examined by qRT-PCR. CCK-8 assay and LDH release assay were adopted to delve into the cell viability and cytotoxicity, respectively. Besides, western blot was utilized to determine mTOR, p-mTOR and PDE4B expression levels. ELISA was executed to detect the levels of tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß) and interleukin 6 (IL-6). Dual-luciferase reporter assay and RIP assay were used to probe the relationship between miR-124-3p and NEAT1 or PDE4B. We demonstrated that, in SH-SY5Y cells treated with MPP+, NEAT1 and PDE4B expression levels were raised, while miR-124-3p expression was repressed; NEAT1 depletion or miR-124-3p overexpression increased the cell viability and suppressed cell injury. Besides, miR-124-3p was confirmed as the direct target of NEAT1, and its down-regulation counteracted the impact of NEAT1 depletion on SH-SY5Y cells. PDE4B was as the downstream target of miR-124-3p, and its overexpression weakens the impact of miR-124-3p on SH-SY5Y cells. Additionally, NEAT1 decoyed miR-124-3p to modulate PDE4B expression. Collectively, in MPP+-induced SH-SY5Y cells, NEAT1 depletion increases cell viability, represses cytotoxicity and reduces inflammatory response by regulating miR-124-3p and PDE4B expression levels, suggesting that NEAT1 may be a promising target for treating PD.

Evaluation of a novel monoclonal antibody mAb109 by immuno-PET/fluorescent imaging for noninvasive lung adenocarcinoma diagnosis.

Monoclonal antibodies are believed to be magic bullets and hold great potential for lots of biological process. About 100 µg of mAb109 was expressed in 5 × 106 cells after 10 days' immunization. 64Cu-NOTA-mAb109 was synthesized with the specific activity of 0.74 MBq/µg and high in vitro stability. The binding affinity of 64Cu-NOTA-mAb109 in A549 cells was determined to be 29.64 nM. 64Cu-NOTA-mAb109 displayed prominent tumor accumulation from 2 h to 60 h p.i. (9.34 ± 0.67 %ID/g). NIRF imaging of Cy5.5-mAb109 showed high accumulation till 9 days p.i., while tumors nearly can not be observed in negative groups, which was confirmed by autoradiography. Immunohistological study confirmed that mAb109 had strong and specific capacity to bind lung adenocarcinoma (concentration to 58 nM). Our study demonstrated mAb109 was a new platform for the development of novel agent for lung adenocarcinoma noninvasive imaging. The resulted 64Cu-NOTA-mAb109/Cy5.5-mAb109 show favorable imaging properties/specificity for A549 tumor and high sensitivity to human lung adenocarcinoma tissues.

Recombinant human calcineurin B inhibits orthotopic hepatocellular carcinoma xenograt growth in mice by promoting apoptosis

Objective: To explore the effects of recombinant human calcineurin B (rhCNB) on hepatocellular carcinoma in mice. Methods: An in vivo mouse model with hepatocellular carcinoma was established, and the mice were randomized into the rhCNB, positive control and vehicle treatments groups. Tumor growth was assessed via bioluminescence using a small animal imaging system. Relative tumor proliferation rate and tumor growth inhibition were calculated. The expression of p53 and caspase-9 proteins in tumors were detected by immunohistochemistry. In vitro, flow cytometry was used to quantify the cell-cycle stages and rate of apoptosis. Western blotting and quantitative real-time PCR assays were used to evaluate the effects of rhCNB on protein and gene expression of CDK1, cyclin B1, p53 and caspase-9. Results: rhCNB at the higher dose significantly reduced tumor growth in vivo and caused tumor cell apoptosis in vitro. The rhCNB at the higher dose was as effective as cisplatin, and was safer. Conclusions: rhCNB has potent pro-apoptotic effects on tumor cells in vivo and in vitro and is well tolerated in vivo.

Associations of MMP-2 and MMP-9 gene polymorphism with ulinastatin efficacy in patients with severe acute pancreatitis.

We aim to explore the associations between matrix metalloproteinase (MMP) MMP-2/MMP-9 gene polymorphism with ulinastatin (UTI) efficacy in treating severe acute pancreatitis (SAP). A total of 276 SAP patients were assigned into the control (n=135) and observation (n=141) groups. PCR-restriction fragment length polymorphism (PCR-RFLP) was used for genotype and allele frequency distribution. Relevance of MMP-2/MMP-9 genotypes with UTI efficacy was analyzed. The observation group showed lowered duration in symptoms (abdominal distension, abdominal pain, tenderness, and rebound tenderness) than the control group. Laboratory analysis (serum calcium, white blood cells, serum amylase, urine amylase, APACHE-II, and Balthazar CTIS scores) were decreased, while serum albumin levels increased after 7th day of therapy. The total effective rate of UTI for patients with MMP-2 C-1306T C/C genotype was higher than those with C/T and T/T genotypes after the 7th day of therapy, which was lower in patients with MMP-9 C-1562T C/C and C/T genotypes than those with T/T genotype. The duration for symptoms in patients with MMP-9 C-1562T T/T genotype was shorter than those with C/C and C/T genotypes, which was less in patients with MMP-2 C-1306T C/C genotype than those with C/T and T/T genotypes. The improvement values of APACHE-II and Balthazar CTIS scores for patients with MMP-2 C-1306T C/C genotype were higher than those with C/T and T/T genotypes, which for patients with MMP-9 C-1562T C/C and C/T genotypes were lower than those with T/T genotype. These results demonstrated that MMP-2/MMP-9 gene polymorphism was associated with UTI efficacy for SAP.

A modified total arch replacement combined with a stented elephant trunk implantation for acute type A dissection under deep hypothermic circulatory arrest and selective antegrade cerebral perfusion.

OBJECTIVES: Since the optimal management of patients with acute aortic dissection is unclear, this study analyzed total arch replacement combined with stented elephant trunk implantation in the treatment of acute type A aortic dissection. METHODS: Between February 2008 and February 2013, 86 consecutive patients admitted to our hospital for acute type A dissection underwent total arch replacement combined with stented elephant trunk implantation under deep hypothermic circulatory arrest. The Bentall, David, and Wheat procedure was performed on 46, 12 and two patients, respectively. Ascending aorta replacement was performed on 26 patients, while two patients in Bentall group and 7 in ascending aorta replacement group underwent coronary artery bypass grafting as a concomitant procedure. RESULTS: Sixty-nine patients were male and 17 patients were female, with an average age of 45.2 ± 2.3 years. The in-hospital mortality rate was 5.8%. Two patients presented with persisting paraplegia. The cardiopulmonary bypass time was 186.3 ± 45.2 minutes and the myocardium ischemia time was 102.6 ± 28.1 minutes. Selective antegrade cerebral perfusion time was 29.4 ± 10.3 minutes. Low-body circulatory arrest time was 18.5 ± 8.4 minutes. Mechanical ventilation time was 80.7 ± 11.3 hours. ICU and hospital stays were 5.3 ± 4.8 and 16.8 ± 5.5 days, respectively. Seven patients underwent reoperation for bleeding. During a mean follow-up of 28.5 months, two patients died and 2 patients were lost to follow-up. Obliteration of the false lumen around the stented graft and at the diaphragmatic level occurred in 97.1% (68 of 70) and 70% (49 of 70) of the patients. CONCLUSIONS: Modified total arch replacement combined with stented elephant trunk implantation using selective antegrade cerebral perfusion is a safe and effective alternative for patients with acute type A dissection and produces satisfactory clinical outcomes in our center.

MiR-223 modulates multidrug resistance via downregulation of ABCB1 in hepatocellular carcinoma cells.

Multidrug resistance (MDR) has become a major impediment to a successful treatment for liver cancer patients, and one of the common reasons for MDR is the activation of ABCB1 gene, leading to the over-expression of P-glycoprotein (P-gp), which conferred cancer cells be resistant to a broad range of anticancer drugs. MicroRNAs (miRNAs) are a class of short, non-coding RNA moleculars that can regulate gene expression at the post-transcriptional level. In the current study, the aim is to explore whether miRNA participates in the regulation of MDR mediated by ABCB1. We found that the expression of ABCB1 was correlated with the doxorubicin IC50 dose in eight hepatocellular carcinoma (HCC) cell lines: Hep3B, HCC3, LM-6, SMMC7721, Huh-7, SK-Hep-1, HepG2 and BEL-7402. Using the bioinformatics, we discovered that there were several miRNAs that can bind to the 3'UTR of ABCB1 gene. Among these candidate miRNAs, miR-223 was chosen for further study. Then, EGFP reporter assay, real-time PCR and Western blot were performed to verify that miR-223 targeted ABCB1 3'UTR directly, and miR-223 downregulated ABCB1 at both mRNA and protein levels. Finally, we found that the over-expression of miR-223 increased the HCC cell sensitivity to anticancer drugs, and the inhibition of miR-223 had the opposite effect. Importantly, the over-expression or silencing of ABCB1 can rescue the cell response to the anticancer drugs mediated by miR-223 over-expression or inhibition, respectively. In conclusion, our findings indicated that miR-223 played an important role in the regulation of MDR mediated by ABCB1, and it suggests that miR-223 may be considered as a therapeutic biomarker for HCC patients who had MDR problems induced by high expression of ABCB1.

[Effects of zilongjin on proliferation and apoptosis of human breast cancer cell line MCF-7].

OBJECTIVE: To study the effect of Zilongjin (ZLJ, a composite Chinese drug) on proliferation and apoptosis of human breast carcinoma cell line MCF-7. METHODS: MCF-7 cells were randomly divided into four groups depending on the culture solution used, the control group, cultured with 1640 medium not contained ZLJ; and the three ZJL groups cultured with medium contained low (1.5 mg/mL), moderate (3 mg/mL) and high (6 mg/mL) dosage of ZLJ crude drug respectively. The changes of cell proliferation were assessed by cell growth curve assay and methyl thiazolyl tetrazolium (MTT) assay. And the cell apoptosis was analyzed by flow cytometry, Hoechst 33342 staining and DNA ladder assay. RESULTS: Compared with that in the normal control, the counts of cells in the three ZLJ groups were decreased significantly (P<0.05) at such time point as 24, 48, 72, 96, 120, and 144 h. Furthermore, apart from the comparison of the growth inhibition rate between the low and moderate dosage group at 24 and 72 h which were found to be no significant difference (P>0.05), the comparison f that among the three ZLJ groups appeared to be significant difference (P<0.05). The inhibitory effect of ZLJ on cell proliferation of MCF-7 was time- and dose-dependent; it could retard cells in G0/G1 cell phase; apoptosis of MCF-7 cell was induced by moderate and high dosage of ZLJ with revealing of apoptotic body and DNA ladder formation. CONCLUSION: ZLJ shows cell proliferation inhibitory and apoptosis inducing effects on human breast cancer cell line MCF-7, and thus to realize its anti-tumor action.

[Experimental study on suppressive effect of zilongjin on growth of human breast cancer cell line MCF-7 in vitro and in vivo].

OBJECTIVE: To investigate the suppressive effect and mechanism of action of Zilongjin (ZLJ, a composite Chinese drug) on the growth of human breast carcinoma cell line MCF-7 in vivo and in vivo. METHODS: The cell survival rate and clone forming efficiency were observed by direct cell counting with trypan blue staining and double layers soft agar test; the p-ERK 1/2 expression was analyzed using Western blotting; 17-beta-estrogen pellet embedding was adopted to make the subcutaneous transplanted tumor MCF-7 cell in BALB/c nude mice for detecting the tumor growth suppressive effect of ZLJ (20 g crude drug/kg). RESULTS: The survival rate of MCF-7 cell was obviously decreased by ZLJ in a time and dose-dependent manner; only few and small clones on soft agar could be found after treated with ZLJ, the inhibition rates of clone formation(%) for 0.75, 1.5, 3, 6 mg/mL of ZLJ were 12.66 +/- 1.54, 88.83 +/- 2.13, 100 and 100, respectively, as compared with that of non-treated. The expression of p-ERK 1/2 was suppressed and the ability of the tumorigenicity in nude mice was reduced effectively by ZLJ. The mean volumes and weights of tumor in the test group and the control group were (0.73 +/- 0.58) cm3 vs (1.36 +/- 0.64) cm3 and (1.02 +/- 0.25) g vs (1.66 +/- 0.09) g respectively, showing significant difference (P<0.05), and the tumor inhibition rate of ZLJ was 38.55%. CONCLUSION: ZLJ shows obviously suppressive actions on malignant proliferation, transformation and tumorigenicity of human breast carcinoma cell line MCF-7; the down-regulation of p-ERK 1/2 protein may involve in these effects.

Limitations in SELDI-TOF MS whole serum proteomic profiling with IMAC surface to specifically detect colorectal cancer.

BACKGROUND: Surface enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF-MS) analysis on serum samples was reported to be able to detect colorectal cancer (CRC) from normal or control patients. We carried out a validation study of a SELDI-TOF MS approach with IMAC surface sample processing to identify CRC. METHODS: A retrospective cohort of 338 serum samples including 154 CRCs, 67 control cancers and 117 non-cancerous conditions was profiled using SELDI-TOF-MS. RESULTS: No CRC "specific" classifier was found. However, a classifier consisting of two protein peaks separates cancer from non-cancerous conditions with high accuracy. CONCLUSION: In this study, the SELDI-TOF-MS-based protein expression profiling approach did not perform to identify CRC. However, this technique is promising in distinguishing patients with cancer from a non-cancerous population; it may be useful for monitoring recurrence of CRC after treatment.

Immunoscintigraphy of local recurrent rectal cancer with 99mTc-labeled anti-CEA monoclonal antibody CL58.

AIM: To explore a specific diagnostic method for local recurrent rectal cancer. METHODS: Immunoscintigraphy with (99m)Tc-labeled anti-CEA monoclonal antibody (MoAb) CL-58 was performed for patients suspected of having a postoperative local recurrent rectal cancer and the findings were compared with the results of conventional imaging and pathology. RESULTS: A total of 36 patients with a suspected local recurrent rectal cancer underwent immunoscintigraphy with (99m)Tc-conjugated CL58. Local recurrence of rectal cancer was identified in 31 patients and established in 30 during operation, endoscopy and pathological examination. No local recurrence was found in 5 patients without specific accumulation of (99m)Tc during the follow-up. Immunoscintigraphy had a positive rate of 86.11%, a specificity of 83.33%, and a sensitivity of 100%. CONCLUSION: Immunoscintigraphy has a highly specific and predictive value for detecting local recurrent rectal cancer, especially after abdominal perineal resection (APR).

A serum proteomic pattern for the detection of colorectal adenocarcinoma using surface enhanced laser desorption and ionization mass spectrometry.

PURPOSE: New serum biomarkers are needed to improve the early detection of colorectal adenocarcinoma. We performed surface enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to screen for differentially expressed proteins in serum and build a proteomic diagnostic pattern for the detection of colorectal adenocarcinoma to improve the prognosis of patients with this disease. EXPERIMENTAL DESIGN: In an attempt to improve current approaches to the serologic diagnosis of colorectal cancer, we analyzed serum samples from subjects with or without colorectal cancer using SELDI-MS. Using a case-control study design, SELDI-MS profile of serum samples from 74 colorectal adenocarcinoma patients were compared with 48 age-and sex-matched healthy subjects using a ProteinChip reader, PBSII-C. Proteomic MS spectra were generated using IMAC3 chips, and protein peaks clustering and classification analyses were performed to build a proteomic pattern that could differentiate patients with colorectal adenocarcinoma from healthy subjects utilizing Biomarker Wizard and Biomarker Patterns software packages, respectively. The constructed pattern was then used to test an independent set of masked serum samples from 60 colorectal cancer patients and 39 healthy subjects. RESULTS: Among the differentially expressed protein peaks identified by SELDI-MS profiling that had the ability to distinguish between patients and healthy subjects, we determined a minimum set of two protein peaks for system training and for developing a decision classification pattern. Masked analysis of an independent set of serum samples showed the diagnostic pattern could differentiate patients with different stages of colorectal cancer from healthy subjects with a sensitivity of 95.00 percent and specificity of 94.87 percent. CONCLUSION: SELDI-TOF-MS profiling of serum proteins combined with bioinformatics tools can be applied to accurately differentiate patients with colorectal cancer from healthy subjects. The high sensitivity and specificity achieved by the constructed clustering analysis algorithm show great potential for the early detection of colorectal cancer.

Nuclear Factor Kappa B Involed in Immunologic Function of Critically Ill Newborns / 实用儿科临床杂志

Objective To observe the immunologic function of critically ill newborn and the relative function of nuclear factor kappa B(NF-?B).Methods The critically ill group contained 50 cases,and 25 cases from healthy newborns were used as control group.Blood samples were collected in each case,levels of cytokine interleukin(IL)-4,interferon(IFN)-?,tumor necrosis factor(TNF)-? and NF-?B were detected.Result Compared with control group,NF-?B of the critically ill newborn activated and the cytokine were disorder,and IL-4 and TNF-? increased,but IFN-? decreased.Conclusions Critically ill newborn exist immune functional disorder.Furthermore,NF-?B activation may be involved in the process in infants with critically illness.

COX-2 expression and tumor angiogenesis in colorectal cancer.

AIM: Cyclooxygenase-2 (COX-2) is one of the rate-limiting enzymes in metabolism of arachidonic acid, and COX-2 inhibitors demonstrate preventive effects on cancer, especially on colorectal cancer. The underlying mechanism remains unclear. The aim of this study was to illustrate the relationship between angiogenesis and COX-2 in carcinogenesis of colorectal cancer. METHODS: One hundred and seventy patients with colorectal cancer were enrolled in our study from January 1993 to September 2001 in School of Oncology, Peking University. COX-2 and VEGF expression were detected with the immunohistochemistry (IHC) technique. IHC assays were carried out with the aid of tissue microarray (TMA) procedure. Specimens from 35 of these patients were examined with reverse transcriptase PCR (RT-PCR). RESULTS: COX-2 and VEGF expressions were stronger in colorectal cancer than those in the corresponding normal tissues, at both protein and mRNA levels. One hundred patients were eligible for analysis after IHC assay of COX-2 and VEGF. The positive rate of VEGF was much higher in COX-2 positive group (47/85) than in COX-2 negative group (chi (2) = 4.181, P = 0.041). The result was further verified by the result of RT-PCR (chi (2) = 8.517, P = 0.003). Correlation coefficient was 0.409 after Spearman correlation analysis (P = 0.015). CONCLUSION: COX-2 may be involved in the course of tumor angiogenesis of colorectal cancer and acts through VEGF.

Role of COX-2 in carcinogenesis of colorectal cancer and its relationship with tumor biological characteristics and patients' prognosis.

AIM: Recent clinical epidemiological studies have demonstrated the preventive effect of non-steroidal anti-inflammatory drugs (NSAIDs) against colorectal cancer. The underlying mechanism might be the inhibition of rate-limiting enzyme cyclooxygenase-2 (COX-2) in metabolism of arachidonic acid. The role of COX-2 in carcinogenesis of colorectal cancer and its relationship with tumor biological characteristics and patients' prognosis still remain unclear. This study was to investigate the role of COX-2 expression in carcinogenesis of colorectal cancer and its relationship with tumor biological characteristics and patients' prognosis. METHODS: A total of 139 colorectal cancers and 19 adenomas surgically treated in School of Oncology, Peking University, from January 1993 to September 2001 were retrospectively studied. COX-2 expression was detected with tissue microarray (TMA) and immunohistochemistry (IHC) procedure. The association between COX-2 expression and clinicopathological features and its influence on patients' prognosis were studied. RESULTS: COX-2 expression was strong in colorectal cancer, moderate in adenoma and weak in normal mucosa, which demonstrated statistically significant difference (chi(2)=46.997, P<0.001). COX-2 expression had no association with clinicopathological features such as gross type, differentiation, invasion depth, vessel emboli and TNM staging. Cox proportional hazards modeling analysis and Log rank test revealed no prognostic role of COX-2 expression in colorectal cancer patients. CONCLUSION: COX-2 may play an important role in the early stage of carcinogenesis, and its expression in colorectal cancer is not associated with clinicopathological features and patients' prognosis.

Rapid screening mitochondrial DNA mutation by using denaturing high-performance liquid chromatography.

AIM: To optimize conditions of DHPLC and analyze the effectiveness of various DNA polymerases on DHPLC resolution, and evaluate the sensitivity of DHPLC in the mutation screening of mitochondrial DNA (mtDNA). METHODS: Two fragments of 16s gene of mitochondrial DNA (one of them F2 is a mutant fragment) and an A3243G mutated fragment were used to analyze the UV detection limit and determine the minimum percentage of mutant PCR products for DHPLC and evaluate effects of DNA polymerases on resolution of DHPLC. Under the optimal conditions, we analyzed the mtDNA mutations from muscle tissues of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and screened blindly for variances in D-loop region of mtDNA from human gastric tumor specimen. RESULTS: Ten A3243G variants were detected in 12 cases of MELAS, no alterations were detected in controls and these results were consistent with the results obtained by analysis of RFLP with ApaI. We also identified 26 D-loop variances in 46 cases of human gastric cancer tissues and 38 alterations in 13 gastric cancer cell lines. The mutation of mtDNA at 80 ng PCR products containing a minimum of 5% mutant sequences could be detected by using DHPLC with UV detector. Moreover, Ampli-Taq Gold polymerase was equally as good as the proofreading DNA polymerase (e.g., Pfu) in eliminating the false positive produced by Taq DNA polymerases. CONCLUSION: DHPLC is a powerful, rapid and sensitive mutation screening method for mtDNA. Proofreading DNA polymerase is more suitable for DHPLC analysis than Taq polymerase.