Effect and Mechanism of RBM8A on Proliferation, Migration and Apoptosis of Human Endometrial Cancer HEC-1A Cells / 肿瘤防治研究

Objective To investigate the effect of silenced RBM8A gene on the biological behavior (proliferation, migration, and apoptosis) of human endometrial cancer HEC-1A cells and its possible mechanism. Methods The hairpin shRNA targeted by the RBM8A gene was designed, and the best shRNA silencing fragment was screened. The recombinant lentiviral interference vector carrying the target gene was constructed and used to infect HEC-1A cells. Cells with stable knockdown of RBM8A gene were screened by puromycin as the experimental group (shRBM8A), while the shRNA of nonsense sequence was designed as the control group (shControl). CCK-8 method was used to detect cell proliferation, and flow cytometry was used to detect cell apoptosis. Transwell assay was used to detect cell migration and invasion. Western blot was used to analyze the expression of apoptosis-related proteins and EMT signal transduction pathway related proteins. Results In comparison with the shControl group, after RBM8A knockdown, HEC-1A cell proliferation was reduced, apoptosis was increased, migration and invasion ability were significantly inhibited (P < 0.05), the expression of apoptosis-related proteins cleaved caspase 9 and caspase 3 increased, EMT-related protein E-cadherin expression increased, and Vimentin expression decreased. Conclusion RBM8A gene silencing can inhibit the proliferation, migration, and invasion and promote the apoptosis of endometrial cancer cells. The inhibition of EMT signal transduction pathway may be its mechanism.

Research advances of the prognostic value of onodera prognostic nutrition index for mali-gnant tumors of digestive systems / 中华消化外科杂志

Onodera prognostic nutrition index (OPNI) is a simple and effective parameter. It is calculated by serum albumin level and peripheral blood lymphocyte count. Initially, OPNI is used to assess preoperative nutritional status and surgical risk. In recent years, researchers have found that OPNI is related to the prognosis of many tumors. Simple and accurate prognosis evaluation can help to select treatment methods for digestive system malignant tumors, determine the best pre-operative treatment time and operation time, and improve the survival rate of patients with diges-tive system malignant tumors. The authors review the related literatures at home and abroad, and summarize the research advances in the prognostic value of OPNI for malignant tumors of digestive systems.

Expression of miR-378 in cervical cancer and its effects on the proliferation and invasion of cancer cells as well as the underlying mechanism / 中国基层医药

Objective:To detect the expression of miR-378 in cervical cancer and investigate its effects on the proliferation and invasion of cancer cells as well as the underlying mechanism.Methods:A total of 185 cervical tissue samples of women who received gynecological examination in Qilu Hospital of Shandong University from January 2012 to January 2016 were included in this study. Reverse transcription-quantitative polymerase chain reaction was performed to determine the expression of miR-378 in cervical tissue and C-33A cells. Western blot assay was performed to detect the expression of different cancer genes ATG12, CCND1 and pRb in C-33A cells. BrdU cell proliferation and Transwell invasion assay were performed to determine cell proliferation and invasion. Target Scan was used to predict and screen miR-378 gene targets and verified by a dual-luciferase reporter assay system.Results:The expression of miR-378 in cervical intraepithelial neoplasia (CIN) III lesioned tissue and cervical cancer tissue was significantly higher than that in normal cervical tissues ( F = 103.091, t = 9.381, 8.936, both P < 0.05). The expression of miR-378 in cervical cancer tissues with positive lymph node metastasis was significantly higher than that in cervical cancer tissues with negative lymph node metastasis ( t = 1.007, P < 0.01). The overexpression of miR-378 in cervical cancer tissues significantly promoted the migration and invasion of C-33A cells ( t = 5.285, P < 0.05), while low expression of miR-378 in cervical cancer tissues significantly inhibited the migration and invasion of HeLa cells ( t = 2.941, P < 0.05). The overexpression of miR-378 in C-33A cells significantly decreased the expression of ATG12, CCND1and pRb ( t = 1.382, 1.431 and 2.086, all P < 0.05). The low expression of miR-378 in C-33A cells significantly increased the expression of ATG12, CCND1 and pRb ( t = 3.961, 3.062 and 2.894, all P < 0.05). Conclusion:miR-378 can greatly promote the metastasis of cervical cancer cells. ATG12, as a direct target of miR-378, provides new insights into the molecular mechanism underlying cervical cancer pathology and therapeutic target.

Mechanistic Differences in the Inhibition of NF-κB by Turmeric and Its Curcuminoid Constituents.

Turmeric extract, a mixture of curcumin and its demethoxy (DMC) and bisdemethoxy (BDMC) isomers, is used as an anti-inflammatory preparation in traditional Asian medicine. Curcumin is considered to be the major bioactive compound in turmeric but less is known about the relative anti-inflammatory potency and mechanism of the other components, their mixture, or the reduced in vivo metabolites. We quantified inhibition of the NF-κB pathway in cells, adduction to a peptide mimicking IκB kinase ß, and the role of cellular glutathione as a scavenger of electrophilic curcuminoid oxidation products, suggested to be the active metabolites. Turmeric extracts (IC50 14.5 ± 2.9 µM), DMC (IC50 12.1 ± 7.2 µM), and BDMC (IC50 8.3 ± 1.6 µM), but not reduced curcumin, inhibited NF-κB similar to curcumin (IC50 18.2 ± 3.9 µM). Peptide adduction was formed with turmeric and DMC but not with BDMC, and this correlated with their oxidative degradation. Inhibition of glutathione biosynthesis enhanced the activity of DMC but not BDMC in the cellular assay. These findings suggest that NF-κB inhibition by curcumin and DMC involves their oxidation to reactive electrophiles, whereas BDMC does not require oxidation. Because it has not been established whether curcumin undergoes oxidative transformation in vivo, oxidation-independent BDMC may be a promising alternative to test in clinical trials.

Curcumin induces secretion of glucagon-like peptide-1 through an oxidation-dependent mechanism.

Curcumin shows antiglycemic effects in animals. Curcumin is chemically unstable at physiological pH, and its oxidative degradation products were shown to contribute to its anti-inflammatory effects. Since the degradation products may also contribute to other effects, we analyzed their role in the antiglycemic activity of curcumin. We quantified curcumin-induced release of glucagon-like peptide 1 (GLP-1) from mouse STC-1 cells that represent enteroendocrine L-cells as a major source of this anti-diabetic hormone. Curcumin induced secretion of GLP-1 in a dose-dependent manner. Two chemically stable analogues of curcumin that do not readily undergo degradation, were less active while two unstable analogues were active secretagogues. Chromatographically isolated spiroepoxide, an unstable oxidative metabolite of curcumin with anti-inflammatory activity, also induced secretion of GLP-1. Stable compounds like the final oxidative metabolite bicyclopentadione, and the major plasma metabolite, curcumin-glucuronide, were inactive. GLP-1 secretion induced by curcumin and its oxidative degradation products was associated with activation of PKC, ERK, and CaM kinase II. Since activity largely correlated with instability of curcumin and the analogues, we tested the extent of covalent binding to proteins in STC-1 cells and found it occurred with similar affinity as N-ethylmaleimide, indicating covalent binding occurred with nucleophilic cysteine residues. These results suggest that oxidative metabolites of curcumin are involved in the antiglycemic effects of curcumin. Our findings support the hypothesis that curcumin functions as a pro-drug requiring oxidative activation to reveal its bioactive metabolites that act by binding to target proteins thereby causing a change in function.

Stability and anti-inflammatory activity of the reduction-resistant curcumin analog, 2,6-dimethyl-curcumin.

The efficacy of the curry spice compound curcumin as a natural anti-inflammatory agent is limited by its rapid reductive metabolism in vivo. A recent report described a novel synthetic derivative, 2,6-dimethyl-curcumin, with increased stability against reduction in vitro and in vivo. It is also known that curcumin is unstable at physiological pH in vitro and undergoes rapid autoxidative transformation. Since the oxidation products may contribute to the biological effects of curcumin, we tested oxidative stability of 2,6-dimethyl-curcumin in buffer (pH 7.5). The rate of degradation was similar to curcumin. The degradation products were identified as a one-carbon chain-shortened alcohol, vanillin, and two isomeric epoxides that underwent cleavage to vanillin and a corresponding hydroxylated cleavage product. 2,6-Dimethyl-curcumin was more potent than curcumin in inhibiting NF-κB activity but less potent in inhibiting expression of cyclooxygenase-2 in LPS-activated RAW264.7 cells. 2,6-Dimethyl-curcumin and some of its degradation products covalently bound to a peptide that contains the redox-sensitive cysteine of IKKß kinase, the activating kinase upstream of NF-κB, providing a mechanism for the anti-inflammatory activity. In RAW264.7 cells vanillin, the chain-shortened alcohol, and reduced 2,6-dimethyl-curcumin were detected as major metabolites. These studies provide new insight into the oxidative transformation mechanism of curcumin and related compounds. The products resulting from oxidative transformation contribute to the anti-inflammatory activity of 2,6-dimethyl-curcumin in addition to its enhanced resistance against enzymatic reduction.

Clinical features and prognosis of cervical adenocarcinoma and adenosquamous carcinoma: an analysis of 237 cases / 浙江大学学报·医学版

OBJECTIVE@#To analyze the clinical features and prognosis of cervical adenocarcinoma (AC) and adenosquamous carcinoma of cervix (ASC).@*METHODS@#The clinical data of 237 patients, including 201 cases of AC and 36 cases of ASC (FIGO stage ⅠB1-ⅡA), who underwent surgery in Qilu Hospital between September 2007 and September 2016 were reviewed. Clinical features of two groups were compared, and Kaplan-Meier survival analysis was performed to evaluate the prognosis.@*RESULTS@#A larger proportion of ASC patients had lymphovascular space invasion compared with AC patients (0.05). The 5-year overall survival rates of AC and ASC groups were 79.4% and 78.3%, and the 5-year recurrence-free survival rates were 77.4% and 73.0%. Among patients received concurrent chemoradiotherapy, the 5-year overall survival rates were 71.0% and 61.4%, and the 5-year recurrence-free survival rates were 68.8% abd 61.1%, respectively. No significant differences were observed in 5-year overall survival rates and recurrence-free survival rates between AC and ASC patients (all >0.05).@*CONCLUSIONS@#Lymphovascular space invasion was more likely to occur in patients with ASC, but there was no significant difference in the prognosis between AC and ASC patients.

The anti-inflammatory activity of curcumin is mediated by its oxidative metabolites.

The spice turmeric, with its active polyphenol curcumin, has been used as anti-inflammatory remedy in traditional Asian medicine for centuries. Many cellular targets of curcumin have been identified, but how such a wide range of targets can be affected by a single compound is unclear. Here, we identified curcumin as a pro-drug that requires oxidative activation into reactive metabolites to exert anti-inflammatory activities. Synthetic curcumin analogs that undergo oxidative transformation potently inhibited the pro-inflammatory transcription factor nuclear factor κB (NF-κB), whereas stable, non-oxidizable analogs were less active, with a correlation coefficient (R2) of IC50versus log of autoxidation rate of 0.75. Inhibition of glutathione biosynthesis, which protects cells from reactive metabolites, increased the potency of curcumin and decreased the amount of curcumin-glutathione adducts in cells. Oxidative metabolites of curcumin adducted to and inhibited the inhibitor of NF-κB kinase subunit ß (IKKß), an activating kinase upstream of NF-κB. An unstable, alkynyl-tagged curcumin analog yielded abundant adducts with cellular protein that were decreased by pretreatment with curcumin or an unstable analog but not by a stable analog. Bioactivation of curcumin occurred readily in vitro, which may explain the wide range of cellular targets, but if bioactivation is insufficient in vivo, it may also help explain the inconclusive results in human studies with curcumin so far. We conclude that the paradigm of metabolic bioactivation uncovered here should be considered for the evaluation and design of clinical trials of curcumin and other polyphenols of medicinal interest.

Molecular cloning and functional characterisation of the tomato E3 ubiquitin ligase SlBAH1 gene.

Emerging evidence suggests that E3 ligases play critical roles in diverse biological processes, including pathogen resistance in plants. In the present study, an ubiquitin ligase gene (SlBAH1) was cloned from a tomato plant, and the functions of the gene were studied. The SlBAH1 gene contained 1002 nucleotides and encodes a protein with 333 amino acids. The SlBAH1 protein contains a SPX domain and a RING domain. SlBAH1 displayed E3 ubiquitin ligase activity in vitro. SlBAH1 was shown to localise in the nucleus, cytoplasm and plasma membrane by a subcellular localisation assay. The expression of SlBAH1 was induced by various hormones and Botrytis cinerea Pers. treatment. SlBAH1-silencing in plants obtained by virus-induced gene silencing (VIGS) technology enhanced resistance to B. cinerea, and the expression of pathogenesis-related (PR) genes, including PR1, PR2, PR4, PR5, and PR7, was significantly increased. These results indicate that the SlBAH1-dependent activation of defence-related genes played a key role in the enhanced fungal resistance observed in the SlBAH1-silenced plants and may be related to the SA-dependent and JA-dependent signalling pathways.

Inhibition of RhoA signaling with increased Bves in trabecular meshwork cells.

PURPOSE: Blood vessel epicardial substance (Bves) is a novel adhesion molecule that regulates tight junction (TJ) formation. TJs also modulate RhoA signaling, which has been implicated in outflow regulation. Given that Bves has been reported in multiple ocular tissues, the authors hypothesize that Bves plays a role in the regulation of RhoA signaling in trabecular meshwork (TM) cells. METHODS: Human TM cell lines NTM-5 and NTM-5 transfected to overexpress Bves (NTM-w) were evaluated for TJ formation, and levels of occludin, cingulin, and ZO-1 protein were compared. Assays of TJ function were carried out using diffusion of sodium fluorescein and transcellular electrical resistance (TER). Levels of activated RhoA were measured using FRET probes, and phosphorylation of myosin light chain (MLC-p), a downstream target of RhoA, was assessed by Western blot analysis. RESULTS: Overexpression of Bves led to increased TJ formation in NTM-5 cells. Increased TJ formation was confirmed by increased occludin, cingulin, and ZO-1 protein. Functionally, NTM-w cells showed decreased permeability and increased TER compared with NTM-5 cells, consistent with increased TJ formation. NTM-w cells also exhibited decreased levels of active RhoA and lower levels of MLC-p than did NTM-5 cells. These findings support a TJ role in RhoA signaling. CONCLUSIONS: Increased Bves in TM cells leads to increased TJ formation with decreased RhoA activation and decreased MLC-p. This is the first report of a regulatory pathway upstream of RhoA in TM cells. In TM tissue, RhoA has been implicated in outflow regulation; thus, Bves may be a key regulatory molecule in aqueous outflow.