Harnessing the Therapeutic Potential of Ginsenoside Rd for Activating SIRT6 in Treating a Mouse Model of Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a prevalent global condition and a common precursor to liver cancer, yet there is currently no specific medication available for its treatment. Ginseng, renowned for its medicinal and dietary properties, has been utilized in NAFLD management, although the precise underlying mechanism remains elusive. To investigate the effectiveness of ginsenoside Rd, we employed mouse and cell models to induce NAFLD using high-fat diets, oleic acid, and palmitic acid. We explored and confirmed the specific mechanism of ginsenoside Rd-induced hepatic steatosis through experiments involving mice with a liver-specific knockout of SIRT6, a crucial protein involved in metabolic regulation. Our findings revealed that administration of ginsenoside Rd significantly reduced the inflammatory response, reactive oxygen species (ROS) levels, lipid peroxide levels, and mitochondrial stress induced by oleic acid and palmitic acid in primary hepatocytes, thereby mitigating excessive lipid accumulation. Moreover, ginsenoside Rd administration effectively enhanced the mRNA content of key proteins involved in fatty acid oxidation, with a particular emphasis on SIRT6 and its target proteins. We further validated that ginsenoside Rd directly binds to SIRT6, augmenting its deacetylase activity. Notably, we made a significant observation that the protective effect of ginsenoside Rd against hepatic disorders induced by a fatty diet was almost entirely reversed in mice with a liver-specific SIRT6 knockout. Our findings highlight the potential therapeutic impact of Ginsenoside Rd in NAFLD treatment by activating SIRT6. These results warrant further investigation into the development of Ginsenoside Rd as a promising agent for managing this prevalent liver disease.

Intervention effects and related mechanisms of glycyrrhizic acid on zebrafish with Hirschsprung-associated enterocolitis.

BACKGROUND: The prevention and treatment of Hirschsprung-associated enterocolitis (HAEC) is a serious challenge in pediatric surgery. Exploring the mechanism of HAEC is conducive to the prevention of this disease. AIM: To explore the possible mechanism of glycyrrhizic acid (GA) and its therapeutic effect on HAEC. METHODS: We developed a model of enteritis induced by trinitrobenzenesulfonic acid (TNBS) in zebrafish, and treated it with different concentrations of GA. We analyzed the effect of GA on the phenotype and inflammation of zebrafish. RESULTS: After treatment with TNBS, the area of the intestinal lumen in zebrafish was significantly increased, but the number of goblet cells in the intestinal lumen was significantly reduced, but these did not increase the mortality of zebrafish, indicating that the zebrafish enteritis model was successfully developed. Different concentrations of GA protected zebrafish with enteritis. In particular, high concentrations of GA were important for the prevention and control of HAEC because it significantly reduced the intestinal luminal area, increased the number of goblet cells in the intestinal lumen, and reduced the levels of interleukin (IL)-1ß and IL-8. CONCLUSION: GA significantly reduced the intestinal luminal area, increased the number of intestinal goblet cells, and decreased IL-1ß and IL-8 in zebrafish, and is important for prevention and control of HAEC.

Effect of modified transanal Soave assisted by laparoscopy in the treatment of Hirschsprung's disease in children and its influencing factors.

OBJECTIVE: To investigate the effect of modified transanal Soave assisted by laparoscopy in children with Hirschsprung's disease (HD). METHODS: The clinical data of 120 children with Hirschsprung's disease admitted to Fujian Children's Hospital from January 2018 to November 2021 were retrospectively analyzed. Based on the surgical methods, 58 children treated with modified transanal Soave were regarded as the modified group and 62 children treated with modified transanal Soave assisted by laparoscopy were divided into the laparoscopic group. The operative indexes, anal function, quality of life and perianal pressure 6 months after surgery, complications within 1 month after surgery, and recovery within 6 months after surgery of the two groups were compared. The risk factors influencing the postoperative recovery of hirschsprung's disease in children were analyzed by univariate and logistic regression analysis. RESULTS: The operation time, intraoperative blood loss, length of hospital stay and gastrointestinal recovery time in the laparoscopic group were lower than those in modified group (P < 0.05). The excellent and good rate of postoperative anal function in laparoscopic group was 87.10%, which was higher than that in modified group (68.97%) (P < 0.05). The proportion of patients with good quality of life in laparoscopic group (90.32%) was higher than that in modified group (74.14%) (P < 0.05). The anal resting pressure and systolic pressure in laparoscopic group were lower than those in modified group (all P < 0.05). The total complication rate of laparoscopic group (6.45%) was lower than that of modified group (22.41%) (P < 0.05). After 6 months, 64 cases (53.33%) were cured and 56 cases (46.67%) were not. After univariate analysis, there were statistically significant differences in enteritis, abdominal distension, and anastomotic stenosis between cured children and uncured children (all P < 0.05). There was no significant difference in other factors (P > 0.05). Logistic regression analysis showed that enteritis, abdominal distension and anastomotic stenosis were the risk factors affecting the recovery of hirschsprung's disease in children (all P < 0.05). CONCLUSIONS: Modified transanal Soave assisted by laparoscopy can improve anal function and quality of life, relieve anal pressure, and have a low complication rate. Enteritis, abdominal distension, and anastomotic stenosis are the factors affecting the recovery of Hirschsprung's disease in children.

Hepatic SIRT6 Modulates Transcriptional Activities of FXR to Alleviate Acetaminophen-induced Hepatotoxicity.

BACKGROUND & AIMS: Excessive acetaminophen (APAP) intake causes oxidative stress and inflammation, leading to fatal hepatotoxicity; however, the mechanism remains unclear. This study aims to explore the protective effects and detailed mechanisms of sirtuin 6 (SIRT6) in the defense against APAP-induced hepatotoxicity. METHODS: Hepatocyte-specific SIRT6 knockout mice, farnesoid X receptor (FXR) knockout mice, and mice with genetic or pharmacological activation of SIRT6 were subjected to APAP to evaluate the critical role of SIRT6 in the pathogenesis of acute liver injury. RNA sequences were used to investigate molecular mechanisms underlying this process. RESULTS: Hepatic SIRT6 expression was substantially reduced in the patients and mice with acute liver injury. The deletion of SIRT6 in mice and mice primary hepatocytes led to high N-acetyl-p-benzo-quinoneimine and low glutathione levels in the liver, thereby enhancing APAP overdose-induced liver injury, manifested as increased hepatic centrilobular necrosis, oxidative stress, and inflammation. Conversely, overexpression or pharmacological activation of SIRT6 enhanced glutathione and decreased N-acetyl-p-benzo-quinoneimine, thus alleviating APAP-induced hepatotoxicity via normalization of liver damage, inflammatory infiltration, and oxidative stress. Our molecular analysis revealed that FXR is regulated by SIRT6, which is associated with the pathological progression of ALI. Mechanistically, SIRT6 deacetylates FXR and elevates FXR transcriptional activity. FXR ablation in mice and mice primary hepatocytes prominently blunted SIRT6 overexpression and activation-mediated ameliorative effects. Conversely, pharmacological activation of FXR mitigated APAP-induced hepatotoxicity in SIRT6 knockout mice. CONCLUSIONS: Our current study suggests that SIRT6 plays a crucial role in APAP-induced hepatotoxicity, and pharmacological activation of SIRT6 may represent a novel therapeutic strategy for APAP overdose-induced liver injury.

Detection and Identification of Serum Peptides Biomarker in Papillary Thyroid Cancer.

BACKGROUND Papillary thyroid cancer (PTC) is currently the most commonly diagnosed endocrine malignancy. In addition, the sex- and age-adjusted incidence of PTC has exhibited a greater increase over the last 2 decades than in many other malignancies. Thus, discovering noninvasive specific serum biomarker to distinguish PTC from cancer-free controls in its early stages remains an important goal. MATERIAL AND METHODS Serum samples from 88 PTC patients and 80 cancer-free controls were randomly allocated into training or validation sets. Serum peptide profiling was performed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) after using weak cation exchange magnetic beads (WCX-MB), and the results were evaluated by use of ClinProTools™ Software. To distinguish PTC from cancer-free controls, quick classifier (QC), supervised neural network (SNN), and genetic algorithm (GA) models were established. The models were blindly validated to verify their diagnostic capabilities. The most discriminative peaks were subsequently identified with a nano-liquid chromatography-electrospray ionization-tandem mass spectrometry system. RESULTS Six peptide ions were identified as the most discriminative peaks between the PTC and cancer-free control samples. The QC model exhibited satisfactory sensitivity and specificity among the 3 models that were validated. Two peaks, at m/z 2671.17 and m/z 1464.68, were identified as fragments of the alpha chain of fibrinogen, while a peak at m/z 1738.92 was a fragment of complement component 4A/B. CONCLUSIONS MS combined with ClinProTools™ software was able to detect peptide biomarkers in PTC patients. In addition, the constructed classification models provided a serum peptidome pattern for distinguishing PTC from cancer-free controls. Both fibrinogen a and complement C4A/B were identified as potential markers for diagnosis of PTC.

Diagnostic accuracy of MALDI-TOF mass spectrometry for non-small cell lung cancer: a meta-analysis.

Objective: To assess the overall accuracy of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying non-small cell lung cancer (NSCLC).Methods: A comprehensive search of PubMed, EMBASE and CNKI databases as well as the reference lists from relevant articles was performed prior to July 2017. Two authors independently screened articles based on inclusion and exclusion criteria and assessed the quality of each study using the Quality Assessment of Diagnostic Accuracy Studies 2 (QADAS-2) tool. Meta-disc 1.4 and Stata12.0 software programs were used for the statistical analysis.Results: Eleven eligible articles comprising 16 studies and representing 935 subjects were included in this meta-analysis. The pooled sensitivity and specificity were 0.84 (95% CI: 0.80-0.87) and 0.77 (95% CI: 0.74-0.80), respectively. The overall diagnostic performance as measured by the area under the curve (AUC) for the summary receiver-operating characteristic (SROC) curve was 0.9380.Conclusions: MALDI-TOF MS has a high diagnostic accuracy for NSCLC.

Upregulation of kallikrein­related peptidase 5 is associated with the malignant behavior of colorectal cancer.

Kallikrein-related peptidase 5 (KLK5) is a serine protease that has exhibited upregulated expression in numerous types of human cancer. The present study assessed KLK5 expression in colorectal cancer (CRC) tissues, in order to determine its association with clinicopathological data and prognosis. The mRNA and protein expression levels of KLK5 were detected using reverse transcription­quantitative polymerase chain reaction, immunohistochemistry and enzyme­linked immunosorbent assay, respectively. KLK5 expression was detected in 48 paraffin­embedded tumor tissue samples and corresponding tumor­free areas within the same specimens, 40 paired normal and CRC frozen tissues, and serum samples from 70 patients with CRC (including 38 serum samples taken pre­ and post­surgery) and 53 healthy individuals. The results demonstrated that KLK5 protein was strongly expressed in CRC; however, its expression was hardly detected in the matched normal mucosal tissue. The KLK5 mRNA expression levels were significantly upregulated in CRC tissues compared with the paired normal tissues, and were higher in Dukes' stage C/D cancer than in stage A/B (P<0.001). Furthermore, sera from patients with CRC exhibited increased KLK5 levels, as compared with that of healthy volunteers (878.02 vs. 391.07 pg/ml; P<0.001). Serum KLK5 levels were also significantly higher in patients prior to surgery compared with post­surgery (909.48±536.72 vs. 644.00±522.87 pg/ml; P<0.001). In addition, increased serum KLK5 levels were associated with CRC lymph node or distant metastasis (P=0.003), tumor­lymph node­metastasis stage (P=0.004), and Dukes' stage (P=0.005). The results of the present study indicated that the mRNA and protein expression levels of KLK5 were significantly upregulated in CRC tissues and sera, and were associated with an advanced tumor stage. Further studies may identify KLK5 as a biomarker of CRC recurrence or treatment response.

Diagnostic value of KLK6 as an ovarian cancer biomarker: A meta-analysis.

Kallikrein-related peptidase 6 (KLK6) is a new potential serum biomarker of ovarian cancer. The aim of the present study was to assess the diagnostic value of KLK6 systematically for ovarian cancer. All the selected studies regarding the changes of KLK6 in ovarian cancer were published prior to April 2015. Five studies involving 485 patients with ovarian cancer, 420 benign cysts and 245 healthy controls met the inclusion criteria. The value of sensitivity, specificity, positive-likelihood ratio (LR+), negative-likelihood ratio (LR-) and area under the receiver operating characteristic curve (ROC) were obtained. All these indices were used to evaluate the diagnostic value of KLK6 for ovarian cancer. The values of sensitivity, specificity, LR+ and LR- (95% confidence interval) of KLK6 were 0.50 (0.47-0.54), 0.91 (0.89-0.93), 7.20 (3.34-15.52) and 0.51 (0.43-0.62), respectively. The area under the summary ROC of KLK6 was 0.86. The index of Q* was 0.79. In conclusion, KLK6 showed high specificity for the diagnosis of ovarian cancer. It can improve the diagnostic accuracy of cancer antigen 125 (CA125). A combined panel of CA125 and KL K6 shows a high diagnostic efficiency for advanced ovarian cancer. Owing to the small number of studies and lack of samples, additional studies meeting the inclusion criteria are required to further analyze the diagnostic value of KLK6 for ovarian cancer.

Liraglutide protects pancreatic ß-cells against free fatty acids in vitro and affects glucolipid metabolism in apolipoprotein E-/- mice by activating autophagy.

The aim of the present study was to determine whether liraglutide (LRG), a long acting glucagon-like peptide 1 analogue, exerted a protective effect on free fatty acid (FFA)­treated pancreatic ß­cells via activating autophagy. INS­1 insulinoma pancreatic islet cell lines were treated with FFA and the levels of cell necrosis, apoptosis and autophagy were detected using an MTT assay, flow cytometry and electron microscopy (ECM). A type 2 diabetes mellitus mouse model was established through treatment of mice with a high­fat diet for 8 weeks and injection of streptozotocin. LRG and autophagy inhibitors were used to investigate the protective effect of LRG on pancreatic ß­cells in vivo. Metabolic indices were measured and pancreatic autophagy was detected. In the INS­1 cells, viability was higher in the FFA + LRG group compared with the FFA group, while the apoptotic rate was lower (P<0.05). The light chain 3B and p62 autophagy­associated proteins were upregulated by LRG, while ATG7 and Beclin1 were downregulated. Autophagy inhibitors reduced the protective effect of LRG in the FFA­treated INS­1 cells. The type 2 diabetes mouse model was successfully established, termed the HF group, in which LRG was observed to reduce body weight and decrease levels of fasting blood glucose, total cholesterol, serum insulin, triglyceride, low density lipoprotein­cholesterol and glycosylated hemoglobin (P<0.05), compared with the HF group. However, chloroquine treatment abrogated these effects (P<0.05, compared with the HF + LRG group; P>0.05, compared with the HF group). Autophagosomes were also observed under ECM in the pancreatic tissues of mice in the HF + LRG group. Therefore, LRG induced autophagy and exerted protective effects on pancreatic ß-cells in vitro and in vivo.

Identification of Novel Inflammatory Cytokines and Contribution of Keratinocyte-Derived Chemokine to Inflammation in Response to Vibrio vulnificus Infection in Mice.

Currently, only tumor necrosis factor alpha (TNF-α) and interleukin family cytokines have been found to be elicited in Vibrio vulnificus (V. vulnificus)-infected animal models and humans. However, multiple other cytokines are also involved in the immune and inflammatory responses to foreign microorganism infection. Antibody array technology, unlike traditional enzyme-linked immunosorbent assay (ELISA), is able to detect multiple cytokines at one time. Therefore, in this study, we examined the proinflammatory cytokine profile in the serum and liver homogenate samples of bacterial-infected mice using antibody array technology. We identified nine novel cytokines in response to V. vulnificus infection in mice. We found that keratinocyte-derived chemokine (KC) was the most elevated cytokine and demonstrated that KC played a very important role in the V. vulnificus infection-elicited inflammatory response in mice, as evidenced by the fact that the blocking of KC by anti-KC antibody reduced hepatic injury in vivo and that KC induced by V. vulnificus infection in AML-12 cells chemoattracted neutrophils. Our findings implicate that KC may serve as a novel diagnostic biomarker and a possible therapeutic target for V. vulnificus infection.

Diagnostic value of total plasma lysophosphatidic acid in ovarian cancer: a meta-analysis.

OBJECTIVE: This study aimed to assess the diagnostic value of lysophosphatidic acid (LPA) in ovarian cancer. METHODS: A systematic review of related studies was performed; sensitivity, specificity, and other measures about the accuracy of serum LPA in the diagnosis of ovarian cancer were pooled using random-effects models. Summary receiver operating characteristic curve analysis was used to summarize the overall test performance. RESULTS: Six studies involving 363 patients with ovarian cancer and 273 healthy control women met the inclusion criteria. The summary estimates for LPA in diagnosing ovarian cancer in the included studies were as follows: sensitivity, 0.94 [95% confidence interval (CI), 0.91-0.96]; specificity, 0.88 (95% CI, 0.83-0.91); and diagnostic odds ratio, 141.59 (95% CI, 52.1-384.63). The area under the curve and Q value for summary receiver operating characteristic curves were 0.97 and 0.92, respectively. CONCLUSIONS: The LPA assay showed high accuracy and sensitivity for the diagnosis of ovarian cancer. The present study was limited by the small number of available studies and sample size; therefore, additional studies with a better design and larger samples are needed to further assess the diagnostic accuracy of LPA.

Correlation between KLK6 expression and the clinicopathological features of glioma.

AIM OF THE STUDY: We measured the impact of changing KLK6 expression levels on the pathological grade of gliomas and on proliferation rate, cell cycle progression, and apoptosis in the U251 glioblastoma cell line. MATERIAL AND METHODS: The expression of KLK6 in 35 brain glioma tissues and adjacent noncancerous tissues was measured using real-time quantitative polymerase chain reaction (PCR) and the relationship between KLK6 expression and pathological grades was analysed. RESULTS: The KLK6 expression in U251 cells was silenced by a specific siRNA, and the effects on proliferation, the cell cycle, and apoptosis were compared to wild type cells. Expression of KLK6 was downregulated in gliomas relative to matched noncancerous tissue. There was no obvious relationship between patient sex, pathological grade, or tumour classification and the expression of KLK6. In the U251 cell line, cell proliferation was enhanced and the fractions of cells in the G2 and S phases were increased by siRNA-mediated KLK6 silencing. CONCLUSIONS: Expression of KLK6 inhibits tumour growth. Decreased KLK6 expression may be a possible risk factor for glioma.

IL-33 promotes airway remodeling and is a marker of asthma disease severity.

OBJECTIVE: To investigate the function of interleukin-33 (IL-33) in the asthmatic airway remodeling and the relationship between IL-33 and asthma severity. METHODS: IL-33 levels, sputum eosinophils percentage (EOS%), pulmonary function and total immunoglobulin (IgE) were measured for 45 patients with asthma and 40 non-allergic controls. Asthma severity was assessed. The expressions of IL-33 and reticular basement membrane (RBM) on bronchial biopsy specimens from eight asthma patients and eight non-allergic controls were observed after hematoxylin-eosin staining (HE) and immunohistochemical staining. In vitro experiments, real-time polymerase chain reactions and western blotting analysis were used to identify the specific effects of IL-33 administration. RESULTS: Serum IL-33 levels in patients with asthma were higher than those in non-allergic controls. Moreover, in asthmatic patients, serum IL-33 levels were negatively correlated to forced expiratory volume in one second (FEV1, % predicted), and positively correlated to asthma severity. Increased expression of IL-33 and RBM thickening were observed on bronchial biopsy specimens obtained from patients with asthma. Serum IL-33 levels were positively correlated to basement membrane thickness. The production of fibronectin1 and type I collagen in human lung fibroblasts (HLF-1) increased at 24 h after IL-33 treatment in vitro. Pre-treatment with anti-ST2 antibody or fluticasone propionate (FP) suppressed the production of fibronectin1 and types I collagen induced by IL-33. CONCLUSIONS: IL-33 is a marker of asthma severity, and may contribute to airway remodeling in asthma by acting on human lung fibroblasts.

Targeting insulin resistance in type 2 diabetes via immune modulation of cord blood-derived multipotent stem cells (CB-SCs) in stem cell educator therapy: phase I/II clinical trial.

BACKGROUND: The prevalence of type 2 diabetes (T2D) is increasing worldwide and creating a significant burden on health systems, highlighting the need for the development of innovative therapeutic approaches to overcome immune dysfunction, which is likely a key factor in the development of insulin resistance in T2D. It suggests that immune modulation may be a useful tool in treating the disease. METHODS: In an open-label, phase 1/phase 2 study, patients (N=36) with long-standing T2D were divided into three groups (Group A, oral medications, n=18; Group B, oral medications+insulin injections, n=11; Group C having impaired ß-cell function with oral medications+insulin injections, n=7). All patients received one treatment with the Stem Cell Educator therapy in which a patient's blood is circulated through a closed-loop system that separates mononuclear cells from the whole blood, briefly co-cultures them with adherent cord blood-derived multipotent stem cells (CB-SCs), and returns the educated autologous cells to the patient's circulation. RESULTS: Clinical findings indicate that T2D patients achieve improved metabolic control and reduced inflammation markers after receiving Stem Cell Educator therapy. Median glycated hemoglobin (HbA1C) in Group A and B was significantly reduced from 8.61%±1.12 at baseline to 7.25%±0.58 at 12 weeks (P=2.62E-06), and 7.33%±1.02 at one year post-treatment (P=0.0002). Homeostasis model assessment (HOMA) of insulin resistance (HOMA-IR) demonstrated that insulin sensitivity was improved post-treatment. Notably, the islet beta-cell function in Group C subjects was markedly recovered, as demonstrated by the restoration of C-peptide levels. Mechanistic studies revealed that Stem Cell Educator therapy reverses immune dysfunctions through immune modulation on monocytes and balancing Th1/Th2/Th3 cytokine production. CONCLUSIONS: Clinical data from the current phase 1/phase 2 study demonstrate that Stem Cell Educator therapy is a safe approach that produces lasting improvement in metabolic control for individuals with moderate or severe T2D who receive a single treatment. In addition, this approach does not appear to have the safety and ethical concerns associated with conventional stem cell-based approaches. TRIAL REGISTRATION: ClinicalTrials.gov number, NCT01415726.

Circulating microRNA-92a and microRNA-21 as novel minimally invasive biomarkers for primary breast cancer.

PURPOSE: MicroRNAs (miRNAs) play an essential role in breast malignant tumor development and progression. The development of clinically validated biomarkers for primary breast cancer (BC) has remained an insurmountable task despite other advances in the field of cancer molecular biology. The objective of this study is to investigate the differential expression of miRNAs and the potential of circulating microRNAs as novel primary breast cancer biomarkers. METHODS: Our analyses were performed on 48 tissue and 100 serum samples of patients with primary BC and a set of 20 control samples of healthy women, respectively. The relative expression of ten candidate miRNAs (miR-106b, miR-125b, miR-17, miR-185, miR-21, miR-558, miR-625, miR-665, miR-92a, and miR-93) from the results of four bioinformatics approaches and literature curation was measured by real-time quantitative reverse transcription PCR (qRT-PCR). RESULTS: The level of miR-92a was significantly lower, while miR-21 was higher, as previous reports, in tissue and serum samples of BC than that of healthy controls (p < 0.001). Logistic regression and receiver operating characteristic curve analyses revealed the significant and independent value (p < 0.001) of the miR-92a and miR-21 expression quantification in serums. Moreover, the comparison with the clinicopathologic data of the BC patients showed that decreased levels of miR-92a and increased levels of miR-21 were associated with tumor size and a positive lymph node status (p < 0.001). CONCLUSIONS: These findings suggest that many miRNAs expressions are altered in BC, whose expression profiling may provide a useful clue for the pathophysiological research. Circulating miR-92a has potential use as novel breast cancer biomarker, which is comparable to miR-21.

Direct serum and tissue assay for EGFR mutation in non-small cell lung cancer by high-resolution melting analysis.

Biological therapy with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have noted promising outcomes for patients with non-small cell lung carcinoma (NSCLC), especially those with mutated EGFR. Tissue EGFR gene mutation testing can predict the benefit of taking a first-line EGFR-TKI, thus, allowing the physician to prescribe the most suitable therapy. Unfortunately, most lung cancer patients, especially NSCLC patients present with advanced disease that is surgically unresectable. The goal of this study was to develop high-resolution melting (HRM) assays to detect EGFR mutations in exons 18 to 21, compare their sensitivity and concordance to direct sequencing, and evaluate the feasibility and reliability of serum as a tissue alternate for routine EGFR mutation screening. EGFR mutations of 126 Formalin-Fixed Paraffin-Embedded (FFPE), 47 fresh frozen tissues and from 47 matched pre-operation serum specimens of NSCLC patients were screened by the HRM assays. EGFR mutations by HRM were confirmed through sequencing. We found 78 EGFR mutations in 70 FFPE tissues, 25 EGFR mutations in 24 fresh frozen tissues, with a mutation rate of 55.56% (70/126) and 51.06% (24/47), respectively. Most mutations were correctly identified by sequencing. EGFR mutations were detected in 22 serum samples from 24 tissue EGFR mutation-positive patients. The concordance rate between serum and tissue in EGFR mutation screening was 91.67%. We conclude that the HRM assay can provide convincing and valuable results both for serum and tissues samples, thus, it is suitable for routine serum EGFR mutation screening for NSCLC patients, especially those surgically unresectable.

Loss of Wnt5a and Ror2 protein in hepatocellular carcinoma associated with poor prognosis.

AIM: To investigate the expression and clinical significance of Wnt member 5a (Wnt5a) and receptor tyrosine kinase-like orphan receptor 2 (Ror2) in hepatocellular carcinoma (HCC). METHODS: In HCC tissues obtained from 85 patients, the protein expressions of Wnt5a, Ror2, ß-catenin, and Ki-67 via immunohistochemical staining using the Envision Plus System. The antibody binding was visualized with 3, 3'-diaminobenzidine tetrahydrochloride (DAB) before brief counterstaining with Mayer's hematoxylin. The degree of immunohistochemical staining was recorded using a semiquantitative and subjective grading system. The mRNA expression of Ror2 was examined by real-time reverse transcription polymerase chain reaction, including nineteen of the 85 HCC and three normal liver tissues. The ratios of Ror2 to the housekeeping gene GAPDH represented the normalized relative levels of Ror2 expression. To determine the prognostic factor, the outcome of the 82 patients was determined by reviewing their medical charts. The overall and disease-free survival rates were estimated using the Kaplan-Meier method and compared with the log-rank test. The prognostic analysis was carried out with univariate and multivariate Cox regressions models. RESULTS: Compared to nontumorous (hepatitis or cirrhotic) tissues, Ror2 mRNA expression was clearly decreased in HCC. Ror2 and Wnt5a protein expressions in the majority of HCC patients (63% and 77%, respectively) was significantly less in tumor tissues, as compared to adjacent nontumorous tissues, and this reduction was correlated with increasing serum α-fetoprotein and tumor stage. In 68% (58/85) of the HCC cases, the expression of ß-catenin in tumor tissues was either downregulated in the cellular membrane, upregulated in the cytoplasm, or both. Survival analysis indicated that Wnt5a and Ror2 protein expressions could be regarded as independent prognostic factors for HCC; HCC patients with decreased Wnt5a or Ror2 protein expression had a poorer prognosis than those with elevated Wnt5a and Ror2 expression (P = 0.016, P = 0.007, respectively). CONCLUSION: Wnt5a and Ror2 may serve as tumor suppressor genes in the development of HCC, and may serve as clinicopathologic biomarkers for prognosis in HCC patients.

[Content of CD4+ CD25+ regulatory T cells in peripheral blood lymphocytes in patients with allergic rhinitis].

OBJECTIVE: To investigate the change of CD4- CD25 regulatory T cells (Tregs) of peripheral blood in allergic rhinitis (AR) patients. METHOD: T lymphocytes of twenty AR patients and eight healthy subjects were separated and the flow cytometry was used to measure the percentage of CD4+ CD25+ Treg. RESULT: Compared with control group, the percentage of Treg and CD4- CD25high Tregs of AR patients was decreased significantly (P < 0.05). CONCLUSION: The proportion of CD4+ CD25+ Tregs decreased in AR patients conspicuously, which might be one of the pathogenesis of AR.

RNA interference-mediated silencing of UBCH10 gene inhibits colorectal cancer cell growth in vitro and in vivo.

1. UbcH10 is the cancer-related E2 ubiquitin-conjugating enzyme, and its overexpression has been demonstrated in a variety of malignancies. The aim of the present study is to silence UbcH10 gene by RNA interference (RNAi) and to observe its inhibitory effect on the colorectal cancer cell growth in vitro and in vivo. 2. We constructed the expression vector pGPU6/GFP/Neo/UbcH10-RNAi (pUbcH10-RNAi), which contained a UbcH10 short hairpin RNA expression cassette. Then the UbcH10 gene silencing cell lines LoVo/UbcH10-RNAi and HT-29/UbcH10-RNAi were established. Reverse transcription-polymerase chain reaction and western blot analysis were used to evaluate the expression of the UbcH10 gene. Cell Counting Kit-8 was used to assess properties of tumour cell growth in vitro. Flow cytometry was used to detect the effect of pUbcH10-RNAi on the cell cycle of colorectal cancer cells. Furthermore, the anti-tumour effects of pUbcH10-RNAi were evaluated in vivo in a nude mouse xenografts model. 3. Results demonstrated that UbcH10 gene expression was significantly decreased in pUbcH10-RNAi treated cells. Colorectal cancer cells growth was markedly suppressed in the pUbcH10-RNAi group compared with control conditions and colorectal cancer cells were arrested in the G2-M phase. In vivo, the downregulation of UbcH10 gene expression by pUbcH10-RNAi also inhibited tumour growth in a nude mice xenograft model. 4. Our study suggests that RNA interference-mediated silencing of UbcH10 gene has anti-tumour activity on colorectal cancer and might have therapeutic potential for the treatment of colorectal cancer.

Association of clinicopathological features with UbcH10 expression in colorectal cancer.

PURPOSE: UbcH10 is the cancer-related E2 ubiquitin-conjugating enzyme, and its overexpression has been demonstrated in a variety of malignancies. The aim of this study is to investigate the association of UbcH10 gene expression with the carcinogenesis and tumor progression of colorectal cancer. METHODS: The expression levels of UbcH10 in human malignant colorectal carcinoma tissues and their adjacent normal tissues were examined using real-time quantitative RT-PCR and immunohistochemical analysis. The correlations of UbcH10 expression to the clinicalpathologic characteristics of the colorectal cancer were analyzed. Cell proliferation and Matrigel invasion assays were performed in HT-29 cells transfected with UbcH10 expression plasmid pcDNA3.1-UbcH10, UbcH10 RNA interference vector pUbcH10-RNAi as well as their control vectors. RESULTS: Our study demonstrated that the expression of UbcH10 in colorectal carcinoma tissues was significantly higher than that in non-cancerous tissues (P < 0.01), and the UbcH10 overexpression was related to the degree of tumor differentiation and lymph node metastasis of colorectal cancer patients (P < 0.05). In vitro, the overexpression of UbcH10 promoted cell proliferation and tumor invasiveness, but the downregulation of UbcH10 expression significantly reduced the growth rate and the invasiveness activity of tumor cell line. CONCLUSIONS: Our study suggests that the overexpression of UbcH10 gene plays a critical role in the carcinogenesis and tumor progression of colorectal cancer. It may be a new marker in diagnosis and prognosis of colorectal cancer, and the inhibition of UbcH10 may be a therapeutic potential for the treatment of colorectal cancer.