METTL14 is Involved in TNF-α-Induced Inflammation in Colorectal Epithelial Cells via Autophagy Modulation.

Ulcerative colitis (UC) is a chronic and relapsing inflammatory bowel disease (IBD) characterized by colorectal inflammation. The N6-methyladenosine (m6A) modification of RNA regulates gene expression through the modulation of RNA metabolism, thus influencing various physiological and pathological processes. The aim of this study was to investigate the biological function of m6A methyltransferase METTL14 in colorectal epithelial cell inflammation. Bioinformatics analysis indicated that METTL14 expression was decreased in UC and was associated with disease severity and immune infiltration. We also noted a downregulation of METTL14 expression and a decrease in the total m6A RNA levels in TNF-α-stimulated Caco-2 cells. Moreover, METTL14 knockdown promoted inflammation and inhibited autophagy in TNF-α-stimulated Caco-2 cells, as indicated by the upregulation of NF-κB signaling and pro-inflammatory cytokine expression as well as LC3B protein downregulation. Treatment with the autophagy activator Torin-1 ameliorated the pro-inflammatory effects of METTL14 silencing. Furthermore, METTL14 knockdown significantly reduced the expression of ATG5. ATG5 overexpression could nullify the pro-inflammatory effect of METTL14 knockdown in TNF-α-stimulated Caco-2 cells. Mechanistically, METTL14 knockdown promoted ATG5 mRNA degradation, and luciferase analysis identified ATG5 as a target of m6A modification by METTL14. Taken together, silencing METTL14 promoted inflammation in Caco-2 cells via the downregulation of ATG5. Our findings revealed the importance of the m6A modification in colonic inflammation and autophagy, indicating that targeting METTL14 might be a potential therapeutic strategy for anti-inflammatory treatment in UC.

Challenging the nexus of power: The gaming dilemma of collaboration between government and enterprises in environmental management.

This study presents a novel approach to analysing environmental governance by Chinese governments through the lens of a tripartite evolutionary game model. The key novelty of our research lies in the explicit incorporation of a reputation mechanism into the evolutionary game analysis, which significantly influences government decision-making, environmental governance strategies, and the dynamics of the relationship between government and polluting enterprises. By assessing the marginal effects of conventional governance and mobilization-based governance on the environmental mechanism, as well as the collusion behaviours between government and polluting enterprises, our study sheds light on previously unexplored aspects of environmental governance. Our findings indicate that the reputation mechanism plays a crucial role within the evolutionary game system of environmental governance, exerting a substantial impact on government decision-making. Furthermore, we demonstrate that policy interventions, such as increasing the cost of penalties under regulatory policies, can encourage compliance strategies among enterprises. Additionally, our research highlights the high sensitivity of reputation signals towards local government's environmental governance strategies, directly impacting their credibility and influencing the adoption of proactive environmental governance strategies. Moreover, we emphasize the role of the central environmental inspection system as a means to facilitate the transmission of environmental governance pressures between central and local governments, ultimately fostering a green development concept and promoting coordinated development between the economy and ecological civilization. Overall, this study provides valuable insights into the intricacies of environmental governance in China and underscores the importance of reputation mechanisms and policy interventions in promoting sustainable development.

Unravelling the veil of appearance anxiety: exploring social media use among Chinese young people.

The purpose of this study is to explore the relationship between appearance anxiety and social media use among Chinese adolescents. Using a grounded theory approach, the study conducted two-round online interviews with ten Chinese university students and subsequently constructed a theoretical model of social media appearance anxiety among Chinese young people. The results of the study indicate that social media has a dual impact on appearance anxiety. On one hand, increased social media engagement amplifies appearance anxiety by shaping aesthetic standards and fostering comparative environments. On the other hand, diverse aesthetic perspectives and authentic presentations on social media partially alleviate appearance anxiety by promoting acceptance of unique appearances and boosting self-confidence. It is emphasized in this study that there should be an in-depth understanding of the dual impact and complicated relationship of social media on the daily lives of Chinese adolescents to further develop relevant strategies that promote healthy social media behavior among youth. Furthermore, this study calls for efforts to actively promote the healthy image and psychological well-being of adolescents while alleviating the negative impact of appearance anxiety and overall mental health. Such efforts are needed to ensure a positive and healthy development for the younger generation.

What makes Chinese adolescents "trapped" in severe mental illness? An interactionist perspective on self and identity.

The aim of this study was to explore the self and identity perspectives among Chinese adolescents with severe mental illness (SMI), with a focus on their illness experience and subjective meaning of a formal diagnosis. Thirty-one Chinese adolescents were interviewed and the interview data were analysed strictly according to principles suggested by the constructivist grounded theory approach. Five theoretical codes emerged in this study, including changes of personal values and beliefs, accumulated persistent developmental challenges and personal stresses, ineffective coping strategies and development, symptoms and development of mental illness, and changed perceptions and understandings of self. A proposed model of "The dynamic interactions of Chinese adolescents' identity and mental illness", was constructed and visualized. The results revealed that adolescents' identity formation is a fluctuating and non-linear process, but tends to be predominantly negative. The negative self, as informed by long-term ineffective coping with accumulated persistent developmental challenges and stressful events, develops towards a more serious status of negative identity and contributes to relapse symptoms, although this impact occurs variably with perceived personal characteristics. Besides, some participants who had achieved a state of "Buddha-like numbness" made a conscious decision to live a seemingly normal life while coexisting with their illness. The study also highlighted the positive aspects of identity formation that can arise from the experience of illness, including an enhanced sense of realism and increased empathy. Our findings will imply much the need for person-centred treatment plan and services that take into account of individual situations.

Insulin Glargine is More Suitable Than Exenatide in Preventing Muscle Loss in Non-Obese Type 2 Diabetic Patients with NAFLD.

AIM: This study investigated the effects of insulin glargine and exenatide on the muscle mass of patients with newly diagnosed type 2 diabetes (T2DM) and nonalcoholic fatty liver disease (NAFLD). METHODS: We performed a post-hoc analysis of our previously study, a 24-week randomized controlled multicenter clinical trial (ClinicalTrials.gov, NCT02303730). Seventy-six patients were randomly assigned 1:1 to receive insulin glargine or exenatide treatment. The changes in psoas muscle area (PMA) (mm2) were obtained with the cross-sectional Dixonfat magnetic resonance images at the fourth lumber vertebra. RESULTS: There were no significant differences in age, BMI, gender, and PMA in insulin glargine and exenatide groups at baseline. After treatment, PMA tended to increase by 13.13 (-215.52, 280.80) mm2 in the insulin glargine group and decrease by 149.09 (322.90-56.39) mm2 in the exenatide group (both p>0.05). Subgroup analysis showed a 560.64 (77.88, 1043.40) (mm2) increase of PMA in the insulin group relative to the Exenatide group in patients with BMI<28 kg/m2 (p0.031) after adjusting for gender, age, and research center. Interaction analysis showed an interaction between BMI and treatment (p0.009). However, no interaction was observed among subgroups with a BMI≥28 kg/m2 or with different genders and ages. CONCLUSION: Compared to exenatide, insulin glargine can relativity increase PMA in patients with T2DM having BMI<28 kg/m2 and NAFLD.

A comparison of NAFLD and MAFLD diagnostic criteria in contemporary urban healthy adults in China: a cross-sectional study.

BACKGROUND: A recently proposed diagnostic criteria of metabolic dysfunction-associated fatty liver disease (MAFLD) is more available for various clinical situations than nonalcoholic fatty liver disease (NAFLD), but understanding about differences between NAFLD and MAFLD in clinical practice remains limited in the general adult urban population in China. METHODS: A total of 795 subjects were recruited from Wu Song Branch of Zhongshan Hospital who participated in the general health assessment. Examination results was obtained through analysis of blood samples and abdominal ultrasonography. Participants were divided into four subgroups according to whether they had NAFLD or MAFLD (NAFLD- MAFLD-, NAFLD + MAFLD-, NAFLD- MAFLD + and NAFLD + MAFLD+). RESULTS: Among the urban healthy adults investigated, 345 people (43.4%) were diagnosed with NAFLD and 356 people (44.8%) with MAFLD. No significant differences in the prevalence, age, fasting blood glucose, glycosylated hemoglobin, liver enzyme examination, percentage of overweight, hypertension or dyslipidaemia were found between NAFLD and MAFLD patients. Patients with MAFLD had worse metabolic disorders than NAFLD + MAFLD- patients. The NAFLD fibrosis score (NFS) of the NAFLD- MAFLD + group was higher than that of the NAFLD + MAFLD- group. Higher proportion of patients in the NAFLD- MAFLD + group have NFS ≥-1.455. CONCLUSION: MAFLD criteria have similar prevalence and patient characteristics compared with previous NAFLD but help to identify a group of patients with high risks of metabolic disorders and liver fibrosis who have been missed with NAFLD, and has superior utility.

Current of injury is an indicator of lead depth and performance during left bundle branch pacing lead implantation.

BACKGROUND: Monitoring of lead depth is crucial to achieve left bundle branch pacing (LBBP) with a low capture threshold and avoid septal perforation, but lacks informative approach. OBJECTIVE: We aimed to prospectively assess the predictive value of current of injury on the occurrence of inadequate left bundle branch (LBB) capture threshold and acute septal perforation. METHODS: Consecutive patients who received LBBP were enrolled. ST-segment elevation ≥ 25% of intrinsic R-wave amplitude on the unipolar intracardiac electrogram was defined as a sign of distinct current of injury. An LBB capture threshold of <1.5 V/0.5 ms was considered acceptable. RESULTS: LBBP was attempted 513 times in 212 patients. The LBB capture threshold was more likely to improve to an acceptable level after 10 minutes in leads with initial (33 of 47 vs 0 of 8, with vs without) and residual (29 of 33 vs 4 of 14, with vs without) current of injury recorded on the tip electrode (P < .0001). Lead perforation during the procedure has occurred in 11 patients who had no current of injury noted on the tip electrode. The ratio of current of injury recorded on the tip electrode to that on the ring electrode was correlated to the lead depth determined by sheath angiography (Spearman correlation coefficient -0.624; P < .0001), and microperforation is highly possible when the ratio is decreased to <1 (sensitivity 100%; specificity 96.6%). CONCLUSION: Current of injury is a useful tool in forecasting LBBP lead depth and septal perforation, and it could facilitate the decision-making process when the initial LBB capture threshold is undesirable.

Silver nanoparticles induce apoptosis via NOX4-derived mitochondrial reactive oxygen species and endoplasmic reticulum stress in colorectal cancer cells.

Aim: To investigate the anticancer mechanisms of silver nanoparticles (AgNPs) in colorectal cancer. Methods: Anticancer effects of AgNPs were determined in colorectal cancer HCT116 cells and xenograft mice using cellular and molecular methods. Results: AgNPs induced mitochondrial reactive oxygen species production, mitochondrial dysfunction and endoplasmic reticulum (ER) stress responses through NOX4 and led to HCT116 cell apoptosis. Pretreatment with DPI or 4-PBA significantly inhibited mitochondrial reactive oxygen species production, apoptosis, ER stress response, NOX4 expression and mitochondrial dysfunction in AgNP-treated HCT116 cells. AgNPs also significantly suppressed HCT116 cell-based xenograft tumor growth in nude mice by inducing apoptosis and ER stress responses. Conclusion: AgNPs exert anticancer effects against colorectal cancer via ROS- and ER stress-related mitochondrial apoptosis pathways.

Transcription factor forkhead box K1 regulates miR-32 expression and enhances cell proliferation in colorectal cancer.

Increased microRNA (miR)-32 expression in colorectal cancer (CRC) tissues enhances CRC cell proliferation, migration, invasion and attenuates CRC cell apoptosis by repressing the expression of phosphatase and tensin homolog (PTEN). Forkhead box K1 (FOXK1) was identified as a potential interacting transcription factor using DNA pull-down assays and mass spectrometry. The present study aimed to elucidate the role of FOXK1 in regulating miR-32 expression in CRC. The expressions of FOXK1, miR-32, transmembrane protein 245 gene (TMEM245) and PTEN were compared between CRC and normal colonic tissues. Levels of miR-32, TMEM245, PTEN and the proliferation and apoptosis of CRC cells were studied using FOXK1-overexpression or knockdown, or by simultaneously interfering with FOXK1 and miR-32 expression. Direct FOXK1 binding to the miR-32 promoter was verified using chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays. The results showed elevated FOXK1, miR-32 and TMEM245 expression, and significantly decreased PTEN expression in CRC, compared with normal colonic tissues. Correlations between the expressions of TMEM245 and miR-32, FOXK1 and miR-32, and FOXK1 and TMEM245 were positive and significant. FOXK1-knockdown led to decreased miR-32 and TMEM245 expression and increased PTEN expression, whereas FOXK1-overexpression had the opposite effect. Overexpressed FOXK1 promoted the malignancy of CRC cells in vitro by stimulating proliferation and reducing apoptosis; whereas FOXK1-depletion suppressed such malignancy and a miR-32 inhibitor partially reversed the effects of FOXK1. The results of ChIP and dual-luciferase reporter assays indicated that FOXK1 directly binds to the promoter of TMEM245/miR-32. Thus, the FOXK1-miR-32-PTEN signaling axis may play a crucial role in the pathogenesis and development of CRC.

Expression profiles of NOD-like receptors and regulation of NLRP3 inflammasome activation in Toxoplasma gondii-infected human small intestinal epithelial cells.

BACKGROUND: Toxoplasma gondii is a parasite that primarily infects through the oral route. Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) play crucial roles in the immune responses generated during parasitic infection and also drive the inflammatory response against invading parasites. However, little is known about the regulation of NLRs and inflammasome activation in T. gondii-infected human small intestinal epithelial (FHs 74 Int) cells. METHODS: FHs 74 Int cells infected with T. gondii were subsequently evaluated for morphological changes, cytotoxicity, expression profiles of NLRs, inflammasome components, caspase-cleaved interleukins (ILs), and the mechanisms of NLRP3 and NLRP6 inflammasome activation. Immunocytochemistry, lactate dehydrogenase assay, reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative RT-PCR, and western blotting techniques were utilized for analysis. RESULTS: Under normal and T. gondii-infected conditions, members of the NLRs, inflammasome components and caspase-cleaved ILs were expressed in the FHs Int 74 cells, except for NLRC3, NLRP5, and NLRP9. Among the NLRs, mRNA expression of NOD2, NLRP3, NLRP6, and NAIP1 was significantly increased in T. gondii-infected cells, whereas that of NLRP2, NLRP7, and CIITA mRNAs decreased significantly in a time-dependent manner. In addition, T. gondii infection induced NLRP3, NLRP6 and NLRC4 inflammasome activation and production of IL-1ß, IL-18, and IL-33 in FHs 74 Int cells. T. gondii-induced NLRP3 inflammasome activation was strongly associated with the phosphorylation of p38 MAPK; however, JNK1/2 had a weak effect. NLRP6 inflammasome activation was not related to the MAPK pathway in FHs 74 Int cells. CONCLUSIONS: This study highlighted the expression profiles of NLRs and unraveled the underlying mechanisms of NLRP3 inflammasome activation in T. gondii-infected FHs 74 Int cells. These findings may contribute to understanding of the mucosal and innate immune responses induced by the NLRs and inflammasomes during T. gondii infection in FHs 74 Int cells.

Gender differences in the efficacy of pioglitazone treatment in nonalcoholic fatty liver disease patients with abnormal glucose metabolism.

BACKGROUND: Pioglitazone is a promising therapeutic method for nonalcoholic fatty liver disease (NAFLD) patients with or without type 2 diabetes. However, there is remarkable variability in treatment response. We analyzed our previous randomized controlled trial to examine the effects of gender and other factors on the efficacy of pioglitazone in treating Chinese nonalcoholic fatty liver disease (NAFLD) patients with abnormal glucose metabolism. METHODS: This is a post hoc analysis of a previous randomized, parallel controlled, open-label clinical trial (RCT) with an original purpose of evaluating the efficacy of berberine and pioglitazone on NAFLD. The total population (n = 185) was randomly divided into three groups: lifestyle intervention (LSI), LSI + pioglitazone (PGZ) 15 mg qd, and LSI + berberine (BBR) 0.5 g tid, respectively, for 16 weeks. The study used proton magnetic resonance spectroscopy (1H-MRS) to assess liver fat content. RESULTS: As compared with LSI, PGZ + LSI treatment further decreased liver fat content in women (- 15.24% ± 14.54% vs. - 8.76% ± 13.49%, p = 0.025), but less decreased liver fat content in men (- 9.95% ± 15.18% vs. - 12.64% ± 17.78%, p = 0.046). There was a significant interaction between gender and efficacy of pioglitazone before and after adjustment for age, smoking, drinking, baseline BMI, BMI change, treatment adherence, baseline liver fat content, and glucose metabolism. CONCLUSION: The study recommends pioglitazone plus lifestyle intervention for Chinese NAFLD female patients with abnormal glucose metabolism. TRIAL REGISTRATION: Role of Pioglitazone and Berberine in Treatment of Non-Alcoholic Fatty Liver Disease, NCT00633282 . Registered on 3 March 2008, https://register.clinicaltrials.gov .

Downregulation of microRNA-126 is inversely correlated with insulin receptor substrate-1 protein expression in colorectal cancer and is associated with advanced stages of disease.

Colorectal cancer (CRC) is a common human malignant tumor, and the fourth most common cause of cancer-associated mortality in China. However, the pathogenesis of CRC is not yet fully understood. The present study aimed to investigate the expression and clinical significance of microRNA (miR)-126 and insulin receptor substrate-1 (IRS-1), as well as the role of miR-126 in the prognosis of patients with CRC. A total of 86 colorectal tissue specimens, including 40 CRC and adjacent normal tissue, 26 colorectal adenoma tissue and 20 normal colorectal tissue samples, were collected for the present study. Reverse transcription-quantitative PCR analysis was performed to determine miR-126 and IRS-1 mRNA expression levels, while western blotting and immunohistochemistry (IHC) analyses were performed to determine IRS-1 protein expression levels. The correlation between miR-126 and IRS-1 expression, as well as the association between altered miR-126 and IRS-1 expression levels and clinicopathological characteristics, and the overall survival time of patients with CRC were assessed. The results demonstrated that miR-126 expression was significantly downregulated, while IRS-1 protein expression was upregulated in CRC tissues compared with that in adjacent normal tissues, colorectal adenoma tissues and normal colorectal tissues, respectively. IHC analysis exhibited strong positive staining of IRS-1 protein in CRC tissues, while absent or weak staining of IRS-1 protein was detected in adjacent normal tissues, colorectal adenoma tissues and normal colorectal tissues. miR-126 expression was inversely correlated with IRS-1 protein expression in CRC tissues (r=-0.420; P<0.05). Furthermore, downregulated miR-126 expression was associated with advanced clinicopathological characteristics of the disease and a shorter overall survival time in patients with CRC. Taken together, the results of the present study suggest that miR-126 downregulation may be a candidate molecular marker predictive of poor prognosis of patients with CRC.

LINC01123 facilitates proliferation, invasion and chemoresistance of colon cancer cells.

Colon cancer is one of the major causes of cancer-related deaths worldwide. Long non-coding RNA (lncRNA) LINC01123 has been suggested to act as an oncogene in non-small cell lung cancer and a prognostic signature in head and neck squamous cell carcinoma. However, its role in colon cancer remains obscure. From TCGA database, LINC01123 was observed to be up-regulated in colon adenocarcinoma (COAD). Subsequently, the up-regulated LINC01123 was also detected in colon cancer cells. Functionally, LINC01123 could enhance cell proliferation, migration, invasion and angiogenesis. Moreover, the chemoresistance of colon cancer cells was verified to be promoted by LINC01123. Afterward, LINC01123 was found to bind with Ago2 and miR-34c-5p. Besides, miR-34c-5p was confirmed to inhibit the cellular process and chemoresistance of colon cancer cells. Then, VEGFA was disclosed to coexist with LINC01123 and miR-34c-5p in RNA-induced silencing complex. And TCGA database suggested that its expression was correlated with different stages of COAD. Moreover, it was uncovered that VEGFA could bind with miR-34c-5p and its expression positively correlated with LINC01123 expression. Finally, LINC01123 was proofed to regulate colon cancer progression and cells chemoresistance via VEGFA. In conclusion, LINC01123/miR-34c-5p/VEGFA axis promotes colon cancer malignancy and cells chemoresistance.

Efficacy of exenatide and insulin glargine on nonalcoholic fatty liver disease in patients with type 2 diabetes.

BACKGROUND: The aim of this study was to investigate the efficacy of exenatide and insulin glargine in patients with newly diagnosed type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD). METHODS: We performed a 24-week randomized controlled multicentre clinical trial. Seventy-six patients were randomly assigned 1:1 to receive exenatide or insulin glargine treatment. The endpoints included changes in liver fat content (LFC), visceral adipose tissue (VAT), and subcutaneous adipose tissue (SAT) measured by magnetic resonance spectroscopy, blood glucose, liver enzymes, lipid profile, body weight, and Fibrosis-4 index (FIB-4). RESULTS: LFC, VAT, SAT, and FIB-4 were significantly reduced after exenatide treatment (ΔLFC, -17.55 ± 12.93%; ΔVAT, -43.57 ± 68.20 cm2 ; ΔSAT, -28.44 ± 51.48 cm2 ; ΔFIB-4, -0.10 ± 0.26; all P < .05). In comparison, only LFC (ΔLFC, -10.49 ± 11.38%; P < .05), and not VAT, SAT, or FIB-4 index (all P > .05), was reduced after insulin glargine treatment. Moreover, exenatide treatment resulted in greater reductions in alanine transaminase (ALT), aspartate transaminase (AST), and gamma glutamyl transpeptidase (GGT) than insulin glargine (P < 0.05). The body weight, waist circumference, postprandial plasma glucose, and low-density lipoprotein cholesterol (LDL-C) in the exenatide group also presented greater reductions than the insulin glargine group (P < .05). The proportion of adverse events were comparable between the two groups. CONCLUSION: Both exenatide and insulin glargine reduced LFC in patients with drug-naive T2DM and NAFLD; however, exenatide showed greater reductions in body weight, visceral fat area, liver enzymes, FIB-4, postprandial plasma glucose, and LDL-C.

Analysis of promoter methylation and epigenetic regulation of miR-32 in colorectal cancer cells.

MicroRNA-32 (miR-32) is upregulated in colorectal cancer (CRC) tissues; its overexpression leads to increased cell proliferation, migration and invasion, as well as reduced apoptosis of CRC cells, at least partly by inhibiting the target gene phosphatase and tensin homolog. However, the mechanisms of its upregulation have remained elusive. In the present study, the effects of methylation and acetylation on the expression of miR-32 were investigated. The promoter methylation status of miR-32 in the CRC cell lines HT-29 and HCT-116 and the normal colonic epithelial cell line NCM460 was investigated by bisulfate sequencing polymerase chain reaction (BSP). The potential role of methylation and histone acetylation in the regulation of miR-32 expression in CRC cells was investigated using the demethylation reagent 5-aza-2'-deoxycytidine (5-Aza-dC), the histone deacetylase inhibitor trichostatin A (TSA) and transfection of DNA methyltransferase 1 (DNMT1) overexpression plasmid. BSP revealed that CpG sites in the miR-32 promoter region of CRC and normal colonic epithelial cells were all hypomethylated, with methylation rates of 0.12, 1.14 and 0.64% in HCT-116, HT-29 and NCM460 cells, respectively. Treatment with 5-Aza-dC and/or TSA and transfection with DNMT1 plasmid did not significantly alter the expression of miR-32. Therefore, the present results suggest that methylation and histone acetylation do not affect miR-32 expression in CRC cells.

Analysis of the promoter region of the human miR-32 gene in colorectal cancer.

The pathogenesis of colorectal cancer (CRC) is poorly understood. MicroRNA (miR)-32 upregulation in CRC tissues was previously reported, where it increased the proliferation, migration and invasion, and reduced apoptosis of CRC cells by inhibiting the expression of phosphatase and tensin homolog (PTEN). However, the mechanism underlying miR-32 upregulation remains unknown. miR-32 is an intronic miRNA located within intron 14 of the transmembrane protein 245 gene (TMEM245). The present study aimed to elucidate the biological pathways underlying miR-32 regulation in CRC. A truncated promoter containing the 5'-flanking region of TMEM245/miR-32 gene was constructed. The promoter region was analyzed by dual luciferase reporter assay in CRC cells. DNA pull-down assay and mass spectrometry (MS) were used to identify proteins binding to the core promoter. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and transcription factor (TF) analyses were used to identify the binding proteins. The -320 to -1 bp fragment of the 5'-flanking region exhibited the highest luciferase activity. The regions spanning -606 to -320 bp exhibited a significant decrease in luciferase activity, compared with the -320 to -1 bp fragment. DNA pull-down assay and MS revealed 403 potential miR-32 promoter binding proteins. GO and KEGG pathway analysis indicated that these proteins were involved in numerous physiological and biochemical processes, including 'structural molecule activity', 'RNA binding', 'small molecule metabolic process' and 'biogenesis'. Furthermore, TF analysis revealed 10 potential interacting TFs, including SMAD family member 1 (SMAD1), signal transducer and activator of transcription 1 (STAT1) and forkhead box K1 (Foxk1). These results suggested that the core promoter region may be located within-320 to -1 bp of the 5'-flanking region of TMEM245/miR-32 gene, while the region from -606 to -320 bp may harbor repressive regulatory elements. The TFs SMAD1, STAT1 and Foxk1 may be involved in the transcriptional regulation of miR-32.

Evaluation of Protective Immune Response Induced by a DNA Vaccine Encoding GRA8 against Acute Toxoplasmosis in a Murine Model.

Toxoplasma gondii is an apicomplexan zoonotic protozoan parasite that infects most species of warm-blooded animals, including humans. The heavy incidence and severe or lethal damage caused by T. gondii infection clearly indicate a need for the development of an effective vaccine. T. gondii GRA8 is a member of the dense granules protein family and is used as a marker of acute infection. In the present study, we evaluated the protective immunity induced by DNA vaccination based on a recombinant eukaryotic plasmid, pDsRed2-GRA8, against acute toxoplasmosis in mice. BALB/c mice were intramuscularly immunized with the pDsRed2-GRA8 plasmid and then challenged by infection with the highly virulent GFP-RH strain of T. gondii. The specific immune responses and protective efficacy against T. gondii of this vaccine were analyzed by measuring cytokine and serum antibody titers, splenocyte proliferation assays, and the survival times of mice after challenge. Our results showed that mice immunized with pDsRed2-GRA8 demonstrated specific humoral and cellular responses, induced higher IgG antibody titers with predominant IgG2a production; increased levels of IL-10, IL-12 (p70), IFN-γ, TNF-α, and splenocyte proliferation; and prolonged survival times compared to those of control mice. The present study showed that DNA immunization with pDsRed2-GRA8 induced humoral and cellular immune responses, and all immunized mice showed greater Th1-type immune responses and longer survival times than those of control mice. These results indicated that T. gondii GRA8 DNA immunization induces a partial protective effect against acute toxoplasmosis.

MicroRNA-206 is involved in the pathogenesis of ulcerative colitis via regulation of adenosine A3 receptor.

Increasing evidence suggests that miRNAs are widely dysregulated in ulcerative colitis (UC), potentially affecting UC pathogenesis, diagnosis, and therapy. microRNA (miR) -206 has been reported to be upregulated in UC; however, its function and role in UC remain unknown. Here, we elucidate the function of miR-206 in the pathogenesis of UC. In patients with active-UC, miR-206 and adenosine A3 receptor (A3AR) levels were significantly upregulated and downregulated, respectively, and were inversely correlated. A3AR was expressed in the colon mucosa (particularly in colon epithelial-cell membranes). In HT-29 cells, miR-206 downregulated A3AR mRNA/protein expression by directly targeting the A3AR 3'-UTR; miR-206 overexpression and knockdown respectively increased and decreased TNF-α-induced nuclear NF-κB/p65, p-IκB-α, IKKα, p-IKKα and IL-8/IL-1ß secretion. However, A3AR-siRNA reversed the miR-206 inhibitory effect. Furthermore, miR-206 increased dextran sodium sulphate-induced colitis severity (i.e., increased bodyweight loss, DAI score, colon shrinkage, and MPO activity), which was partially ameliorated by miR-206-antagomir treatment. miR-206-agomir treatment potently suppressed A3AR expression and increased NF-κB signalling and downstream cytokine (TNF-α/IL-8/IL-1ß) expression in the mouse colon, in contrast to miR-206-antagomir administration. Taken together, our results demonstrated that miR-206 has a proinflammatory role in UC by downregulating A3AR expression and activating NF-κB signalling.

MicroRNA-16 is putatively involved in the NF-κB pathway regulation in ulcerative colitis through adenosine A2a receptor (A2aAR) mRNA targeting.

MicroRNAs (miRNAs) act as important post-transcriptional regulators of gene expression by targeting the 3'-untranslated region of their target genes. Altered expression of miR-16 is reported in human ulcerative colitis (UC), but its role in the development of the disease remains unclear. Adenosine through adenosine A2a receptor (A2aAR) could inhibit nuclear factor-kappaB (NF-κB) signaling pathway in inflammation. Here we identified overexpression of miR-16 and down-regulation of A2aAR in the colonic mucosa of active UC patients. We demonstrated that miR-16 negatively regulated the expression of the A2aAR at the post-transcriptional level. Furthermore, transfection of miR-16 mimics promoted nuclear translocation of NF-κB p65 protein and expression of pro-inflammatory cytokines, IFN-γ and IL-8 in colonic epithelial cells. Treatment with miR-16 inhibitor could reverse these effects in cells. The A2aAR-mediated effects of miR-16 on the activation of the NF-κB signaling pathway were confirmed by the A2aAR knockdown assay. Our results suggest that miR-16 regulated the immune and inflammatory responses, at least in part, by suppressing the expression of the A2aAR to control the activation of the NF-κB signaling pathway.

Serum retinol binding protein 4 is negatively related to estrogen in Chinese women with obesity: a cross-sectional study.

BACKGROUND: The association between serum Retinol Binding Protein 4 (RBP4) and obesity is still controversial. Serum RBP4 levels varies by gender, and estradiol may play a role in the difference. To investigate the participation of sex hormones in the association of RBP4 and obesity in humans, we measured serum RBP4, BMI, and sex hormones in 87 women from the outpatient. METHODS: Eighty-seven subjects of Chinese women origin from the outpatient (aged 40.22 ± 15.54 years) were enrolled. Subjects with diseases affecting the metabolic state or not suitable to participate in this study were excluded. Anthropometrics and laboratory tests, including lipid profile, luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin (PRL), estradiol (E2),progesterone (PROG), testosterone (TESTO), and dehydroepiandrosterone (DHEA) were conducted. Serum RBP4 was detected by an enzyme immunoassay kit and validated by quantitative Western blotting. RESULTS: Circulating RBP4 levels were positively associated with BMI, waist circumference, waist-to-hip ratio (WHR), systolic and diastolic (SBP), diastolic blood pressure (DBP), triglycerides (TG), low high-density lipoprotein cholesterol(LDL-c), and testosterone (TESTO) in the total group. While only in obese individuals, serum RBP4 levels were negatively associated with E2. The highest value was in the subjects with both obesity and the low estrogen level. Multiple linear regression analysis revealed that RBP4 correlated independently with TG, TC and insulin in all subjects, TC in non- obese individuals. However, E2 were significantly associated with serum RBP4 only in obese individuals. CONCLUSIONS: RBP4 could be a marker of obesity-related factors; estrogen was negatively related to RBP4 and might be one of the influential factors.