The identification of N6-methyladenosine-related miRNAs predictive of hepatocellular carcinoma prognosis and immunotherapy efficacy.

BACKGROUND: Hepatocellular carcinoma (HCC) has a high degree of malignancy and poor prognosis. N6-methyladenosine (m6A) modifications and microRNAs (miRNAs) play pivotal roles in tumorigenesis and development. However, the role of m6A-related miRNAs in HCC has not been clarified yet. This study aimed to identify the role of m6A-miRNAs in HCC prognosis through bioinformatics analysis. METHODS: The clinicopathological information and RNA sequencing data of 369 HCC tumor tissues and 49 tumor-adjacent tissues were downloaded from the TCGA database. A total of 23 m6A regulators were extracted to evaluated the m6A-related miRNAs using Pearson's correlation analysis. Then, we selected prognosis-related m6A-miRNAs using a univariate Cox regression model and used the consensus cluster analysis to explore the characteristics of the m6A-miRNAs. The coefficient of the least absolute shrinkage and selection operator (LASSO) Cox regression was applied to construct a prognostic risk score model. The receiver operated characteristic (ROC) analysis was applied to evaluate the prognostic value of the signature. The biological functions of targeted genes were predicted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Then, to validate the potential predictive value for prognosis, the miRNA expression profiles from the GSE76903 and GSE6857 were used. Single sample Gene Set Enrichment Analysis (ssGSEA) and Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE) were applied to assess the immune microenvironment of HCC. Additionally, a meta-analysis was used to verify the prognostic value of the m6A-microRNAs. RT-PCR was applied to validated the expression of miRNAs in HCC tissues. Cell viability, transwell assay and RNA m6A dot blot assays of HCC cells was applied to access the function of miR-17-5p. RESULTS: The expression of 48 m6A-related miRNAs was identified and 17 prognostic m6A-miRNAs was discovered. The expression profile of those 17 miRNAs was divided into three clusters, and these clusters were associated with the tumor microenvironment (TME) and prognosis. The nine m6A-related miRNA signature was associated with the prognosis of HCC, the AUC of the ROC was 0.771(TCGA dataset), 0.788(GSE76903) and 0.646(GSE6857). The TME and the expression of immune checkpoint molecules were associated with the risk score. The meta-analysis also validated the prognostic value of the m6A-related miRNAs (miR182-5p (HR:1.58, 95%CI:1.04-2.40) and miR-17-5p (HR:1.58, 95%CI: 1.04-2.40)). The expression of miR-17-5p was upregulated in HCC tissues and miR-17-5p showed an oncogenic role in HCC cells. CONCLUSION: The clinical innovation is the use of m6A-miRNAs as biomarkers for predicting prognosis regarding immunotherapy response in HCC patients.

Dickkopf-1 has an Inhibitory Effect on Mesenchymal Stem Cells to Fibroblast Differentiation.

BACKGROUND: Mesenchymal stem cells (MSCs) are bone marrow stem cells which play an important role in tissue repair. The treatment with MSCs will be likely to aggravate the degree of fibrosis. The Wnt/ß-catenin signaling pathway is involved in developmental and physiological processes, such as fibrosis. Dickkopfs (DKKs) are considered as an antagonist to block Wnt/ß-catenin signaling pathway by binding the receptor of receptor-related protein (LRP5/6). DKK1 was chosen in attempt to inhibit fibrosis of MSCs by lowering activity of Wnt/ß-catenin signaling pathway. METHODS: Stable MSCs were randomly divided into four groups: MSCs control, MSCs + transforming growth factor-ß (TGF-ß), MSCs + DKK1, and MSCs + TGF-ß + DKK1. Flow cytometry was used to identify MSCs. Cell viability was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide test. Immunofluorescence was used to detect protein expression in the Wnt/ß-catenin signaling pathways. Western blotting analysis was employed to test expression of fibroblast surface markers and, finally, real-time reverse transcription polymerase chain reaction was employed to test mRNA expression of fibroblast surface markers and Wnt/ß-catenin signaling proteins. RESULTS: Cultivated MSCs were found to conform to the characteristics of standard MSCs: expression of cluster of differentiation (CD) 73, 90, and 105, not expression of 34, 45, and 79. We found that DKK1 could maintain the normal cell morphology of MSCs. Western blotting analysis showed that fibroblast surface markers were expressed in high quantities in the group MSCs + TGF-ß. However, the expression was lower in the MSCs + TGF-ß + DKK1. Immunofluorescence showed high expression of all Wnt/ß-catnin molecules in the MSCs + TGF-ß group but expressed in lower quantities in MSCs + TGF-ß + DKK1 group. Finally, mRNA expression of fibroblast markers vimentin, α-smooth muscle actin and Wnt/ß-catenin signaling proteins ß-catenin, T-cell factor, and glycogen synthase kinase-3ß was significantly increased in MSCs + TGF-ß group compared to control (P < 0.05). Expression of the same fibroblast markers and Wnt/ß-catenin was decreased to regular quantities in the MSCs + TGF-ß + DKK1 group. CONCLUSIONS: DKK1, Wnt/ß-catenin inhibitors, blocks the Wnt/ß-catenin signaling pathway to inhibit the process of MSCs fibrosis. It might provide some new ways for clinical treatment of certain diseases.

Effectiveness and safety of oseltamivir for treating influenza: an updated meta-analysis of clinical trials.

BACKGROUND: Oseltamivir has been widely used to treat patients with influenza; however, its effects have been debated. Recently, a new meta-analysis on the controversial topic of oseltamivir's effectiveness found that the drug reduces the duration of influenza symptoms and the risk of hospitalization, while increasing the risk of nausea and vomiting. Unfortunately, the analysis did not include articles published in Chinese. Thus, we performed an updated meta-analysis by adding more studies in China. METHODS: The terms 'oseltamivir,' 'influenza,' and 'effect' were used to search for publications in both English and Chinese. Only controlled clinical trials were included. We used the weighted mean difference (WMD) or relative risk (RR), together with the 95% confidence interval (95% CI), to estimate the effects of oseltamivir. RESULTS: A total of 12 studies including 107 712 patients were eligible for analysis. Oseltamivir significantly reduced the duration of fever (WMD, -20.48; 95% CI, -28.43, -12.53) and influenza-like symptoms (WMD, -19.39; 95% CI, -32.94, -5.84). The rates of hospitalization (RR, 0.79; 95% CI, 0.68, 0.90), antibiotics usage (RR, 0.56; 95% CI, 0.42, 0.74), otitis media (RR, 0.78; 95% CI, 0.65, 0.93), and nonspecific complications (RR, 0.58; 95% CI, 0.35, 0.95) were significantly decreased among patients taking oseltamivir. No significant difference was observed with respect to the risk of adverse reactions. CONCLUSION: Oseltamivir can effectively alleviate the symptoms of influenza and reduce hospitalization, antibiotic usage, and the risk of otitis media without significantly increasing the rate of adverse drug reactions.

Is tuberculosis treatment really free in China? A study comparing two areas with different management models.

OBJECTIVE: China has implemented a free-service policy for tuberculosis. However, patients still have to pay a substantial proportion of their annual income for treatment of this disease. This study describes the economic burden on patients with tuberculosis; identifies related factors by comparing two areas with different management models; and provides policy recommendation for tuberculosis control reform in China. METHODS: There are three tuberculosis management models in China: the tuberculosis dispensary model, specialist model and integrated model. We selected Zhangjiagang (ZJG) and Taixing (TX) as the study sites, which correspond to areas implementing the integrated model and dispensary model, respectively. Patients diagnosed and treated for tuberculosis since January 2010 were recruited as study subjects. A total of 590 patients (316 patients from ZJG and 274 patients from TX) were interviewed with a response rate of 81%. The economic burden attributed to tuberculosis, including direct costs and indirect costs, was estimated and compared between the two study sites. The Mann-Whitney U Test was used to compare the cost differences between the two groups. Potential factors related to the total out-of-pocket costs were analyzed based on a step-by-step multivariate linear regression model after the logarithmic transformation of the costs. RESULTS: The average (median, interquartile range) total cost was 18793.33 (9965, 3200-24400) CNY for patients in ZJG, which was significantly higher than for patients in TX (mean: 6598.33, median: 2263, interquartile range: 983-6688) (Z = 10.42, P < 0.001). After excluding expenses covered by health insurance, the average out-of-pocket costs were 14304.4 CNY in ZJG and 5639.2 CNY in TX. Based on the multivariable linear regression analysis, factors related to the total out-of-pocket costs were study site, age, number of clinical visits, residence, diagnosis delay, hospitalization, intake of liver protective drugs and use of the second-line drugs. CONCLUSION: Under the current "free of diagnosis and treatment" policy, the financial burden remains heavy on tuberculosis patients. Policy makers need to consider appropriate steps to lessen the burden of out-of-pocket costs for tuberculosis patients in China and how best to improve service delivery for poor patients.

Obesity-associated gene FTO rs9939609 polymorphism in relation to the risk of tuberculosis.

BACKGROUND: Obesity is known to affect cell-mediated immune responses. Recent studies have revealed that genetic polymorphisms in the fat mass and obesity associated (FTO) gene are related to human obesity. We hypothesize that this gene may also play a role in the risk of immune-related infectious diseases such as tuberculosis. METHODS: This case-control study included 1625 pulmonary tuberculosis cases and 1570 unaffected controls recruited from the Jiangsu province in China. Single nucleotide polymorphisms (SNPs), rs9939609 and rs8050136, in the FTO gene were genotyped using TaqMan allelic discrimination assays. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using the unconditional logistic regression model. RESULTS: We observed a significant association between the genetic polymorphism rs9939609 and tuberculosis risk. Compared with the common genotype TT, individuals carrying AA had a significantly increased risk, with an OR of 3.77 (95% CI: 2.26-6.28). After adjusting for potential confounders, the relationship remains significant. An additive model showed that carriers of an allele A had a 26% increased risk of tuberculosis compared with the T allele (OR: 1.26, 95% CI: 1.08-1.48). Compared with the common haplotype rs9939609T-rs8050136C, the haplotype rs9939609A-rs8050136C was related to an increased risk of tuberculosis (OR = 6.09, 95% CI: 3.27-12.34). CONCLUSIONS: The FTO polymorphism rs9939609 is associated with a risk of pulmonary tuberculosis in the Chinese population.

Genetic polymorphisms of IFNG and IFNGR1 in association with the risk of pulmonary tuberculosis.

OBJECTIVE: Genetic host factors play an important role in controlling individual's susceptibility to the pathogen. This study aims to explore the single and joint effect of genetic polymorphisms of interferon-gamma (IFNG) and its receptor (IFNGR1) in association with the pulmonary tuberculosis in a Chinese Han population. METHODS: This population-based case control study consisted of 1434 pulmonary tuberculosis patients and 1412 healthy controls. Six tag SNPs in IFNG/IFNGR1 were genotyped using TaqMan allelic discrimination technology. The logistic regression model was carried out to analyze the associations between the genotypes and haplotypes and the risk of tuberculosis by calculating the odds ratio (OR) and 95% confidence interval (CI). RESULTS: After the Bonferroni correction for multiple comparisons, three SNPs (rs2234711, rs1327475 and rs7749390) in IFNGR1 gene were observed to be significantly associated with the altered risks of tuberculosis. For the SNP rs2234711, individuals carrying C allele (vs. T) showed a decreased risk, with the adjusted OR(95% CI) of 0.82(0.76-0.91). The additive model revealed that each additional allele contributed about 14% decreased risk (OR: 0.86, 95% CI: 0.77-0.95). Moreover, we observed a strong linkage disequilibrium between rs2234711 and rs3799488. Compared with the common rs2234711C-rs3799488C haplotype, the haplotype rs2234711T-rs3799488C contributed to a significant increase in the risk of tuberculosis (adjusted OR: 1.24, 95% CI: 1.09-1.41). CONCLUSIONS: Our results suggest that genetic polymorphisms in IFNGR1 gene are involved in the risk of tuberculosis in the Chinese population. Future studies should include a comprehensive sequencing analysis to identify the specific causative sequence variants underlying the observed associations.