The Expression of miR-377-3p in Patients with DKD and the Regulatory Mechanism of miR-377-3p on the Inflammatory Response of HK-2 Cells Through TGF-ß.

Objective: The purpose of the study was to investigate the expression levels and correlation of inflammatory factors such as miR-377-3p and TGF-ß in patients with diabetic kidney disease (DKD), and to investigate the regulatory mechanism of transfection of miR-377-3p on the inflammatory response of HK-2 cell induced by high glucose. Methods: According to UACR, patients were divided into normal albuminuria group (Con, n = 29), microalbuminuria group (Micro, n = 31) and macroalbuminuria group (Macro, n = 30), analyzed the correlation and influencing factors between DKD and inflammatory factor. HK-2 cells were randomly divided into four groups: normal control group (NC), high glucose group (HG), miR-377-3p overexpression group (MIN), and miR-377-3p inhibition group (IN). After transfection of miR-377-3p mimics and inhibitors, the contents of TGF-ß, IL-6 and IL-18 were detected by RT-PCR and Western blot. Results: The levels of miR-377-3p, TGF-ß, IL-6 and IL-18 in both Micro group and Macro group were significantly higher than those in Con group (P < 0.05); Pearson correlation analysis showed that miR-377-3p was positively correlated with UACR, TG, TGF-ß, IL-6 and IL-18, and negatively correlated with GFR (P < 0.05). Cell experiment: RT-PCR and Western blot results showed that miR-377-3p, TGF-ß, IL-6 and IL-18 in HG group were significantly higher than those in NC group (P < 0.05). After transfection with miR-377-3p inhibitor, the levels of miR-377-3p, TGF-ß, IL-6 and IL-18 in IN group were significantly decreased compared with HG group and MIN group. Conclusion: miR-377-3p expression was elevated both in serum of DKD patients and in HK-2 cells with high glucose induced injury, overexpression of miR-377-3p exacerbates the damage to HK-2 cells and promotes the progression of DKD. Silencing miR-377-3p can potentially regulate the levels of inflammatory factors in HK-2 cells by targeting downregulation of TGF-ß expression, thereby mitigating the damage to HK-2 cells and delaying the development of diabetic kidney disease.

The predictive value of miR-377 and phospholipase A2 in the early diagnosis of diabetic kidney disease and their relationship with inflammatory factors.

OBJECTIVE: The value of novel biomarkers for DKD has received increasing attention, and there is an urgent need for novel biomarkers with sensitivity, specificity and ability to detect kidney damage.miR-377 regulates many basic biological processes, plays a key role in tumor cell proliferation, migration and inflammation, and can also increase the expression of matrix proteins and fibronectin, leading to renal tubulointerstitial inflammation and renal fibrosis. Lipoprotein-associated phospholipase A2, as an inflammatory marker, is involved in the pathological process of microalbuminuria production and renal function decline, and is a predictive factor of microalbuminuria production and renal function decline, and can be used as an indicator to evaluate the progression of DKD.The aim of this study was to investigate the effects of miR-377 and phospholipase A2 on the development of diabetic kidney disease through regulation of inflammatory factors and the mechanism of action. METHODS: 80 diabetic patients were divided into two groups according to urinary albumin-to-creatinine ratio (UACR): diabetic normal proteinuria group (n = 42) and diabetic proteinuria group (n = 38). Forty-three healthy people were selected as the normal control group. The serum levels of TGF-ß, IL-6, and IL-18 were measured by ELISA, miR-377 was detected by qPCR, and the serum levels of phospholipase A2 were detected by electrochemiluminescence. Analyze the correlation of study group indicators, ROC curve was used to evaluate the diagnostic efficacy of miR-377 and phospholipase A2 in diabetic kidney disease. RESULTS: The average levels of serum TGF-ß, IL-6, IL-18, miR-377 and phospholipase A2 in diabetic proteinuria group were significantly higher than those in normal control group and diabetic proteinuria normal group(P < 0.05). miR-377, phospholipase A2 were significantly correlated with inflammatory factors such as glomerular filtration rate and TGF-ß. miR-377 and phospholipase A2 are independent predictors of diabetic kidney disease. The area under the curve of miR-377 and phospholipase A2 in the normal diabetic proteinuria group and the diabetic proteinuria group were 0.731 and 0.744, respectively. CONCLUSION: miR-377 and phospholipase A2 have good diagnostic efficiency for the early diagnosis of diabetic kidney disease. They can be used as early biomarkers.miR-377 and phospholipase A2 were positively correlated with inflammatory factors and involved in the occurrence and development of diabetic kidney disease.

Promotion of IL­17/NF­κB signaling in autoimmune thyroid diseases.

IL-17 and other cytokines have a number of immunomodulatory effects on thyroid cells. The present study investigated the changes and correlations amongst IL-17, NF-κB, IL-6, IL-10, interferon-γ (IFN-γ), TNF-α, IL-2 and IL-4 in patients with different autoimmune thyroid diseases in order to further clarify the pathogenesis of autoimmune thyroid disease. A total of 82 patients with autoimmune thyroid diseases (41 with Graves' disease and 41 with Hashimoto's thyroiditis) and 53 healthy controls were enrolled. All relevant thyroid hormones were detected by electrochemiluminescence analyzer. The serum levels of IL-17 and other cytokines were detected using flow cytometry, NF-κB was detected by ELISA, reverse transcription-quantitative PCR was used to detect the protein expression of various mRNAs, and the correlations between IL-17 and these factors were analyzed. Significant differences occurred amongst all groups. NF-κB, TNF-α, IL-6, IL-17 and their mRNA levels were significantly higher in the healthy controls compared with those in the patients; whereas IFN-γ and IL-10 levels were significantly lower in the healthy controls compared with those in the patients . Correlation analysis showed that the expression levels of IL-17 and its mRNA were significantly positively correlated with the expression levels of NF-κB, IL-6, thyroid peroxidase antibody, thyroid gland globulin, thyroglobulin antibody, TNF-α and IFN-γ, and were also significantly negatively correlated with IL-10 . These findings suggested that IL-17 was elevated in patients with autoimmune thyroid disease and that IL-17 could activate the NF-κB signaling pathway, stimulate the production and release of inflammatory factors such as TNF-α, IL-6 and IFN-γ and participate in the pathogenesis of autoimmune thyroid injury.

Changes of ACE2 in different glucose metabolites and its relationship with COVID-19.

BACKGROUND: To study the changes and effects of angiotensin-converting enzyme 2 (ACE2)/angiotensin 1-7 (Ang1-7) and ACE/AngII in people with different glucose metabolisms and to explore the possible mechanisms underlying the severity of COVID-19 infection in diabetic patients. METHODS: A total of 88 patients with type 2 diabetes, 72 patients with prediabetes (impaired fasting glucose, 30 patients; impaired glucose regulation, 42 patients), and 50 controls were selected. Changes and correlations of ACE2, Ang1-7 and other indicators were detected among the three groups. Patients were divided into four groups according to the course of diabetes: <1 year, 1-5 years, 5-10 years, and >10 years. ACE2 and Ang1-7 levels were compared and analyzed. RESULTS: ACE2 and Ang1-7 increased with the severity of diabetes (P0 < .05 or P < .01). The levels of ACE2 and Ang1-7 in the longer course group were lower than those in the shorter course group, whereas the levels of ACE, Ang II, and interleukin-6 (IL-6) gradually increased (P < .05). Pearson correlation analysis showed that ACE2 was positively correlated with IL-6, FBG, and 2hPBG levels in the prediabetes group. In the diabetic group, ACE2 was positively correlated with Ang1-7 and negatively correlated with ACE, AngII, IL-6, and C-reactive protein levels. Multiple linear regression analysis showed that IL-6 and ACE were the main factors influencing ACE2 in the diabetic group. CONCLUSION SUBSECTIONS: ACE2/Ang1-7 and ACE/AngII systems are activated, and inflammatory cytokine release increases in prediabetes. With the prolongation of the disease course, the effect of ACE2/Ang1-7 decreased gradually, while the effect of ACE/AngII increased significantly. Dysfunctions of ACE2/Ang1-7 may be one of the important mechanisms underlying the severity of COVID-19 infection in patients with diabetes.

Machine Learning Screens Potential Drugs Targeting a Prognostic Gene Signature Associated With Proliferation in Hepatocellular Carcinoma.

Background: This study aimed to screen potential drugs targeting a new prognostic gene signature associated with proliferation in hepatocellular carcinoma (HCC). Methods: CRISPR Library and TCGA datasets were used to explore differentially expressed genes (DEGs) related to the proliferation of HCC cells. Differential gene expression analysis, univariate COX regression analysis, random forest algorithm and multiple combinatorial screening were used to construct a prognostic gene signature. Then the predictive power of the gene signature was validated in the TCGA and ICGC datasets. Furthermore, potential drugs targeting this gene signature were screened. Results: A total of 640 DEGs related to HCC proliferation were identified. Using univariate Cox analysis and random forest algorithm, 10 hub genes were screened. Subsequently, using multiplex combinatorial screening, five hub genes (FARSB, NOP58, CCT4, DHX37 and YARS) were identified. Taking the median risk score as a cutoff value, HCC patients were divided into high- and low-risk groups. Kaplan-Meier analysis performed in the training set showed that the overall survival of the high-risk group was worse than that of the low-risk group (p < 0.001). The ROC curve showed a good predictive efficiency of the risk score (AUC > 0.699). The risk score was related to gene mutation, cancer cell stemness and immune function changes. Prediction of immunotherapy suggetsted the IC50s of immune checkpoint inhibitors including A-443654, ABT-888, AG-014699, ATRA, AUY-922, and AZ-628 in the high-risk group were lower than those in the low-risk group, while the IC50s of AMG-706, A-770041, AICAR, AKT inhibitor VIII, Axitinib, and AZD-0530 in the high-risk group were higher than those in the low-risk group. Drug sensitivity analysis indicated that FARSB was positively correlated with Hydroxyurea, Vorinostat, Nelarabine, and Lomustine, while negatively correlated with JNJ-42756493. DHX37 was positively correlated with Raltitrexed, Cytarabine, Cisplatin, Tiotepa, and Triethylene Melamine. YARS was positively correlated with Axitinib, Fluphenazine and Megestrol acetate. NOP58 was positively correlated with Vorinostat and 6-thioguanine. CCT4 was positively correlated with Nerabine. Conclusion: The five-gene signature associated with proliferation can be used for survival prediction and risk stratification for HCC patients. Potential drugs targeting this gene signature deserve further attention in the treatment of HCC.

Erratum: CircCCNB1 silencing acting as a miR-106b-5p sponge inhibited GPM6A expression to promote HCC progression by enhancing DYNC1I1 expression and activating the AKT/ERK signaling pathway: Erratum.

[This corrects the article DOI: 10.7150/ijbs.66915.].

CircCCNB1 silencing acting as a miR-106b-5p sponge inhibited GPM6A expression to promote HCC progression by enhancing DYNC1I1 expression and activating the AKT/ERK signaling pathway.

Background: Circular RNAs (circRNAs), which generally act as microRNA (miRNA) sponges to competitively regulate the downstream target genes of miRNA, play an essential role in cancer biology. However, few studies have been reported on the role of circRNA based competitive endogenous RNA (ceRNA) network in hepatocellular carcinoma (HCC). Herein, we aimed to screen and establish the circRNA/miRNA/mRNA networks related to the prognosis and progression of HCC and further explore the underlying mechanisms of tumorigenesis. Methods: GEO datasets GSE97332, GSE108724, and GSE101728 were utilized to screen the differentially expressed circRNAs (DE-circRNAs), DE-miRNAs, and DEmRNAs between HCC and matched para-carcinoma tissues. After six RNA-RNA predictions and five intersections between DE-RNAs and predicted RNAs, the survival-related RNAs were screened by the ENCORI analysis tool. The ceRNA networks were constructed using Cytoscape software, based on two models of up-regulated circRNA/down-regulated miRNA/up-regulated mRNA and down-regulated circRNA/up-regulated miRNA/down-regulated mRNA. The qRT-PCR assay was utilized for detecting the RNA expression levels in HCC cells and tissues. The apoptosis, Edu, wound healing, and transwell assays were performed to evaluate the effect of miR-106b-5p productions on the proliferation, invasion, and metastasis of HCC cells. In addition, the clone formation, cell cycle, and nude mice xenograft tumor assays were used to investigate the influence of hsa_circ_0001495 (circCCNB1) silencing and overexpression on the proliferation of HCC cells in vitro and in vivo. Furthermore, the mechanism of downstream gene DYNC1I1 and AKT/ERK signaling pathway via the circCCNB1/miR-106b-5p/GPM6A network in regulating the cell cycle was also explored. Results: Twenty DE-circRNAs with a genomic length less than 2000bp, 11 survival-related DE-miRNAs, and 61 survival-related DE-mRNAs were screened out and used to construct five HCC related ceRNA networks. Then, the circCCNB1/miR-106b-5p/GPM6A network was randomly selected for subsequent experimental verification and mechanism exploration at in vitro and in vivo levels. The expression of circCCNB1 and GPM6A were significantly down-regulated in HCC cells and cancer tissues, while miR-106b-5p expression was up-regulated. After transfections, miR-106b-5p mimics notably enhanced the proliferation, invasion, and metastasis of HCC cells, while the opposite was seen with miR-105b-5p inhibitor. In addition, circCCNB1 silencing promoted the clone formation ability, the cell cycle G1-S transition, and the growth of xenograft tumors of HCC cells via GPM6A downregulation. Subsequently, under-expression of GPM6A increased DYNC1I1 expression and activated the phosphorylation of the AKT/ERK pathway to regulate the HCC cell cycle. Conclusions: We demonstrated that circCCNB1 silencing promoted cell proliferation and metastasis of HCC cells by weakening sponging of oncogenic miR-106b-5p to induce GPM6A underexpression. DYNC1I1 gene expression was up-regulated and further led to activation of the AKT/ERK signaling pathway.

[Advances of long-acting recombinant protein therapeutics].

Some of the recombinant protein therapeutics with short half-life requires high frequent dose or injection, which results in poor patient compliance. This challenge has prompted the development of long-acting recombinant proteins in recent years. Four strategies and methods, including chemical modification, protein engineering, fusion proteins and protein glycosylation are used to modify protein molecule and finally obtain improved pharmacokinetics (PK) properties. This article reviews the four strategies of half-life extension and presents a detailed list of long-acting therapeutics on US, EU and China markets.

Relationship between XspI Site Polymorphisms of LDL-R Gene and Serum IL-2 and IL-10 in Patients with Hypercholesterolemia.

BACKGROUND: Relationship has been identified in sporadic reports between polymorphisms and hypercholesterolemia. However, the relationship between inflammatory cytokines and polymorphism of low-density lipoprotein receptor (LDL-R) gene in hypercholesterolemia is unclear. This study aimed to explore the relationship and significance between polymorphisms of LDL-R gene and serum Interleukin-2 (IL-2), IL-10 in patients with hypercholesterolemia. METHODS: PCR-RFLP and direct DNA sequencing assay were employed to determine polymorphism of LDL-R gene in 900 patients with hypercholesterolemia and 400 healthy cases. ELISA was applied to assay serum concentration of IL-2 and IL-10. Blood lipid indexes were tested in all cases. RESULTS: Compared with the healthy controls, level of IL-2 increased significantly, while IL-10 decreased significantly (P < 0.05). Correlation analysis showed that IL-2 was positively correlated with total cholesterol (TC), LDL-c, and genotype (r = 0.542, 0.410, 0.598, P < 0.05) and negatively correlated with HDL-c (r = -0.352, P < 0.05). Negative relationship also was found between TC, LDL-c, genotype, and IL-10 (r = -0.452, -0.390, -0.613, P < 0.05), and positive correlation between HDL-c and IL-10 (r = 0.398, P < 0.05). Multiple linear regression showed that genotypes and TC were independent factors affecting the levels of IL-2 and IL-10 (P < 0.05). CONCLUSION: IL-2 and IL-10 were related to gene polymorphisms of LDL-R, which might be involved in the development and progress of hypercholesterolemia.

Association of RBP4 levels with increased arterial stiffness in adolescents with family history of type 2 diabetes.

BACKGROUND: The aim of this study was to explore the impact of family history of type 2 diabetes (FH2D) on arterial stiffness in young people and its relationship to adipocytokines. METHODS: This case-control study included 52 adolescents (male/female 28/24) with FH2D (FH2D+) and 40 adolescents (male/female 21/19) without FH2D (FH2D-). Anthropometric measurements, including height, weight, waist circumference (WC), and blood pressure, were obtained. Blood samples were collected, fasting plasma glucose (FPG), serum lipids, Retinol Binding Protein 4 (RBP4), C reactive protein (CRP), adiponectin and visfatin were examined. Brachial-ankle pulse wave velocity (baPWV) was used to evaluate arterial stiffness. Visceral fat area (VFA) was measured by computerized tomography. RESULTS: Compared with FH2D- group, FH2D+ group had a significantly higher oral glucose tolerance test (OGTT) 2-hour insulin, RBP4 and baPWV levels, a lower adiponectin and glucose infusing rate (GIR) (P<0.05). BaPWV was positively correlated with age, systolic blood pressure (SBP), diastolic blood pressure (DBP), 2-hour (OGTT) insulin, RBP4, and VFA, and negatively correlated with GIR in FH2D+ group. After multivariate analysis, age, SBP, RBP4 and VFA maintained an independent association with baPWV in FH2D+ group (P<0.05), while only age, SBP, and VFA were independent predictors of baPWV in FH2D- group (P<0.05). CONCLUSIONS: These findings led to the conclusion that RBP4 level was associated with increased arterial stiffness in young subjects with family history of type 2 diabetes.

Phenotypic and molecular characteristics of carbapenem-non-susceptible Enterobacteriaceae from a teaching hospital in Wenzhou, southern China.

Carbapenem resistance in Enterobacteriaceae is increasing and has become a matter of great concern. The aim of this study was to characterize carbapenem-non-susceptible Enterobacteriaceae from a teaching hospital. A total of 49 carbapenem-non-susceptible Enterobacteriaceae clinical isolates recovered in 2007-2010 from the First Affiliated Hospital of Wenzhou Medical College were analyzed by antimicrobial susceptibility testing. The carbapenemase phenotype, outer membrane protein profiles, and clonal relatedness were investigated using the modified Hodge test, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) of Klebsiella pneumoniae was also performed. ß-Lactamase genes were examined by PCR and sequencing, and the transferability of carbapenemase genes was determined by a conjugation experiment. The rates of imipenem, meropenem, and ertapenem resistance were 59.2%, 40.8%, and 96.0%, respectively. Thirty isolates exhibited carbapenemase activity, and 32 isolates carried carbapenemase genes. Furthermore, 10 and 9 clinical isolates posessed AmpC ß-lactamase and extended-spectrum ß-lactamase (ESBL) genes, respectively. Eight of 32 carbapenemase-producing isolates were proved to be carried by conjugative plasmids, and there was porin loss in 34.7% (17/49) of the isolates. PFGE analysis demonstrated that 9 KPC-2-producing Serratia marcescens belonged to a clonal strain, suggesting the clonal dissemination of these KPC-2-bearing isolates among different wards. The MLST of K. pneumoniae revealed that two KPC-2 producers were ST11. This study suggests that KPC-2-type carbapenemase is the main contributor to carbapenems resistance in carbapenemase-producing Enterobacteriaceae, and that ESBL, AmpC ß-lactamase overproduction, and porin loss contribute to the resistance level among these isolates; in carbapenemase-non-producing Enterobacteriaceae, ESBL, AmpC enzyme, and porin loss contribute to the carbapenems resistance of Enterobacteriaceae, especially the ertapenem resistance of Enterobacter cloacae.

Construction and characterization of a bacterial artificial chromosome library for the A-genome of cotton (G. arboreum L.).

A bacterial artificial chromosome (BAC) library for the A-genome of cotton has been constructed from the leaves of G. arboreum L cv. Jianglinzhongmian. It is used as elite A-genome germplasm resources in the present cotton breeding program and has been used to build a genetic reference map of cotton. The BAC library consists of 123,648 clones stored in 322 384-well plates. Statistical analysis of a set of 103 randomly selected BAC clones indicated that each clone has an average insert length of 100.2 kb per plasmid, with a range of 30 to 190 kb. Theoretically, this represents 7.2 haploid genome equivalents based on an A-genome size of 1697 Mb. The BAC library has been arranged in column pools and superpools allowing screening with various PCR-based markers. In the future, the A-genome cotton BAC library will serve as both a giant gene resource and a valuable tool for map-based gene isolation, physical mapping and comparative genome analysis.