RRM2 regulates osteogenesis of mouse embryo fibroblasts via the Wnt/ß­catenin signaling pathway.

Osteoporosis is a widespread bone metabolic disease characterized by reduced bone mass and bone microstructure deterioration. Ribonucleotide reductase M2 (RRM2) is a key enzyme in DNA synthesis and repair. The present study investigated the effect of RRM2 on osteogenesis of mouse embryo fibroblasts (MEFs) and its molecular mechanism. Bioinformatics analysis revealed that RRM2 expression was increased during osteogenesis of MEFs triggered by bone morphogenetic protein 9. Subsequently, MEFs were used as a mesenchymal stem cell model and osteogenic inducing medium was used to induce osteogenic differentiation. RRM2 protein expression was measured by western blotting during osteogenic differentiation induction of MEFs. RRM2 levels in MEFs were upregulated and downregulated by RRM2-overexpressing recombinant adenovirus and small interfering RNA-RRM2, respectively. Bone formation markers (RUNX family transcription factor 2, osterix, distal-less homeobox 5, collagen type I α1 chain, osteopontin and osteocalcin) were detected by reverse transcription-quantitative (RT-q) PCR and alkaline phosphatase (ALP) and Alizarin Red S staining were examined. The protein expression levels of ß-catenin and the ratio of phosphorylated (p-)GSK-3ß to GSK-3ß were detected by western blotting and the RNA expression of downstream related target genes (ß-catenin, axis inhibition protein 2 (AXIN2), transcription factor 7 like 2, lymphoid enhancer binding factor 1, c-MYC and Cyclin D1) in the Wnt/ß-catenin signaling pathway was measured by RT-qPCR. RRM2 protein expression increased as the osteogenic differentiation induction period was extended. RRM2 overexpression increased osteogenic marker RNA expression, ALP activity, bone mineralization, the protein expression levels of ß-catenin, the ratio of p-GSK-3ß to GSK-3ß and the RNA expression of downstream related target genes in the Wnt/ß-catenin signaling pathway, whereas RRM2 knockdown had the opposite effect. The findings of the present study revealed that RRM2 overexpression enhanced osteogenic differentiation, while RRM2 knockdown reduced osteogenic differentiation. RRM2 may regulate osteogenic differentiation of MEFs via the canonical Wnt/ß-catenin signaling pathway, providing a possible therapeutic target for osteoporosis.

Identification of Hub mRNAs and lncRNAs in Atrial Fibrillation Using Weighted Co-expression Network Analysis With RNA-Seq Data.

Atrial fibrillation (AF)/paroxysmal AF (PAF) is the main cause of cardiogenic embolism. In recent years, the progression from paroxysmal AF to persistent AF has attracted more and more attention. However, the molecular mechanism of the progression of AF is unclear. In this study, we performed RNA sequencing for normal samples, paroxysmal AF and persistent AF samples to identify differentially expressed gene (DEG) and explore the roles of these DEGs in AF. Totally, 272 differently expressed mRNAs (DEmRNAs) and 286 differentially expressed lncRNAs (DElncRNAs) were identified in paroxysmal AF compared to normal samples; 324 DEmRNAs and 258 DElncRNAs were found in persistent atrial fibrillation compared with normal samples; and 520 DEmRNAs and 414 DElncRNAs were identified in persistent AF compared to paroxysmal AF samples. Interestingly, among the DEGs, approximately 50% were coding genes and around 50% were non-coding RNAs, suggesting that lncRNAs may also have a crucial role in the progression of AF. Bioinformatics analysis demonstrated that these DEGs were significantly related to regulating multiple AF associated pathways, such as the regulation of vascular endothelial growth factor production and binding to the CXCR chemokine receptor. Furthermore, weighted gene co-expression network analysis (WGCNA) was conducted to identify key modules and hub RNAs and lncRNAs to determine their potential associations with AF. Five hub modules were identified in the progression of AF, including blue, brown, gray, turquoise and yellow modules. Interestingly, blue module and turquoise module were significantly negatively and positively correlated to the progression of AF respectively, indicating that they may have a more important role in the AF. Moreover, the hub protein-protein interaction (PPI) networks and lncRNA-mRNA regulatory network were constructed. Bioinformatics analysis on the hub PPI network in turquoise was involved in regulating immune response related signaling, such as leukocyte chemotaxis, macrophage activation, and positive regulation of α-ß T cell activation. Our findings could clarify the underlying molecular changes associated fibrillation, and provide a useful resource for identifying AF marker.

Associations between Seven Common Cytokine Gene Polymorphisms and Coronary Artery Disease: Evidence from a Meta-Analysis.

BACKGROUND: Associations of polymorphisms in interferon-gamma (IFNG), interleukin-10 (IL-10), and interleukin-18 (IL-18) with coronary artery disease (CAD) have already been investigated by many studies, yet the findings of these studies have been somewhat inconsistent. OBJECTIVES: The aim of this meta-analysis was to better clarify associations between polymorphisms in IFNG/IL-10/IL-18 and CAD by combing the results of all relevant articles. METHODS: Eligible articles were searched from PubMed, EMBASE, WOS, and CNKI. We used Review Manager to combine the results of indi-vidual studies. RESULTS: Fifty-one studies were included in this meta-analysis. Combined results revealed that IFNG rs243056, IL-10rs1800871, IL-18rs187238, IL-18rs1946518, and IL-18rs1946519 polymorphisms were all significantly associated with CAD in the general population. We also obtained similar significant results for IFNG rs243056, IL-10rs1800871, IL-10 rs1800896, IL-18rs187238, IL-18rs1946518, and IL-18rs1946519 polymorphisms in Asians in further subgroup analyses. CONCLUSIONS: Collectively, this meta-analysis proved that IFNG rs243056, IL-10rs1800871, IL-10 rs1800896, IL-18rs187238, IL-18rs1946518, and IL-18rs1946519 polymorphisms may confer susceptibility to CAD for Asians.

The feasibility of using high frequency ultrasound to assess nerve ending neuropathy in patients with diabetic foot.

OBJECTIVES: The nerve ending problem is one of the major causes for diabetic feet. In this work, we explored the feasibilities of using high frequency ultrasound (US) in nerve ending problem evaluation for patients with diabetic foot. METHODS: The endings of the medial branch of deep peroneal nerves (mbDPN) were interrogated by US, and the nerve conduction characters were studied in a cohort of 19 clinically diagnosed diabetic feet patients and a control group of healthy volunteers. RESULTS: Distinct echoic appearances were consistently detected between the mbDPN nerves of diabetic feet patients and healthy volunteers. In healthy volunteers, hypoechoic bands were readily observed at the anatomical locations of mbDPNs. However, these hypoechoic bands of the mbDPNs were not clear in the diabetic feet patients, and the surfaces of the mbDPNs appeared obscure and irregular in these patients relative to those of healthy volunteers. In addition, the US echoes of mbDPN in patients with diabetic feet were more heterogeneous than those in healthy volunteers. The mean diameters of mbDPNs were 1.3±0.4 mm in patients with diabetic foot and 0.8±0.2 mm in the control group (P<0.05). Finally, results from the nerve conduction studies (NCS) showed abnormalities in patients with diabetic feet syndrome. CONCLUSION: High frequency US can be a useful modality for evaluating nerve ending problems in diabetic feet patient; and the mbDPN enlargement, obscurity, surface irregularity and heterogeneity in echo can serve as the markers indicating nerve ending problems in the diabetic feet patients under ultrasound interrogation.

[Reversal of multidrug resistance in hepatocellular cell line HepG2R by mdr1-antisense RNA].

OBJECTIVE: To investigate whether multidrug resistance gene 1(mdr1) could reverse multidrug resistance (MDR) in HepG2R cells. METHODS: An adenovirus vector, Adeno-asmdr, containing the antisense RNA driven by AFP promoter, was construct. Adeno-EGFP was transfected into HepG2, an AFP producing cell line, L02, a normal human liver cell line, and HeLa, a human cervical cancer cell line, the EGFP transcription level was detected by RT-PCR. Adeno-asmdr was transfected into HepG2R cells, the expression of P-gp170 was detected by western blotting, apoptosis was detected using TUNEL and flow cytometry, cell cycle was analyzed by flow cytometry. RESULTS: EGFP was highly expressed in HepG2 cells, however, its expression in L02 or HeLa cells was very weak. Western blot showed that the P-gp170 was marked down-regulated 48h after transfection with Adeno-asmdr, and the expression of P-gp170 was detectable at least 7d post-transfection. Compared with control cells, Adeno-asmdr transfected HepG2R cells were more sensitive to different chemicals, as indicated by TUNEL staining and flow cytometry. Chemical treatment arrested the cells in S and G0/M phase. CONCLUSION: The recombinant adenoviral vector, Adeno-asmdr, can block the expression of mdr1, and reverse MDR in HepG2R cells.

[Reversal of adriamycin resistance of hepatocellular carcinoma by targeting it with recombined adenovirus carrying antisense multidrug resistance gene 1 RNA].

OBJECTIVE: To investigate if an adenovirus vector carrying antisense multidrug resistance gene 1 (MDR1) could reverse multidrug resistance (MDR) of HepG2/ adriamycin (ADM) cells in tumors transplanted in athymic mice. METHODS: An adenovirus vector carrying AFP promoter and antisense MDR1 was constructed. HepG2 MDR cells (HepG2/ADM) were induced by graded resistance to ADM and were subcutaneously inoculated into athymic mice to construct the transplanted tumor. After adeno-asmdr1 was injected, the volume of the transplanted tumor and the apoptotic body in the xenograft tumor cells were observed and reverse transcriptase polymerase chain reaction was employed to investigate the expression of the mdr1-mRNA from the mouse transplanted tumor cells. RESULTS: Following injection with adeno-asmdr1, the tumor volumes in this mice group did not increase. However the tumor volume in the PBS plus ADM group did increase significantly (P less than 0.05). In the tumor xenograft cells, mdr1 mRNA in the xenografts was assessed by RT-PCR and found to be reduced at week 1, and at week 4 in the ADM+asmdr1 group, but it was stable in the ADM group. It was only 20% in the ADM+asmdr1 group compared to the ADM group at the 4th week. Evidence of apoptosis was observed in the tumor xenograft cells treated with adeno-asmdr1, but there was rarely any apoptosis in the group treated with ADM and PBS. CONCLUSION: Adenovirus carrying antisense mdr1 RNA can partially reverse the MDR of HepG2/ADM cells and inhibit tumor growth by down-regulating mdr1 mRNA resulting in tumor cell apoptosis.

The analysis of epidemiological characteristics of road traffic crashes in a mountain city in western China.

OBJECTIVE: To study the epidemiological characteristics and preventive methods of road traffic crashes in a mountain city in western China through sampling investigation of traffic crashes in different regions of Chongqing city in recent years. METHODS: Two police teams of traffic management in downtown, two teams in suburb, one team in county and one freeway team were randomly selected, and road traffic crashes between 1988 and 1997 were investigated and analyzed. RESULTS: A total of 13121 road traffic crashes with 6201 crashes with casualties were analyzed. The incidence of crashes was higher in May, June and July, and on Friday and Wednesday and at 8:00-12:00 and 14:00-18:00 within a day. Casualties were 44.0% in pedestrians and 42.5% in passengers of total casualties. The deaths and severe injuries in pedestrians were 59.1% of total deaths and 56.4% of total severe injuries. The age of drivers and passengers were mainly at 18-30 years, followed by 31-40 years. People over 60 years old accounted for 24.1% of total pedestrian casualty. Head injury was the most common reason for deaths. The main reasons for these crashes were improper driving and violating traffic laws. Violating traffic laws by pedestrian was one of the main reasons for pedestrian casualty. CONCLUSIONS: The crashes are related to the characteristics of geography, climate, society activity of people, and the sense of traffic safety, the basic traffic construction and management in Chongqing. The traffic casualty of pedestrian is a big problem in Chongqing. To prevent and decrease road traffic injuries effective methods should be worked out and propaganda on traffic safety and traffic management should be strengthened according to different characteristics of different regions. To strengthen the first aid and treatment of cranium-brain injury and chest-back injury is also helpful in decreasing traffic deaths.

Protective effects of fibronectin on vascular endothelial cells during trauma in rats.

OBJECTIVE: To investigate the protective effects of fibronectin (Fn) on vascular endothelial cells. METHODS: Block reticuloendothelial system model compound with injury of tail vein and femur fractures in rats were used. The level of plasma Fn and surface Fn of peritoneal macrophage cells, the numbers of circulating endothelial cells and the injured tail vein were observed. RESULTS: Fn increased the level of plasma Fn and peritoneal macrophage cells surface Fn,but decreased the number of circulating endothelial cells after infusing Fn 1.7 mg/10 g before injury. Under the scanning electron microscope, only a local focus injury to vascular endothelial cells in the rats was observed after Fn was infused, but a piecemeal injury and detachment of vascular endothelial cells were observed in rats without giving Fn. CONCLUSIONS: Fn has obvious protective effects on vascular endothelial cells in rats.