Construction and validation of a three-microRNA signature as prognostic biomarker in patients with hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), a common type of primary liver cancer, is one of the most aggressive malignant tumors worldwide. Although overall survival (OS) rates for HCC has significantly improved in recent years, however, the exact predictive value of microRNA (miRNA) for the prognosis of HCC has not yet been recognized. Here, we aimed to identify potential prognostic miRNAs involved in HCC by bioinformatics analysis and validated expression levels through quantitative polymerase chain reaction (qPCR) and GEO database. The RNA expression profiles and corresponding clinical information of HCC were available from The Cancer Genome Atlas (TCGA) datasets. Differentially expression and standardization analysis of miRNAs, Kaplan-Meier curve and time dependent ROC curve were performed by using R tools. Differentially expressed miRNAs (DEmiRNAs) and clinical parameters involved in the OS of HCC were confirmed by Cox regression models. And functional enrichment analysis was used to establish functions of the targeted genes of DEmiRNAs. A total of 300 DEmiRNAs were significantly related with HCC, of which 40 were down-regulated and 260 were up-regulated. A total of 344 patients with DEmiRNAs, status, overall survival (OS) time were randomized into training group (172) and test group (172). Multivariate Cox regression analyses revealed that 3 miRNA (hsa-miR-139-3p, hsa-miR-760, hsa-miR-7-5p) had independent prognostic significance for the OS of HCC in both training and test group. Moreover, according to Kaplan Meier analysis, the OS of HCC patients with high-risk score was shorter in validation and entire series. The time dependent ROC curve demonstrated high accuracy of the signature for OS. Besides, target genes of three miRNAs were analyzed by functional enrichment analysis and 20 genes associated with OS were verified by using Kaplan-Meier method. Compared with normal and benign group, the relative expression level of hsa-miR-139-3p was significantly decreased, while hsa-miR-7-5p and hsa-miR-760 were distinctly increased in the plasma of HCC patients. The same results were observed in the independent cohort. Collectively, our research suggested that three-miRNA signature could serve as an independent prognostic indicator for HCC patients.

Efficacy of combined orthodontic-periodontic treatment for patients with periodontitis and its effect on inflammatory cytokines: A comparative study.

INTRODUCTION: In this study, we aimed to investigate the efficacy of combined orthodontic-periodontic treatment in the treatment of patients with periodontitis and its effects on the levels of inflammatory cytokines. METHODS: A total of 117 patients with periodontitis were randomly assigned to the basic group (receiving basic periodontic treatment, n = 58) and the combined group (receiving combined orthodontic-periodontic treatment, n = 59). In addition, 52 healthy people without periodontal disease were selected as the normal group. Probing depth, tooth mobility, plaque index, clinical attachment level, and sulcus bleeding index were recorded. ELISA was applied to detect gingival crevicular fluid (GCF) and serum levels of inflammatory cytokines. A 2-year clinical follow-up was conducted. RESULTS: Before treatment, the periodontal parameters (probing depth, tooth mobility, plaque index, clinical attachement level, and sulcus bleeding index) and GCF and serum levels of inflammatory cytokines (high-sensitivity C-reactive protein, interleukin-1ß, interleukin-5, interleukin-6, interleukin-8, tumor necrosis factor-α, and prostaglandin E2) in the combined and basic groups were higher than those in the normal group. After 6 and 18 months of treatment, the periodontal parameters and GCF and serum levels of inflammatory cytokines decreased in the combined and basic groups. The periodontal parameters and the GCF and serum levels of inflammatory cytokines in the combined group were significantly lower than those in the basic group after 18 months of treatment. The combined group had a lower recurrence rate compared with the basic group. CONCLUSIONS: Combined orthodontic-periodontic treatment had good clinical efficacy in the treatment of periodontitis and could effectively decrease the levels of inflammatory cytokines.

ß-Catenin overexpression is associated with gefitinib resistance in non-small cell lung cancer cells.

BACKGROUND: Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) presents great challenges in the treatment of non-small cell lung cancer (NSCLC) patients, while the mechanisms are still not well understood. The ß-catenin signaling pathway has been found to be associated with chemoresistance and can activate the EGFR and its downstream pathways. This study aimed to investigate the role of ß-catenin in acquired resistance to EGFR-TKIs in NSCLC cell lines. METHODS: The expression and transcriptional activity of ß-catenin were measured in both the NSCLC cell line PC9 and its sub-line PC9/AB(2) which has acquired resistance to gefitinib. Knockdown and overexpression of ß-catenin in the PC9/AB(2) and PC9 cells were performed. The cell survival rate and the activation of the EGFR and its downstream pathways were detected in the two cell lines after transfection. RESULTS: Nuclear translocation of ß-catenin was increased in the PC9/AB(2) cells and the baseline expression of members of the ß-catenin signaling pathway was also higher in the PC9/AB(2) cells. Knocking down the expression of ß-catenin increased the sensitivity of the PC9/AB(2) cells to gefitinib by blocking the activation of the EGFR downstream pathways, while ß-catenin overexpression improved PC9 cells resistance to gefitinib by enhancing the activation of the EGFR and its downstream signaling. CONCLUSION: ß-catenin plays an important role in acquired resistance to EGFR-TKIs in NSCLC cell lines and may be a potential therapeutic target for NSCLC patients who have failed to respond to targeted therapy.

[Effects of 1, 25 (OH)2D3 on bleomycin-induced pulmonary fibrosis in mice].

OBJECTIVE: To observe the effects of 1, 25(OH)2D3 on bleomycin-induced pulmonary fibrosis in mice and to explore its mechanisms. METHODS: Ninety male C57BL/6 mice, 6 to 8 weeks old, were randomly divided into 3 groups according to the table of random numbers: a control group, a model group and a treatment group(n = 30 each). Bleomycin was injected to the mice in the latter 2 groups by single intratracheal injection to duplicate the pulmonary fibrosis model, while the control group was injected with saline. From the next day, the mice in the treatment group received 1, 25 (OH) 2D3 (0.5 µg·kg(-1)·d(-1)) diluted in olive oil by gavage daily, while the other groups were treated with equivalent olive oil. Ten mice in each group were killed randomly on day 14, 21 and 28 after surgery respectively. Pulmonary alveolitis and fibrosis were evaluated by using hematoxylin-eosin and Masson stain method. The content of hydroxyproline was measured by acid hydrolysis method. The mRNA levels of collagen1α1, α-SMA, Wnt3a, Wnt4, and Wnt7a in the lung tissues were measured by real-time RT-PCR, while the protein expression of α-SMA and ß-catenin were assessed by immunohistochemistry. RESULTS: Pulmonary alveolitis at day 14, 21 and fibrosis at day14, 21, 28 in the treatment group were remarkably reduced compared to the model group (all P < 0.05). Compared with the model group, the treatment group showed decreased content of hydroxyproline, decreased mRNA levels of collagen1α1, α-SMA, Wnt3a, Wnt4 and decreased protein expression of α-SMA and ß-catenin at the 3 time points (all P < 0.05). The content of hydroxyproline and the mRNA levels of collagen1α1, α-SMA, Wnt3a, Wnt4, Wnt7a in the treatment group at 28 d were 0.67 ± 0.14, 1.66 ± 0.34, 1.37 ± 0.41, 1.43 ± 0.27, 1.29 ± 0.19, 1.18 ± 0.20, respectively, all of which were significantly lower than those in the model group (1.10 ± 0.16, 3.50 ± 0.74, 2.68 ± 0.61, 2.60 ± 0.58, 2.23 ± 0.45, 1.93 ± 0.36, respectively). Protein expression of α-SMA and ß-catenin in the treatment group were 0.44 ± 0.13 and 0.25 ± 0.05, respectively, which were also significantly lower than those of the model group(0.98 ± 0.20, 0.58 ± 0.06, respectively). CONCLUSION: 1, 25 (OH) 2D3 was shown to reduce pulmonary fibrosis induced by bleomycin in mice, and its mechanisms might be associated with Wnt signaling suppression.

Transmission electron microscopy of sputum deposition in the diagnosis of pulmonary alveolar proteinosis.

OBJECTIVE: To clarify the diagnostic value of sputum in pulmonary alveolar proteinosis (PAP) through transmission electron microscopy (TEM) of sputum deposition (SD). METHODS: Eleven SD samples and 9 bronchoalveolar lavage (BAL) sediments from a PAP group including 11 patients were observed by TEM and compared with sputum direct smear, BAL cytology, and lung biopsy histopathology. Eleven healthy adults were chosen as controls. RESULTS: The 11 sputum smears from the PAP group showed no diagnostic component, but TEM of SD revealed 7 of 11 samples had many myelin-like lamellar bodies with degeneration in the cytoplasm of macrophages, alveolar epithelial cells, and extracellular spaces, which suggested PAP. Especially, 2 patients on whom lung biopsy could not be performed and who failed to be diagnosed by BAL fluid were finally diagnosed by TEM of SD. TEM of BAL sediments showed 7 of 9 cases had diagnostic myelin-like lamellar bodies. No statistical significance was found between BAL fluid and SD by TEM. The control group didn't show diagnostic components by cytology or TEM of SD. CONCLUSION: TEM of SD is an important noninvasive diagnostic method especially for patients against lung biopsy and BAL.

[The ultrastructural features of sputum deposition and its value in the diagnosis of pulmonary alveolar proteinosis].

OBJECTIVE: To investigate the ultrastructural features of sputum deposition (SD) and its value in the diagnosis of pulmonary alveolar proteinosis (PAP). METHODS: Seven patients with PAP diagnosed by lung biopsy and cytology were enrolled in this study. The patients consisted of 5 men and 2 women, whose median age was 48 years (range 36 to 73). SD and bronchoalveolar lavage fluid (BALF) sediment were made into ultrathin sections and observed under transmission electron microscope (TEM), respectively. Seven cases of control group composed of 4 men and 3 women whose median age was 49 years (range 39 to 68) including 3 cases of bacterial pneumonia, two cases of COPD and 2 cases of exudative pulmonary tuberculosis. Each SD was made into ultrathin section, and compared with the experimental group. RESULTS: In PAP group, Periodic acid-schiff (PAS) staining was performed on 7 sputum smears and none of them was tested positive for any components with diagnostic interest. Four cases from the 7 paraffin-embed sections of BALF sediment by microscopic examination suggested PAP. Under TEM, BALF sediment showed that many lamellar bodies existed in and outside alveolar epithelial cells, and 5 specimens were consistent with PAP diagnosis. Compared with BALF sediment, SD had apparent degeneration with more myelin phagosomes in the cytoplasm of macrophages, more lamellar bodies in alveolar epithelial cells, and lots of lamellar bodies in the shape of concentric circle in the extracellular spaces. Four from the 7 SD samples were consistent with the diagnosis of PAP. No significant difference was found between SD and BALF in the diagnosis of PAP by electronic examination (P > 0.05). In the 7 cases of control group no drifting osmiophilic lamellar bodies in extracellular space were detected. CONCLUSIONS: The osmiophilic lamellar bodies with diagnostic value were found in SD and BALF of patients with PAP. TEM of SD in combination with clinical manifestations and radiologic findings can make a definitive diagnosis of PAP, especially for those patients who have contraindications to lung biopsy and lung lavage.

Necroptosis Plays a Crucial Role in Vascular Injury during DVT and Is Enhanced by IL-17B.

Background: This study investigated whether vascular endothelial necroptosis is involved in deep vein thrombosis (DVT) and how IL-17B facilitates necroptosis signaling. Methods: The DVT mouse model was induced by ligation of the IVC. The cross-sectional area of thrombus increases and the thrombus occupied the entire venous lumen at 48 h after ligation. Meanwhile, the increased expression of p-RIP3/RIP3 was most pronounced at 48 h after ligation, and the p-MLKL/MLKL peaked at 72 h. Results: Based on Illumina sequencing and KEGG pathway analyses, the activated RIP3/MLKL is associated with increased IL-17B. With thrombus formation, IL-17B was upregulated and enhanced the expression of RIP3 and MLKL in the IVC wall, as well as their phosphorylation levels (all P < 0.05, the comparison group consisted of the control group, DVT group, DVT/IL-17B group, and DVT/anti-IL-17B group). The p-RIP3/RIP3 and p-MLKL/MLKL ratios were reduced by anti-IL-17B. Similarly, the weight and cross-sectional area of the thrombi were increased by IL-17B and decreased by the IL-17B antibody. IL-17B had a smaller effect on thrombosis in knockout mice compared with WT mice. In vitro, the IL-17B protein expression and the level of RIP3 and MLKL phosphorylation increased high in the OGD cells, accompanied by increased expression of IL-6 and TNF-α. IL-17B enhanced the expression of IL-6 and TNF-α but had little effect on the IL-6 and TNF-α after transfected with siRIP3 or siMLKL. Similarly, the plasma IL-17B, IL-6, and TNF-α were significantly increased after thrombosis in WT mice, and enhanced by IL-17B. But IL-17B did not increase the plasma IL-6 and TNF-α in knockout mice. Conclusions: In conclusion, those results suggest that vascular endothelial necroptosis plays a crucial role in vascular injury and IL-17B could enhance the necroptosis pathway.

Umbilical cord blood mononuclear cell therapy induces clinical remission of steroid-dependent or -resistant ulcerative colitis patients.

To compare the efficacy and safety of umbilical cord blood mononuclear cells (CBMNC) and azathioprine (AZA) in the treatment of patients with steroid-dependent or -resistant ulcerative colitis. One hundred and six patients diagnosed with steroid-dependent or -resistant ulcerative colitis were studied retrospectively, including 36 patients treated with CBMNC and 70 treated with AZA. To reduce confounding bias due to retrospective nature of this study, the propensity score matching system was applied to equipoise the pretreatment data of two groups. After matching, 35 matched pairs (1:1) were created. The ratios of clinical remission, clinical response and endoscopic mucosal healing, Mayo score, and major complications were compared between two groups at weeks 8, 16, and 36 after treatment. The results demonstrated that the ratios of clinical remission (80% vs. 57%, P < 0.05) and mucosal healing (74% vs. 51%, P < 0.05) were significantly higher in CBMNC-treated patients compared with those in AZA-treated patients at week 8. The erythrocyte sedimentation rate was significantly decreased in CBMNC group compared with that in AZA-treated group (14.5 ± 3.9 mm/h vs. 18.0 ± 5.7 mm/h, P < 0.01) at week 8. In AZA group, 2 patients had neutropenia and 3 patients had elevated alanine aminotransferase levels, whereas no obvious side-effects were observed in CBMNC-treated group. Our results reveal that CBMNC therapy appears to be an effective and safe strategy for patients with steroid-dependent or -resistant ulcerative colitis. Further prospective studies are needed to define the potential roles and mechanisms of CBMNC in the treatment of refractory ulcerative colitis.

Reduction of intraocular pressure and improvement of vision after cataract surgeries in angle closure glaucoma with concomitant cataract patients.

OBJECTIVE: This study is to compare the efficacy of three different cataract surgeries in eyes with angle closure glaucoma (ACG) with concomitant cataract. METHODS: A retrospective comparative analysis of 106 ACG patients (112 eyes) with concomitant cataract was conducted between February, 2012 and February, 2014. Clinical outcomes of ACG patients with concomitant cataract underwent phacoemulsification and intraocular lens implantation (group A, n = 34, 36 eyes, angle closure < 180°); combined phacoemulsification, intraocular lens implantation, and goniosynechialysis (group B, n = 43, 45 eyes, angle closure, 180°~270°); and combined phacoemulsification, intraocular lens implantation, and trabeculectomy (group C, n = 29, 31 eyes, angle closure > 270°) were compared during a 6-month follow-up. RESULTS: There were no statistical differences among the 3 groups in pre-operative or post-operative average visual acuity (VA), intraocular pressure (IOP), anterior chamber depth (ACD), and angle opening distance (AOD) (all P > 0.05). Post-operative VA, IOP, ACD, AOD and the degree of angle opening in the 3 groups were all improved as compared with pre-operative levels (all P < 0.05). No statistical difference was detected among the 3 groups in the incidence of complications (χ(2) = 0.376, P = 0.829). CONCLUSION: Phacoemulsification alone, combined phacoemulsification/goniosynechialysis, and combined phacoemulsification/trabeculectomy provide safe, effective, predictable, and stable options of cataract surgery for treatment of ACG with concomitant cataract.

Preventive effects of vitamin D treatment on bleomycin-induced pulmonary fibrosis.

Patients with pulmonary fibrosis often have low vitamin D levels, the effects of which are largely unknown. We here report that early vitamin D supplementation significantly reduced the severity of pulmonary fibrosis and inflammatory cell accumulationin in the bleomycin-induced pulmonary fibrosis mouse model on supplementary days 14, 21 and 28 (P < 0.001). Vitamin D supplementation also prevented some ultrastructural changes in response to bleomycin administration, including basement membrane thickening, interstitial fibrin deposition and microvilli flattening or disappearance on days 14, 21 and 28, and lamellar body swelling or vacuolation on days 21 and 28. The bleomycin group had rising hydroxyproline level on days 14, 21 and 28, whereas the vitamin D treatment group showed consistently lower hydroxyproline level but still higher than that of the control group (P < 0.001). Our immunohistochemistry and densitometry analyses showed less staining for α-smooth muscle actin, a myofibroblast marker, in the vitamin D group compared to the bleomycin group (P < 0.001). Thus, vitamin D treatment could prevent bleomycin-induced pulmonary fibrosis by delaying or suppressing ultrastructural changes, as well as attenuating hydroxyproline accumulation and inhibiting myofibroblastic proliferation. These data further our understanding of the roles of vitamin D in pulmonary fibrogenesis and in the treatment of pulmonary fibrosis.