Prognostic value of inflammation and immune-related gene NOD2 in clear cell renal cell carcinoma.

Inflammation and immune responses play important roles in cancer development and prognosis. We identified 59 upregulated inflammation- and immune-related genes (IIRGs) in clear cell renal cell carcinoma (ccRCC) from The Cancer Genome Atlas database. Among the upregulated IIRGs, nucleotide binding oligomerization domain 2 (NOD2), PYD and CARD domain (PYCARD) were also confirmed to be upregulated in the Oncomine database and in three independent GEO data sets. Tumor immune infiltration resource database analysis revealed that NOD2 and PYCARD levels were significantly positively correlated with infiltration levels of B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages and dendritic cells. Multivariate Cox hazards regression analysis indicated that based on clinical variables (age, gender, tumor grade, pathological TNM stage), NOD2, but not PYCARD, was an independent, unfavorable ccRCC prognostic biomarker. Functional enrichment analyses (GSEA) showed that NOD2 was involved in innate immune responses, inflammatory responses, and regulation of cytokine secretion. Meanwhile, mRNA and protein levels of NOD2 were elevated in four ccRCC cell lines (786-O, ACHN, A498 and Caki-1), and its knockdown significantly inhibited IL-8 secretion, thereby inhibiting ccRCC cell proliferation and invasion. Furthermore, results showed that miR-20b-5p targeted NOD2 to alleviate NOD2-mediated IL-8 secretion. In conclusion, NOD2 is a potential prognostic biomarker for ccRCC and the miR-20b-5p/NOD2/IL-8 axis may regulate inflammation- and immune-mediated tumorigenesis in ccRCC.

Lifestyle factors, metabolic factors and socioeconomic status for pelvic organ prolapse: a Mendelian randomization study.

BACKGROUND: Previous observational studies have reported that lifestyle factors, metabolic factors and socioeconomic status are associated with the development of female pelvic organ prolapse (POP); however, whether these associations are causal remains unclear. The current study aimed to assess the causal effect of lifestyle factors, metabolic factors and socioeconomic status on POP risk. METHODS: We conducted a two-sample Mendelian randomization (MR) study based on summary-level data from the largest available genome-wide association studies (GWAS) to evaluate whether lifestyle factors, metabolic factors and socioeconomic status are causally related to POP. We used single nucleotide polymorphisms that are strongly associated with exposure at the genome-wide significance level (P < 5 × 10-8) as instrumental variables from genome-wide association studies. The method of random-effect inverse-variance weighting (IVW) was used as the primary analysis method, supplemented with the weighted median, MR-Egger and the MR pleiotropy residual sum and outlier applied to verify the MR assumptions. Two-step MR was conducted to investigate potential intermediate factors that are on the causal pathway from exposure to POP. RESULTS: There were associations with POP for genetically predicted waist-to-hip ratio (WHR) (odds ratio (OR) 1.02, 95% confidence interval (CI) 1.01-1.03 per SD-increase, P < 0.001), WHR adjusted for body mass index (WHRadjBMI) (OR 1.017, 95% CI 1.01-1.025 per SD-increase, P < 0.001) and education attainment (OR 0.986, 95% CI 0.98-0.991 per SD-increase) in the meta-analysis. Additionally, genetically predicted coffee consumption (OR per 50% increase 0.67, 95% CI 0.47-0.96, P = 0.03), vigorous physical activity (OR 0.83, 95% CI 0.69-0.98, P = 0.043) and high-density lipoprotein cholesterol (HDL-C) (OR 0.91, 95% CI 0.84-0.98 per SD-increase, P = 0.049) were inversely associated with POP in the FinnGen Consortium. The mediation analysis showed that the indirect effects of education attainment on POP were partly mediated by WHR and WHRadjBMI, with a mediated proportion of 27% and 13% in the UK Biobank study, respectively. CONCLUSIONS: Our study provides MR evidence of a robust causal association of WHR, WHRadjBMI and education attainment with POP.

Deficiency of mindin reduces renal injury after ischemia reperfusion.

BACKGROUND: Acute renal injury (AKI) secondary to ischemia reperfusion (IR) injury continues to be a significant perioperative problem and there is no effective treatment. Mindin belongs to the mindin/F-spondin family and involves in inflammation, proliferation, and cell apoptosis. Previous studies have explored the biological functions of mindin in liver and brain ischemic injury, but its role in AKI is unknown. METHOD: To investigate whether mindin has a pathogenic role, mindin knockout (KO) and wild-type (WT) mice were used to establish renal IR model. After 30 min of ischemia and 24 h of reperfusion, renal histology, serum creatinine, and inflammatory response were examined to assess kidney injury. In vitro, proinflammatory factors and inflammatory signaling pathways were measured in mindin overexpression or knockdown and vector cells after hypoxia/reoxygenation (HR). RESULTS: Following IR, the kidney mindin level was increased in WT mice and deletion of mindin provided significant protection for mice against IR-induced renal injury as manifested by attenuated the elevation of serum creatinine and blood urea nitrogen along with less severity for histological alterations. Mindin deficiency significantly suppressed inflammatory cell infiltration, TNF-α and MCP-1 production following renal IR injury. Mechanistic studies revealed that mindin deficiency inhibits TLR4/JNK/NF-κB signaling activation. In vitro, the expression levels of TNF-α and MCP-1 were increased in mindin overexpression cells compared with vector cells following HR. Moreover, TLR4/JNK/NF-κB signaling activation was elevated in the mindin overexpression cells in response to HR stimulation while mindin knockdown inhibited the activation of TLR4/JNK/ NF-κB signaling after HR in vitro. Further study showed that mindin protein interacted directly with TLR4 protein. And more, mindin protein was confirmed to be expressed massively in renal tubule tissues of human hydronephrosis patients. CONCLUSION: These data demonstrate that mindin is a critical modulator of renal IR injury through regulating inflammatory responses. TLR4/JNK/NF-κB signaling most likely mediates the biological function of mindin in this model of renal ischemia.

Significant Prognostic Value of the Autophagy-Related Gene P4HB in Bladder Urothelial Carcinoma.

While hundreds of consistently altered autophagy-related genes (ARGs) have been identified in cancers, their prognostic value in bladder urothelial carcinoma (BUC) remains unclear. In the present study, we collected 232 ARGs from the Human Autophagy Database (HADb), and identified 37 differentially expressed ARGs in BUC based on The Cancer Genome Atlas (TCGA) database. Kaplan-Meier survival analysis based on the Gene Expression Profiling Interactive Analysis (GEPIA) database revealed that among the 37 differentially expressed ARGs, prolyl 4-hydroxylase, beta polypeptide (P4HB), and regulator of G protein signaling 19 (RGS19) were significantly negatively correlated with overall survival (OS) and disease-free survival (DFS). Overexpression of P4HB and RGS19 in BUC was further validated using independent data sets, including those from the Oncomine and Gene Expression Omnibus (GEO) databases. cBioPortal and UALCAN analyses indicated that altered P4HB and RGS19 mRNA expression was significantly associated with mutations and clinical characteristics (nodal metastasis and cancer stage). Moreover, co-expression network analysis and gene set enrichment analysis (GSEA) predicted that the potential functions of P4HB and RGS19 are involved in the endoplasmic reticulum (ER) stress response, cytokine-mediated signaling pathway and inflammatory response. More importantly, multivariate Cox proportional hazards regression analysis demonstrated that P4HB, but not RGS19, is an independent and unfavorable BUC biomarker based on clinical characteristics (age, gender, cancer stage, and pathological TNM stage). Finally, we validated that the mRNA and protein expression levels of P4HB were upregulated in four bladder cancer cell lines (T24, J82, EJ, and SW780) and found that knockdown of P4HB dramatically inhibited the invasion and proliferation of bladder cancer cells. In summary, our study screened ARGs and identified P4HB as a biomarker that can predict the progression and prognosis of BUC and may provide a better understanding of the autophagy regulatory mechanisms involved in BUC.

The long non-coding RNA SNHG1 promotes bladder cancer progression by interacting with miR-143-3p and EZH2.

The long non-coding RNA (lncRNA) SNHG1 has been shown to be implicated in the progression of multiple human carcinomas. Nevertheless, the biological functions and potential mechanism of SNHG1 in bladder cancer (BC) are uncharacterized. In the present study, SNHG1 was found to be substantially up-regulated in BC tissues and cells and was intimately correlated with the TNM stage, lymphatic invasion, metastasis and recurrence-free survival in BC patients. Down-regulation of SNHG1 dramatically attenuated the proliferation, migration and invasion of BC cells, whereas the ectopic overexpression of SNHG1 had the opposite effects in vitro. The in vivo experimental results also indicated that SNHG1 down-regulation hampered the tumour growth and metastasis of BC cells. Mechanistic investigations revealed that SNHG1 enhances HK2 expression by serving as an endogenous sponge to regulate miR-143-3p in the cytoplasm of BC cells. In the nucleus, SNHG1 could interact with EZH2 and regulate the histone methylation of the CDH1 promoter, altering the biological behaviours of BC cells. Overall, these findings elucidate an oncologic role of SNHG1 in BC and provide a new therapeutic strategy against BC.

The lncRNA SNHG5-mediated miR-205-5p downregulation contributes to the progression of clear cell renal cell carcinoma by targeting ZEB1.

Recent findings have unraveled the critical functions of the long noncoding RNA (lncRNA) SNHG5 in human malignancies. Nevertheless, the role and mechanism of SNHG5 in clear cell renal cell carcinoma (ccRCC) are still elusive. In our study, substantially higher abundance of SNHG5 was observed in ccRCC specimens and cell lines, and increased SNHG5 expression was intimately correlated with tumor size, tumor-node-metastasis (TNM) stage, lymph node invasion, and distant metastases in patients with ccRCC. SNHG5 knockdown obviously suppressed the proliferative, migratory, and invasive capabilities of ccRCC cells, whereas SNHG5 overexpression induced the opposite effects. Mechanistically, SNHG5 activated the transcription of ZEB1, which exerts a pivotal role in modulation of epithelia-mesenchymal transition (EMT) and tumor metastasis. SNHG5 was then shown to act as an endogenous sponge for miR-205-5p, which targets ZEB1 in ccRCC. Moreover rescue experiments revealed that SNHG5 promotes ccRCC cell proliferation, migration, and invasion in a miR-205-5p-dependent manner. Additionally, in vivo assays further indicated that overexpression or silencing of SNHG5 in ccRCC cells promoted or suppressed the tumorigenesis and metastasis, respectively. Altogether, the present data provide the first evidence that the lncRNA SNHG5 has an oncogenic role in ccRCC through the SNHG5/miR-205-5p/ZEB1 signaling axis and represents a novel potential therapeutic regimen against ccRCC.

Integrative analysis of the lncRNA-associated ceRNA network reveals lncRNAs as potential prognostic biomarkers in human muscle-invasive bladder cancer.

BACKGROUND: Long noncoding RNAs (lncRNAs) play important roles in competing endogenous RNA (ceRNA) networks involved in the development and progression of various cancers, including muscle-invasive bladder cancer (MIBC). PURPOSE: This study aims to construct the lncRNA-associated ceRNA network and identify lncRNA signatures correlated with the clinical features of MIBC tissue samples from The Cancer Genome Atlas (TGCA) database. METHODS: The differential expression profiles of MIBC associated lncRNAs, miRNAs and mRNAs were obtained from TCGA. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to determine the principal functions of significantly dysregulated mRNAs. The dysregulated lncRNA-associated ceRNA network of MIBC was constructed based on the bioinformatics data, and the correlations between lncRNA expression and clinical features were analyzed using a weighted gene coexpression network analysis (WGCNA). Six cancer specific lncRNAs from the ceRNA network were randomly selected to detect their expression in 32 paired MIBC tissue samples and 5 bladder cancer cell lines using quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The ceRNA network was constructed with 30 lncRNAs, 13 miRNAs and 32 mRNAs. Seventeen lncRNAs in the ceRNA network correlated with certain clinical features, and only 1 lncRNA (MIR137HG) correlated with the overall survival (OS) of patients with MIBC (log-rank test P<0.05). GO and KEGG analyses revealed roles for the potential mRNA targets of MIR137HG in epithelial cell differentiation and the peroxisome proliferator-activated receptor (PPAR) and tumor necrosis factor (TNF) signaling pathways. The expression data from TCGA were highly consistent with the verification results of the MIBC tissue samples and bladder cancer cell lines. CONCLUSION: These findings improve our understanding of the regulatory mechanism of the lncRNA-miRNA-mRNA ceRNA network and reveal potential lncRNAs as prognostic biomarkers of MIBC.

Cell-penetrating peptide conjugates of gambogic acid enhance the antitumor effect on human bladder cancer EJ cells through ROS-mediated apoptosis.

BACKGROUND: Gambogic acid (GA) is the main active ingredient of resin gamboges and possesses anti-cancer activity toward various human cancer cells. However, clinical application of GA has been limited by its poor aqueous solubility and dose-limiting toxicities. Cell-penetrating peptides (CPPs) are widely used to deliver anti-cancer drugs into cancer cells and to enhance the water solubility of drugs. PURPOSE: The object of this study was to synthesize peptide-drug conjugates in which the cell-penetrating peptide TAT (trans-activator of transcription) was conjugated to GA and evaluated the anti-cancer activity of this GA-CPP conjugate (GA-TAT) in EJ bladder cancer cells. METHODS: GA is built onto the TAT, and the GA-TAT conjugates are cleaved from the solid support and purified via HPLC. The equilibrium solubility of GA-TAT was measured using the shake-flask method. The effects of GA-TAT on EJ cell viability and proliferation were determined by MTT assay, Edu assay and colony formation assay, respectively. After treated with 1.0 µM GA-TAT for 24 h, the apoptosis rate of EJ cells were detected by Acridine orange/ethidium bromide (AO/EB) assay and flow cytometry assay. The proteins of caspase-3 (processing), caspase-9 (processing), Bcl-2 and Bax were analyzed by Western blotting, and the intracellular reactive oxygen species (ROS) production was evaluated by a reactive oxygen species assay. RESULTS: In contrast to free GA, the solubility of GA-TAT in water was significantly improved. Meanwhile, GA-TAT significantly increased EJ cellular uptake, toxicity and apoptosis. Mechanistic analysis revealed that GA-TAT enhanced the anti-cancer effect of GA against EJ cells through ROS-mediated apoptosis. The results were demonstrated that GA-TAT increased the ROS level in EJ cells, and N-acetyl-L-cysteine (NAC; a well-known ROS scavenger) inhibited GA-TAT-induced ROS generation and apoptosis. Additionally, GA-TAT activated caspase-3 and caspase-9 and down-regulated the Bcl-2/Bax ratio, but these effects were largely rescued by NAC. CONCLUSION: GA-TAT has outstanding potential for promoting tumor apoptosis and exhibits promise for use in bladder cancer therapy.

[MiR-124 suppresses the proliferation of human prostate cancer PC3 cells by targeting PKM2].

OBJECTIVE: To explore the mechanism of miR-124 inhibiting the proliferative activity of prostate cancer PC3 cells. METHODS: Luciferase reporter gene assay was used to examine the specific binding ability of miR-124 to PKM2 mRNA 3'-UTR. After miR-124 was transfected mimic to PC3 cells, the expression levels of PKM2 mRNA and protein were detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot, respectively. The effects of miR-124 mimic and PKM2 siRNA on the proliferative activity of the PC3 cells were determined by MTT assay. RESULTS: The expressions of PKM2 mRNA and protein were upregulated (5.12 +/- 0.35) times and (4.05 +/- 0.20) times respectively in the PC3 cells as compared with those in the RWPE-1 cells (P < 0.05). Luciferase reporter gene assay demonstrated that miR-124 targeted PKM2 3'-UTR. At 24 hours after transfection with miR-124 mimic, the PKM2 protein expression in the PC3 cells was downregulated (0.16 +/- 0.04) times (P < 0.05), while the PKM2 mRNA level was not changed significantly (P > 0.05), as compared with the control group. MTT assay showed that both miRNA-124 mimic and PKM2 siRNA could inhibit the proliferation of the PC3 cells, but the former exhibited a greater inhibitory effect than the latter. After transfection with miR-124 mimic and PKM2 siRNA, the cell growth rates were (66.20 +/- 5.10)% vs (82.10 +/- 6.35)% at 24 hours (P < 0.05) and (49.34 +/- 2.37)% vs (70.10 +/- 5.80)% at 48 hours (P < 0.05). CONCLUSION: miR-124 can suppress the proliferation of PC3 cells by regulating the PKM2 gene.

Stabilization of Snail by HIF-1α and TNF-α is required for hypoxia-induced invasion in prostate cancer PC3 cells.

Hypoxia has been involved in the development of tumor by regulating the expression of invasiveness-associated genes. However, the specific function of hypoxia in cancer cell invasion is still unclear. The aim of the present study was to determine the role of hypoxia in invasion of prostate cancer PC3 cells and to investigate the underlying mechanisms. We found that hypoxia significantly increased the invasive activity of PC3 cells, via up-regulation of the expression of hypoxia inducible factor 1α (HIF-1α) and the autocrine production of tumor necrosis factor α (TNF-α). More important, TNF-α cooperated with HIF-1α in promoting stabilization of Snail, a transcriptional repressor of E-cadherin expression, which lead to the up-regulation of invasiveness-associated genes MMP-9, fibronectin and vimentin. Snail silencing by specific siRNA significantly inhibited hypoxia-induced invasion of PC3 cells, indicating an essential role of Snail in conferring the malignant phenotype to cancer cells under hypoxic conditions. In conclusion, our data demonstrate that hypoxia promoted the invasiveness of prostate cancer PC3 cells via HIF-1α- and TNF-α-induced stabilization of Snail, suggesting a signaling mechanism involving HIF-1α/TNF-α/Snail that mediates invasiveness hypoxic tumor cells in the absence of neoangiogenesis.

The attenuation of cockroach allergy by DNA vaccine encoding cockroach allergen Bla g 2.

Bla g 2 is one of the most potent cockroach allergens. No effective treatment or vaccination strategies are yet available. We evaluated the prophylactic efficacy of Bla g 2 DNA vaccination in a mouse model of allergic airway inflammation. C57/BL6 mice were given Bla g 2 DNA vaccine prior to sensitization with recombinant Bla g 2 (rBla g 2) antigens, followed by nebulized rBla g 2 challenge. Bla g 2 vaccine could express at both transcriptional and translational levels in mammalian cells. Moreover, Bla g 2 vaccine significantly reduced the total inflammatory cell infiltrate and eosinophilia in bronchoalveolar lavage fluid, and markedly decreased allergen-induced inflammatory infiltrates in the lungs and Bla g 2-specific IgE in serum upon challenge with rBla g 2. Importantly, Bla g 2 vaccine could induce the production of antigen-specific IFN-γ and downregulated Th2 pro-inflammatory cytokines IL-4, IL-5, and IL-13. Thus, DNA vaccination showed protective efficacy against a clinically relevant allergen, Bla g 2.

Administering human adipose-derived mesenchymal stem cells to prevent and treat experimental arthritis.

Rheumatoid arthritis is a chronic autoimmune disease and affecting approximately 1% of the population. Human adipose-derived mesenchymal stem cells (hASCs) were recently found to suppress effector T cell and inflammatory responses and, thus, to have beneficial effects in various autoimmune diseases. In this study, we examined whether hASCs could play a protective and/or therapeutic role in collagen-induced arthritis (CIA). We showed that hASCs both prevented and treated CIA by significantly reducing the incidence and severity of experimental arthritis. We further demonstrated that treatment with hASCs inhibited the production of various inflammatory mediators, decreased antigen-specific Th1/Th17 cell expansion, and induced the production of anti-inflammatory cytokine interleukin-10. Moreover, hASCs could induce the generation of antigen-specific Treg cells with the capacity to suppress collagen-specific T cell responses.

The therapeutic efficacy of human adipose tissue-derived mesenchymal stem cells on experimental autoimmune hearing loss in mice.

Autoimmune inner ear disease is characterized by progressive, bilateral although asymmetric, sensorineural hearing loss. Patients with autoimmune inner ear disease had higher frequencies of interferon-γ-producing T cells than did control subjects tested. Human adipose-derived mesenchymal stem cells (hASCs) were recently found to suppress effector T cells and inflammatory responses and therefore have beneficial effects in various autoimmune diseases. The aim of this study was to examine the immunosuppressive activity of hASCs on autoreactive T cells from the experimental autoimmune hearing loss (EAHL) murine model. Female BALB/c mice underwent ß-tubulin immunization to develop EAHL; mice with EAHL were given hASCs or PBS intraperitoneally once a week for 6 consecutive weeks. Auditory brainstem responses were examined over time. The T helper type 1 (Th1)/Th17-mediated autoreactive responses were examined by determining the proliferative response and cytokine profile of splenocytes stimulated with ß-tubulin. The frequency of regulatory T (Treg) cells and their suppressive capacity on autoreactive T cells were also determined. Systemic infusion of hASCs significantly improved hearing function and protected hair cells in established EAHL. The hASCs decreased the proliferation of antigen-specific Th1/Th17 cells and induced the production of anti-inflammatory cytokine interleukin-10 in splenocytes. They also induced the generation of antigen-specific CD4(+) CD25(+) Foxp3(+) Treg cells with the capacity to suppress autoantigen-specific T-cell responses. The experiment demonstrated that hASCs are one of the important regulators of immune tolerance with the capacity to suppress effector T cells and to induce the generation of antigen-specific Treg cells.

[Construction of a tissue engineering skin with epidermal stem cells].

OBJECTIVE: To constitute a composite skin substitute that can proliferate well with epidermal stem cells and fibroblasts on collagen sponge. METHODS: Epidermal stem cells were selected by rapid attachment to collagen IV for 10-15 min and cultured on 3T3 feeder layers. Collagen was extracted from rat tail. The matrix lattice was fabricated by freeze-dryer and cross-linked with glutaraldehyde. Fibroblasts were inoculated on collagen sponge and cultured for 4 days prior to inoculation of epidermal stem cells to construct composite skin substitute. The composite skin substitute were examined by means of histology, immunohistochemistry and electron microcopy, the histologic appearance was similar to that of normal epidermis. RESULTS: The epidermal stem cells formed large colonies at 7-8 days, expressed K19 antigen. The percentages of cells at G0/G1 phase of cell cycle and the percentage of alpha6 briCD71dim cells in ESC groups were higher than those in the control group. The skin substitute had epidermis and dermis, the histologic appearance was similar to that of normal skin. The artificial skin expressed keratin antigen by immunocytochemical methods. CONCLUSIONS: Epidermal stem cells proliferated well and differentiated properly on this artificial skin dermis which contained fibroblasts. It seemed that the composite skin to be a good equivalent.

An experimental study on the repair of full skin loss of nude mice with composite graft of epidermal stem cells.

This study is to constitute a composite skin substitute with epidermal stem cells (ESCs) and fibroblasts on collagen sponge. ESCs were selected by rapid attachment to collagen IV for 10 min. Collagen was extracted from rat's tail. The matrix lattice was fabricated by freeze-dryer and cross-linked with glutaraldehyde. Fibroblasts were inoculated on collagen sponge and cultured for 1 week prior to inoculation of ESCs. Having cultured for 2 weeks in submerged culture, the bioengineered tissue was raised to the air-liquid interface and cultured for 2 weeks. The artificial skin was then grafted onto full skin loss wounds of nude mice. Collagen sponge membrane lacking cell inoculation and an artificial skin with epidermal cells (ECs) and fibroblasts were used as controls. The wounds were observed daily. Tissue samples were harvested and examined by means of histology, immunohistochemistry and electron microscopy. The wounds in the test group healed at a significantly faster rate than controls, with good skin appearance and minimal scar formation. The control group showed delayed wound healing and intensive wound contraction as compared to the test group. Thus the skin substitute with ESCs seemed to be a good equivalent.