Electroacupuncture Improving Intestinal Barrier Function in Rats with Irritable Bowel Syndrome Through Regulating Aquaporins.

BACKGROUND: Intestinal mucosal barrier dysfunction plays a crucial role in the pathogenesis of irritable bowel syndrome with diarrhea (IBS-D). In order to explore the mechanism of electroacupuncture (EA) treatment on intestinal mucosal barrier, this study observed the effect of EA on aquaporins (AQPs), tight junctions (TJs), NF-κB pathway and the gut microbiota in IBS-D rats. METHODS: The IBS-D model was established by acetic acid enema combined with chronic restraint method. The effects of EA on the treatment of IBS-D were examined by the abdominal withdrawal reflex score, Bristol's fecal character score, fecal water content, small intestine propulsion rate and HE staining. AQPs, TJs and inflammation-related molecular mechanisms were explored. The fecal samples were applied for 16S rRNA sequencing to assess the effect of EA intervention to the intestinal bacterial abundance. RESULTS: EA reduced intestinal sensitization, restored intestinal motility and improved inflammatory cell infiltration. Furthermore, EA improved intestinal inflammation and flora environment significantly, inhibited NF-κB signaling and inflammatory factors (IL-1ß and TNF-α). It can also increase the gene and protein expression of AQPs (AQP1, AQP3, and AQP8) and the gene levels of TJs (ZO-1 and Occludin). CONCLUSION: EA has an inhibitory effect on the NF-κB signaling pathway, and regulates the proteins of AQP1, AQP3, AQP8, and TJs to restore the balance of water metabolism and intestinal permeability in IBS-D, which also restored the function of the intestinal mucosa by regulating the intestinal flora.

Role of doublecortin-like kinase 1 and leucine-rich repeat-containing G-protein-coupled receptor 5 in patients with stage II/III colorectal cancer: Cancer progression and prognosis.

BACKGROUND: Cancer stem cells (CSCs) are a subpopulation of cancer cells with the potential of self-renewal and differentiation. CSCs play critical roles in tumorigenesis, recurrence, metastasis, radiation tolerance and chemoresistance. AIM: To assess the expression patterns and clinical potential of doublecortin-like kinase 1 (DCLK1) and leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5), as prognostic CSC markers of colorectal cancer (CRC). METHODS: The expression of DCLK1 and Lgr5 in CRC tissue sections from 92 patients was determined by immunohistochemistry. Each case was evaluated using a combined scoring method based on signal intensity staining (scored 0-3) and the proportion of positively stained cancer cells (scored 0-3). The final staining score was calculated as the intensity score multiplied by the proportion score. Low expression of DCLK1 and Lgr5 was defined as a score of 0-3; high expression of DCLK1 and Lgr5 was defined as a score of ≥ 4. Specimens were categorized as either high or low expression, and the correlation between the expression of DCLK1 or Lgr5 and clinicopathological factors was investigated. RESULTS: DCLK1 and Lgr5 expression levels were significantly positively correlated. CRC patients with high DCLK1, Lgr5 and DCLK1/Lgr5 expressions had poorer progression-free survival and overall survival. Moreover, high expression of DCLK1 was an independent prognostic factor for recurrence and overall survival in patients with CRC by multivariate analysis (P = 0.026 and P = 0.049, respectively). CONCLUSION: DCLK1 may be a potential CSC marker for the recurrence and survival of CRC patients.

Integrin ß4 promotes invasion and anoikis resistance of papillary thyroid carcinoma and is consistently overexpressed in lymphovascular tumor thrombus.

Although the majority of papillary thyroid cancers (PTC) are indolent, a subset of PTCs behaves aggressively due to extensive invasion and distant metastasis. Integrin ß4, a member of the integrin family, has been shown to enhance the progression in some malignancies; however, its role in PTC remains unclear. Here, we demonstrated that ß4 overexpression was associated with extrathyroid extension, lymph node metastasis, high TNM stage, and poor overall survival based on The Cancer Genome Atlas cohort. Immunohistochemistry showed that ß4 expression was significantly upregulated in the tumors with infiltrating growth pattern, as well as those with positive lymphovascular invasion. Moreover, ß4 was invariably overexpressed in the lymphovascular tumor thrombi, which has not been reported before. After shRNA-induced knockdown of ß4 in vitro, the migration, invasion and scratch repair ability of the tumor cells were significantly reduced. Furthermore, ß4 reduction decreased anchorage-independent growth and increased anoikis. The bioinformatics analysis revealed that approximately 70 pathways were significantly dysregulated in the high ß4 expression group. The MAPK pathway and propanoate metabolism were located in the network center of those pathways. Taken together, our results suggest that ß4 could promote the tumor's aggressiveness by enhancing invasion and antagonizing anoikis. The upregulated expression of ß4 in the tumor thrombi is intrinsically linked to its role in strengthening the anoikis resistance.

Direct in vivo application of induced pluripotent stem cells is feasible and can be safe.

Increasing evidence suggests the consensus that direct in vivo application of induced pluripotent stem cells (iPSCs) is infeasible may not be true. Methods: Teratoma formation and fate were examined in 53 normal and disease conditions involving brain, lung, liver, kidney, islet, skin, hind limb, and arteries. Results: Using classic teratoma generation assays, which require iPSCs to be congregated and confined, all mouse, human, and individualized autologous monkey iPSCs tested formed teratoma, while iPSC-derived cells did not. Intravenously or topically-disseminated iPSCs did not form teratomas with doses up to 2.5×108 iPSCs/kg and observation times up to 18 months, regardless of host tissue type; autologous, syngeneic, or immune-deficient host animals; presence or absence of disease; disease type; iPSC induction method; commercial or self-induced iPSCs; mouse, human, or monkey iPSCs; frequency of delivery; and sex. Matrigel-confined, but not PBS-suspended, syngeneic iPSCs delivered into the peritoneal cavity or renal capsule formed teratomas. Intravenously administered iPSCs were therapeutic with a dose as low as 5×106/kg and some iPSCs differentiated into somatic cells in injured organs. Disseminated iPSCs trafficked into injured tissue and survived significantly longer in injured than uninjured organs. In disease-free animals, no intravenously administered cell differentiated into an unwanted long-lasting cell or survived as a quiescent stem cell. In coculture, the stem cell medium and dominant cell-type status were critical for iPSCs to form cell masses. Conclusion: Teratoma can be easily and completely avoided by disseminating the cells. Direct in vivo iPSC application is feasible and can be safe.

Nox2 and Nox4 regulate self-renewal of murine induced-pluripotent stem cells.

Reactive oxygen species (ROS) and redox homeostasis have a pivotal role in the maintenance of stem cell pluripotency and in stem cell self-renewal; however, the mechanisms by which ROS regulate the self-renewal of stem cells have not been thoroughly studied. Here, we evaluated the role of the ROS produced by NADPH oxidase 2 (Nox2) and NADPH oxidase 4 (Nox4) in the self-renewal and stemness of murine induced-pluripotent stem cells (miPSCs). Targeted silencing of Nox2 or Nox4 reduced both NADPH oxidase activity and intracellular ROS levels, as well as alkaline phosphatase activity, the total number of miPSCs, the expression of insulin-like growth factor-1 (IGF-1), IGF-1 receptor, and the phosphorylation of extracellular signal regulated kinase (ERK) 1/2. Nox2/Nox4 overexpression or low, nontoxic concentration of H2 O2 increased cell proliferation in miPSCs. Furthermore, expression of the stemness genes Sox2 and Oct4 was lower in Nox2/Nox4-deficient miPSCs, and higher in Nox2/Nox4-overexpressing miPSCs, than in miPSCs with normal levels of Nox2/Nox4 expression. Collectively, these results suggest that Nox2- and Nox4-derived ROS contribute to stem cell pluripotency maintenance and self-renewal. © 2016 IUBMB Life, 68(12):963-970, 2016.

Nox2 contributes to the arterial endothelial specification of mouse induced pluripotent stem cells by upregulating Notch signaling.

Reactive oxygen species (ROS) have a crucial role in stem-cell differentiation; however, the mechanisms by which ROS regulate the differentiation of stem cells into endothelial cells (ECs) are unknown. Here, we determine the role of ROS produced by NADPH oxidase 2 (Nox2) in the endothelial-lineage specification of mouse induced-pluripotent stem cells (miPSCs). When wild-type (WT) and Nox2-knockout (Nox2(-/-)) miPSCs were differentiated into ECs (miPSC-ECs), the expression of endothelial markers, arterial endothelial markers, pro-angiogenic cytokines, and Notch pathway components was suppressed in the Nox2(-/-) cells but increased in both WT and Nox2(-/-) miPSCs when Nox2 expression was upregulated. Higher levels of Nox2 expression increased Notch signaling and arterial EC differentiation, and this increase was abolished by the inhibition of ROS generation or by the silencing of Notch1 expression. Nox2 deficiency was associated with declines in the survival and angiogenic potency of miPSC-ECs, and capillary and arterial density were lower in the ischemic limbs of mice after treatment with Nox2(-/-) miPSC-ECs than WT miPSC-EC treatment. Taken together, these observations indicate that Nox2-mediated ROS production promotes arterial EC specification in differentiating miPSCs by activating the Notch signaling pathway and contributes to the angiogenic potency of transplanted miPSC-derived ECs.

Bach1 Represses Wnt/ß-Catenin Signaling and Angiogenesis.

RATIONALE: Wnt/ß-catenin signaling has an important role in the angiogenic activity of endothelial cells (ECs). Bach1 is a transcription factor and is expressed in ECs, but whether Bach1 regulates angiogenesis is unknown. OBJECTIVE: This study evaluated the role of Bach1 in angiogenesis and Wnt/ß-catenin signaling. METHODS AND RESULTS: Hind-limb ischemia was surgically induced in Bach1(-/-) mice and their wild-type littermates and in C57BL/6J mice treated with adenoviruses coding for Bach1 or GFP. Lack of Bach1 expression was associated with significant increases in perfusion and vascular density and in the expression of proangiogenic cytokines in the ischemic hindlimb of mice, with enhancement of the angiogenic activity of ECs (eg, tube formation, migration, and proliferation). Bach1 overexpression impaired angiogenesis in mice with hind-limb ischemia and inhibited Wnt3a-stimulated angiogenic response and the expression of Wnt/ß-catenin target genes, such as interleukin-8 and vascular endothelial growth factor, in human umbilical vein ECs. Interleukin-8 and vascular endothelial growth factor were responsible for the antiangiogenic response of Bach1. Immunoprecipitation and GST pull-down assessments indicated that Bach1 binds directly to TCF4 and reduces the interaction of ß-catenin with TCF4. Bach1 overexpression reduces the interaction between p300/CBP and ß-catenin, as well as ß-catenin acetylation, and chromatin immunoprecipitation experiments confirmed that Bach1 occupies the TCF4-binding site of the interleukin-8 promoter and recruits histone deacetylase 1 to the interleukin-8 promoter in human umbilical vein ECs. CONCLUSIONS: Bach1 suppresses angiogenesis after ischemic injury and impairs Wnt/ß-catenin signaling by disrupting the interaction between ß-catenin and TCF4 and by recruiting histone deacetylase 1 to the promoter of TCF4-targeted genes.

Chromosomal imbalances revealed in primary renal cell carcinomas by comparative genomic hybridization.

Renal cell carcinoma (RCC) accounts for approximately 3% of all new cancer cases. Although the classification of RCC is based mainly on histology, this method is not always accurate. We applied comparative genomic hybridization (CGH) to determine genomic alterations in 46 cases of different RCC histological subtypes [10 cases of clear cell RCC (CCRCC), 13 cases of papillary RCC (PRCC), 12 cases of chromophobe RCC (CRCC), 9 cases of Xp11.2 translocation RCC (Xp11.2RCC), 2 cases of undifferentiated RCC (unRCC)], and investigated the relationships between clinical parameters and genomic aberrations. Changes involving one or more regions of the genome were seen in all RCC patients; DNA sequence gains were most frequently (>30%) seen in chromosomes 7q, 16p, and 20q; losses from 1p, 3p, 13q, 14q, and 8p. We conclude CGH is a useful complementary method for differential diagnosis of RCC. Loss of 3p21-25, 15q, and gain of 16p11-13 are relatively particular to CCRCC vs. other types of RCC. Gain of 7p13-22, 8q21-24, and loss of 18q12-ter, 14q13-24, and Xp11-q13/Y are more apparent in PRCC, and gain of 8q21-24 is characteristic of type 2 PRCC vs. type 1 PRCC. Loss of 2q12-32, 10p12-15, and 11p11-15, 13p are characteristic of CRCC, and gain of 3p and loss of 11p11-15 and 13p are significant differentiators between common CRCC and CRCC accompanied by sarcomatous change groups. Gain of Xp11-12 is characteristic of the Xp11.2RCC group. Based on Multivariate Cox regression analysis, aberration in 5 chromosome regions were poor prognostic markers of RCC, and include the gain of chromosome 12p12-ter (P = 0.034, RR = 3.502, 95% CI 1.097-11.182), 12q14-ter (P = 0.002, RR = 5.115, 95% CI 1.847-14.170), 16q21-24 (P = 0.044, RR = 2.629, 95% CI 1.027-6.731), 17p12-ter (P = 0.017, RR = 3.643, 95% CI 1.262-10.512) and the loss of 18q12-23 (P = 0.049, RR = 2.911, 95% CI 1.006-8.425), which may provide clues of new genes involved in RCC tumorigenesis.

Chromophobe renal cell carcinoma with and without sarcomatoid change: a clinicopathological, comparative genomic hybridization, and whole-exome sequencing study.

Chromophobe renal cell carcinomas (CRCC) with and without sarcomatoid change have different outcomes; however, fewstudies have compared their genetic profiles. Therefore, we identified the genomic alterationsin CRCC common type (CRCC C) (n=8) and CRCC with sarcomatoid change (CRCC S) (n=4) using comparative genomic hybridization (CGH) and whole-exome sequencing. The CGH profiles showed that the CRCC C group had more chromosomal losses (72 vs. 18) but fewer chromosomal gains (23 vs. 57) than the CRCC S group. Losses of chromosomes 1p, 8p21-23, 10p16-20, 10p12-ter, 13p20-30, and 17p13 and gains of chromosomes 1q11, 1q21-23, 1p13-15, 2p23-24, and 3p21-ter differed between the groups. Whole-exome sequencing showed that the mutational status of 270 genes differed between CRCC (n=12) and normal renal tissues (n=18). In the functional enrichment analysis, the missense-mutated genes were classified into 6 biological processes (38 functions) and 5 pathways. The biological processes included cell adhesion, cell motility, ATP metabolism, sensory perception, carbohydrate and lipid metabolism and transport. The pathways included ATP-binding cassette transporter, extracellular matrix-receptor interaction, olfactory transduction, chondroitin sulfate biosynthesis, and hypertrophic cardiomyopathy. Whole-exome sequencing analysis revealed that the missense mutation statuses of 49 genes differed between the CRCC C and CRCC S groups. Furthermore, genetic alterations in metastasis suppressor 1, serine peptidase inhibitor Kazal type 8, transient receptor potential cation channel super family M member 6, Rh family B glycoprotein, and mannose receptor C type 1 were located in different chromosomal regions. These alterations may provide clues regarding CRCC tumorigenesis and provide a basis for future targeted therapies.

Unclassified renal cell carcinoma: a clinicopathological, comparative genomic hybridization, and whole-genome exon sequencing study.

Unclassified renal cell carcinoma (URCC) is a rare variant of RCC, accounting for only 3-5% of all cases. Studies on the molecular genetics of URCC are limited, and hence, we report on 2 cases of URCC analyzed using comparative genome hybridization (CGH) and the genome-wide human exon GeneChip technique to identify the genomic alterations of URCC. Both URCC patients (mean age, 72 years) presented at an advanced stage and died within 30 months post-surgery. Histologically, the URCCs were composed of undifferentiated, multinucleated, giant cells with eosinophilic cytoplasm. Immunostaining revealed that both URCC cases had strong p53 protein expression and partial expression of cluster of differentiation-10 and cytokeratin. The CGH profiles showed chromosomal imbalances in both URCC cases: gains were observed in chromosomes 1p11-12, 1q12-13, 2q20-23, 3q22-23, 8p12, and 16q11-15, whereas losses were detected on chromosomes 1q22-23, 3p12-22, 5p30-ter, 6p, 11q, 16q18-22, 17p12-14, and 20p. Compared with 18 normal renal tissues, 40 mutated genes were detected in the URCC tissues, including 32 missense and 8 silent mutations. Functional enrichment analysis revealed that the missense mutation genes were involved in 11 different biological processes and pathways, including cell cycle regulation, lipid localization and transport, neuropeptide signaling, organic ether metabolism, and ATP-binding cassette transporter signaling. Our findings indicate that URCC may be a highly aggressive cancer, and the genetic alterations identified herein may provide clues regarding the tumorigenesis of URCC and serve as a basis for the development of targeted therapies against URCC in the future.

Xp11 translocation renal cell carcinoma in adults: a clinicopathological and comparative genomic hybridization study.

To study the clinicopathological and genomic characteristics of Xp11.2 translocation renal cell carcinoma (Xp11.2 RCC) in adults, we analyzed 9 Xp11.2 RCCs, confirmed by transcription factor E3 (TFE3) immunohistochemistry, in patients aged ≥20 years. TFE3 expression was also determined in 12 cases of alveolar soft part sarcoma (ASPS) served as a positive control. Comparative genomic hybridization (CGH) was used to investigate genomic imbalances in all Xp11.2 RCC cases. Most of our Xp11.2 RCC patients (5/9) presented with TNM stages 3-4, and 6 patients died 10 months to 7 years after their operation. Histologically, Xp11.2 RCC was composed of a mixed papillary nested/alveolar growth pattern (8/9). Immunostaining showed that all Xp11.2 RCC and ASPS cases had strong TFE3 expression and high positive ratios for p53 and vimentin. However, there were significant differences in the expression of AMACR (p<0.001), AE1/AE3 (p=0.002), and CD10 (p=0.024) between the 2 diseases. CGH profiles showed chromosomal imbalances in all 9 Xp11.2 RCC cases; gains were observed in chromosomes Xp11 (6/9), 7q20-25, 12q25-31 (5/9), 7p16-24 (4/9), 8p12-13, 8q20-21, 16q20-22, 17q25-26, 20q22-23 (4/9), and losses occurred frequently on chromosomes 3p12-16, 9q31-32, 14q22-24 (4/9). Our Conclusions show Xp11.2 RCC that occur in adults may be aggressive cancers, the expressions of AMACR, CD10, AE1/AE3 are helpful in the differential diagnosis between Xp11.2 RCC and ASPS, and CGH assay is a useful complementary method for confirming the diagnosis of Xp11.2 RCC.

Oxidative stress inhibits adhesion and transendothelial migration, and induces apoptosis and senescence of induced pluripotent stem cells.

Oxidative stress caused by cellular accumulation of reactive oxygen species (ROS) is a major contributor to disease and cell death. However, how induced pluripotent stem cells (iPSC) respond to different levels of oxidative stress is largely unknown. Here, we investigated the effect of H2 O2 -induced oxidative stress on iPSC function in vitro. Mouse iPSC were treated with H2 O2 (25-100 µmol/L). IPSC adhesion, migration, viability, apoptosis and senescence were analysed. Expression of adhesion-related genes, stress defence genes, and osteoblast- and adipocyte-associated genes were determined by reverse transcription polymerase chain reaction. The present study found that H2 O2 (25-100 µmol/L) decreased iPSC adhesion to matrix proteins and endothelial cells, and downregulated gene expression levels of adhesion-related molecules, such as integrin alpha 7, cadherin 1 and 5, melanoma cell adhesion molecule, vascular cell adhesion molecule 1, and monocyte chemoattractant protein-1. H2 O2 (100 µmol/L) decreased iPSC viability and inhibited the capacity of iPSC migration and transendothelial migration. iPSC were sensitive to H2 O2 -induced G2/M arrest, senescence and apoptosis when exposed to H2 O2 at concentrations above 25 µmol/L. H2 O2 increased the expression of stress defence genes, including catalase, cytochrome B alpha, lactoperoxidase and thioredoxin domain containing 2. H2 O2 upregulated the expression of osteoblast- and adipocyte-associated genes in iPSC during their differentiation; however, short-term H2 O2 -induced oxidative stress did not affect the protein expression of the pluripotency markers, octamer-binding transcription factor 4 and sex-determining region Y-box 2. The present results suggest that iPSC are sensitive to H2 O2 toxicity, and inhibition of oxidative stress might be a strategy for improving their functions.

NADPH oxidase 4 mediates insulin-stimulated HIF-1α and VEGF expression, and angiogenesis in vitro.

Acute intensive insulin therapy causes a transient worsening of diabetic retinopathy in type 1 diabetes patients and is related to VEGF expression. Reactive oxygen species (ROS) have been shown to be involved in HIF-1α and VEGF expression induced by insulin, but the role of specific ROS sources has not been fully elucidated. In this study we examined the role of NADPH oxidase subunit 4 (Nox4) in insulin-stimulated HIF-1α and VEGF expression, and angiogenic responses in human microvascular endothelial cells (HMVECs). Here we demonstrate that knockdown of Nox4 by siRNA reduced insulin-stimulated ROS generation, the tyrosine phosphorylation of IR-ß and IRS-1, but did not change the serine phosphorylation of IRS-1. Nox4 gene silencing had a much greater inhibitory effect on insulin-induced AKT activation than ERK1/2 activation, whereas it had little effect on the expression of the phosphatases such as MKP-1 and SHIP. Inhibition of Nox4 expression inhibited the transcriptional activity of VEGF through HIF-1. Overexpression of wild-type Nox4 was sufficient to increase VEGF transcriptional activity, and further enhanced insulin-stimulated the activation of VEGF. Downregulation of Nox4 expression decreased insulin-stimulated mRNA and protein expression of HIF-1α, but did not change the rate of HIF-1α degradation. Inhibition of Nox4 impaired insulin-stimulated VEGF expression, cell migration, cell proliferation, and tube formation in HMVECs. Our data indicate that Nox4-derived ROS are essential for HIF-1α-dependent VEGF expression, and angiogenesis in vitro induced by insulin. Nox4 may be an attractive therapeutic target for diabetic retinopathy caused by intensive insulin treatment.

[Notch1 mRNA and protein expression in human breast cancer and normal mammary gland tissues].

OBJECTIVE: To explore the Notch1 mRNA and protein expression in human breast cancers and normal mammary tissues, and their relationship with the clinical indicators of breast cancers were analyzed. METHODS: Notch1 gene of human breast invasive ductal carcinoma (IDC) and normal mammary gland tissues were amplified by RT-PCR, and the expression of Notch1 protein was detected by immunohistochemical Streptavidin-Biotin Complex (SP) stain in 60 IDC, 30 ductal carcinoma in situ (DCIS) and 60 normal mammary tissues. RESULTS: Notch1 gene of human IDC and normal mammary tissues both could express in a transcription level; the positive rates of Notch1 protein expression in normal mammary tissues and DCIS were 55% and 70%. Respectively, which did not differ statistically (P > 0.05), while the positive rate in IDC was 90%, significantly higher than that of the normal mammary tissues and DCIS (P < 0.05). The high expression of Notch1 protein in IDC correlate significantly with lymph node metastasis, pathological grades and TNM stages. CONCLUSIONS: Notch1 protein was over expressed in breast IDC. A high Notch1 protein expression is considered associating with the evolution and malignant transformation of the breast tumor. The expression of Notch1 gene maybe impact the effect of on the progression of breast cancers.