Interleukin-22 Ameliorates Dextran Sulfate Sodium-Induced Colitis through the Upregulation of lncRNA-UCL to Accelerate Claudin-1 Expression via Sequestering miR-568 in Mice.

Background: Bioactive compound such as interleukin-22 (IL-22) treatment is regarded as a sufficient treatment for ulcerative colitis (UC). It has been found that long noncoding RNAs (lncRNAs) expressed in many inflammatory diseases, including UC. We aimed to verify the treatment effect of bioactive compounds including IL-22 and lncRNAs in UC on colitis mice. Methods: UC mice were induced using DSS, followed by IL-22 or PBS intraperitoneally (i.p.) injection. Then, the histopathological parameters of the mice were determined. Then, RNA sequencing was performed to screen the differential lncRNAs. Quantitative real-time PCR (qRT-PCR) and lentivirus identified lncRNA-Ulcerative Colitis lncRNA (lncRNA-UCL) were regarded as the molecular regulator of the colitis mice. The correlation with lncRNA-UCL and mmu-miR-568 was validated using RNA-pulldown. Meanwhile, claudin-1 was predicted and confirmed as the target molecule of mmu-miR-568 using dual-luciferase assay. Results: IL-22 could significantly improve the histopathological features and decrease proinflammatory cytokine production in UC mice induced by DSS. It also can stimulate intestinal epithelial cell (IEC) reproduction and prevention of apoptosis. lncRNA-UCL was significantly downregulated in UC mice caused by DSS, while IL-22 treatment effectively reversed this effect. In terms of mechanism, lncRNA-UCL regulates intestinal epithelial homeostasis by sequestering mmu-miR-568 and maintaining close integrated protein expression, such as claudin-1. Conclusions: We have demonstrated the incredible role of bioactive compound, such as IL-22, in alleviating DSS-induced colitis symptoms via enhancing lncRNA-UCL expression. It can be regulated using tight junction (TJ) protein.

Asperosaponin VI protects mice from sepsis by regulating Hippo and Rho signaling pathway.

OBJECTIVE: To explore the novel protective effect of Asperosaponin VI (AVI) on sepsis and its potential mechanism. METHODS: In in vitro experiments, bone marrow mononuclear cells and THP-1-derived cells were used to evaluate the viability of AVI treatment. Besides, the quantitative real-time PCR and Western blot were adopted to explore the protective effect of AVI on LPS-induced inflammation. For in vivo work, the effect of AVI on mice was evaluated by using both CLP-induced and the LPS-induced sepsis mice model. The fluctuation of anal temperature and the behavior of mice were recorded after surgery. Further, the content of bacteria in peritoneal lavage fluid was detected, as well as the levels of ALT, AST, LD and LDH in serum with ELISA. H&E staining and real-time PCR were used to evaluate the histopathology of liver, spleen and lung. Finally, relevant signaling pathways were detected by Western blot, real-time PCR and immunohistochemistry. RESULTS: AVI inhibited the expression of inflammatory factors in both CLP-induced and LPS-induced sepsis mice models, and reduced the number of bacteria in abdominal lavage fluid. The preventive treatment with AVI alleviated sepsis-induced organ injuries, reduced inflammatory responses, which was through inhibiting Hippo and Rho signaling pathway. CONCLUSIONS: This study indicated that AVI effectively protected mice from sepsis by down-regulating the activation of Hippo signaling and Rho family, and reducing inflammation and organ damage. However, conventional treatment was using antibiotics, and its mechanism was different with AVI.

The Regulatory Role of GBF1 on Osteoclast Activation Through EIF2a Mediated ER Stress and Novel Marker FAM129A Induction.

Bone-resorbing activities of osteoclasts (OCs) are highly dependent on actin cytoskeleton remodeling, plasma membrane reorganization, and vesicle trafficking pathways, which are partially regulated by ARF-GTPases. In the present study, the functional roles of Golgi brefeldin A resistance factor 1 (GBF1) are proposed. GBF1 is responsible for the activation of the ARFs family and vesicular transport at the endoplasmic reticulum-Golgi interface in different stages of OCs differentiation. In the early stage, GBF1 deficiency impaired OCs differentiation and was accompanied with OCs swelling and reduced formation of mature OCs, indicating that GBF1 participates in osteoclastogenesis. Using siRNA and the specific inhibitor GCA for GBF1 knockdown upregulated endoplasmic reticulum stress-associated signaling molecules, including BiP, p-PERK, p-EIF2α, and FAM129A, and promoted autophagic Beclin1, Atg7, p62, and LC3 axis, leading to apoptosis of OCs. The present data suggest that, by blocking COPI-mediated vesicular trafficking, GBF1 inhibition caused intense stress to the endoplasmic reticulum and excessive autophagy, eventually resulting in the apoptosis of mature OCs and impaired bone resorption function.

Targeted inhibition of ATP5B gene prevents bone erosion in collagen-induced arthritis by inhibiting osteoclastogenesis.

Bone resorption by osteoclasts is an energy consuming activity, which depends on mitochondrial ATP. ATP5B, a mitochondrial ATP synthase beta subunit, is a catalytic core involved in producing ATP. Here, we investigated the contribution of ATP5B in osteoclast differentiation and joint destruction. ATP5B (LV-ATP5B) targeting or non-targeting (LV-NC) siRNA containing lentivirus particles were transduced into bone marrow macrophage derived osteoclasts or locally administered to arthritic mouse joints. Inhibition of ATP5B reduced the expression of osteoclast related genes and proteins, suppressed bone resorption by significantly impairing F-actin formation and decreased the levels of adhesion-associated proteins. In addition, ATP5B deficiency caused osteoclast mitochondrial dysfunction and, impaired the secretion of vacuole protons and MMP9. Importantly, inhibition of ATP5B expression, protected arthritis mice from joint destructions although serum levels of inflammatory mediators (TNF-α, IL-1ß) and IgG2α antibodies were unaffected. These results demonstrate an essential function of ATP5B in osteoclast differentiation and bone resorption, and suggest it as a potential therapeutic target for protecting bones in RA.

Asperosaponin VI protects against bone destructions in collagen induced arthritis by inhibiting osteoclastogenesis.

BACKGROUND: Bone destructive diseases like rheumatoid arthritis (RA), osteoporosis and bone metastatic tumors are mainly mediated by over-activated osteoclasts. Asperosaponin VI (AVI), isolated from the rhizome of Dipsacus asper, belongs to triterpenoid saponins. It has multiple physiological activities but its effects on RA, especially on osteoclast differentiation and activation are still unclear. PURPOSE: Explore the protective role of AVI on collagen induced arthritis (CIA) in vivo and RANKL induced osteoclastogenesis in vitro. METHODS: The effects of AVI on cell viability and RANKL-induced osteoclastogenesis, actin ring formation, bone resorption activity as well as on osteoclast specific gene and protein expression were tested using bone marrow derived monocytes (BMMs). Paws from CIA mice were used for micro-CT, HE and TRAP staining, real-time PCR and western blot. Sera were used for cytokine analysis by ELISA. The signaling pathways were detected using western blot, real-time PCR and immunofluorescence assay. RESULTS: AVI significantly inhibited RANKL-induced osteoclast formation and bone resorption activity by suppressing the formation of actin ring. It also inhibited the expression of various osteoclatogenesis marker genes and signaling pathways. AVI protected arthritis in vivo by suppressing inflammation and bone loss. CONCLUSION: AVI exerts its anti-osteoclastogenic activity both in vitro and in vivo by inhibiting RANKL-induced osteoclast differentiation and function. Thus, our studies demonstrate a potential therapeutic role for AVI in preventing or inhibiting RANKL-mediated osteolytic bone diseases.

Regulatory role of Golgi brefeldin A resistance factor-1 in amyloid precursor protein trafficking, cleavage and Aß formation.

ß-amyloid peptide (Aß) deposition derived from sequential cleavage of the amyloid precursor protein (APP) through the amyloidogenic pathway is an important characteristic feature of Alzheimer's disease (AD). During this process, cellular trafficking plays a crucial role. A large Sec7-domain containing ADP-ribosylation factor guanine nucleotide exchange factor (ARF-GEF), Golgi brefeldin A resistance factor 1 (GBF1) has been reported to initiate the ADP-ribosylation factor (Arf) activation cascade at trans-Golgi network, which plays a crucial function at the endoplasmic reticulum-Golgi interface. In this study, we investigated the role of GBF1 in APP transmembrane transport and Aß formation. Using APP/PS1 (presenilin 1) overexpressing transgenic mice, we demonstrate that GBF1 has upregulated the expression of APP, indicating a role for GBF1 in APP physiological process. Knocking down of GBF1 using small interfering has significantly increased the intracellular but not the surface expression of APP. In contrast, overexpression of wild-type (WT) and guanine nucleotide exchange factor (GEF) in the activated form but not the GEF deficient mutation induced continuous activation of GBF1, which subsequently increased the surface level of APP. Interestingly, inhibition of GBF1 by c(BFA) also impaired APP trafficking and induced endoplasmic reticulum (ER) stress in SH-SY5Y cells. Our results thus for identified the role of GBF1 in APP trafficking and cleavage, and provide evidence for GBF1 as a possible therapeutic target in AD.

Blocking the recruitment of naive CD4+ T cells reverses immunosuppression in breast cancer.

The origin of tumor-infiltrating Tregs, critical mediators of tumor immunosuppression, is unclear. Here, we show that tumor-infiltrating naive CD4+ T cells and Tregs in human breast cancer have overlapping TCR repertoires, while hardly overlap with circulating Tregs, suggesting that intratumoral Tregs mainly develop from naive T cells in situ rather than from recruited Tregs. Furthermore, the abundance of naive CD4+ T cells and Tregs is closely correlated, both indicating poor prognosis for breast cancer patients. Naive CD4+ T cells adhere to tumor slices in proportion to the abundance of CCL18-producing macrophages. Moreover, adoptively transferred human naive CD4+ T cells infiltrate human breast cancer orthotopic xenografts in a CCL18-dependent manner. In human breast cancer xenografts in humanized mice, blocking the recruitment of naive CD4+ T cells into tumor by knocking down the expression of PITPNM3, a CCL18 receptor, significantly reduces intratumoral Tregs and inhibits tumor progression. These findings suggest that breast tumor-infiltrating Tregs arise from chemotaxis of circulating naive CD4+ T cells that differentiate into Tregs in situ. Inhibiting naive CD4+ T cell recruitment into tumors by interfering with PITPNM3 recognition of CCL18 may be an attractive strategy for anticancer immunotherapy.

ZKSCAN1 gene and its related circular RNA (circZKSCAN1) both inhibit hepatocellular carcinoma cell growth, migration, and invasion but through different signaling pathways.

There is increasing evidence that circular RNA (circRNA) are involved in cancer development, but the regulation and function of human circRNA remain largely unknown. In this study, we demonstrated that ZKSCAN1, a zinc finger family gene, is expressed in both linear and circular (circZKSCAN1) forms of RNA in human hepatocellular carcinoma (HCC) tissues and cell lines. Here, we analyzed a cohort of 102 patients and found that expression of both ZKSCAN1mRNA and circZKSCAN1 was significantly lower (P < 0.05) in the HCC samples compared with that in matched adjacent nontumorous tissues by reverse transcription PCR (RT-PCR). The low expression level of ZKSCAN1 was only associated with tumor size (P = 0.032), while the cirZKSCAN1 levels varied in patients with different tumor numbers (P < 0.01), cirrhosis (P = 0.031), vascular invasion (P = 0.002), or microscopic vascular invasion (P = 0.002), as well as with the tumor grade (P < 0.001). Silencing both ZKSCAN1mRNA and circZKSCAN1 promoted cell proliferation, migration, and invasion. In contrast, overexpression of both forms of RNA repressed HCC progression in vivo and in vitro. Silencing or overexpression of both forms of RNA did not interfere with each other. RNA-seq revealed a very different molecular basis for the observed effects; ZKSCAN1mRNA mainly regulated cellular metabolism, while circZKSCAN1 mediated several cancer-related signaling pathways, suggesting a nonredundant role for ZKSCAN1mRNA and circRNA. In conclusion, our results revealed two post-translational products (ZKSCAN1mRNA and circZKSCAN1) that cooperated closely with one another to inhibit growth, migration, and invasion of HCC. cirZKSCAN1 might be a useful marker for the diagnosis of HCC.

miR-142-5p and miR-130a-3p are regulated by IL-4 and IL-13 and control profibrogenic macrophage program.

Macrophages play a pivotal role in tissue fibrogenesis, which underlies the pathogenesis of many end-stage chronic inflammatory diseases. MicroRNAs are key regulators of immune cell functions, but their roles in macrophage's fibrogenesis have not been characterized. Here we show that IL-4 and IL-13 induce miR-142-5p and downregulate miR-130a-3p in macrophages; these changes sustain the profibrogenic effect of macrophages. In vitro, miR-142-5p mimic prolongs STAT6 phosphorylation by targeting its negative regulator, SOCS1. Blocking miR-130a relieves its inhibition of PPARγ, which coordinates STAT6 signalling. In vivo, inhibiting miR-142-5p and increasing miR-130a-3p expression with locked nucleic acid-modified oligonucleotides inhibits CCL4-induced liver fibrosis and bleomycin-induced lung fibrosis in mice. Furthermore, macrophages from the tissue samples of patients with liver cirrhosis and idiopathic pulmonary fibrosis display increased miR-142-5p and decreased miR-130a-3p expression. Therefore, miR-142-5p and miR-130a-3p regulate macrophage profibrogenic gene expression in chronic inflammation.

Nerve fibers in breast cancer tissues indicate aggressive tumor progression.

Emerging evidence has indicated nerve fibers as a marker in the progression of various types of cancers, such as pancreatic cancer and prostate cancer. However, whether nerve fibers are associated with breast cancer progression remains unclear. In this study, we evaluated the presence of nerve fibers in 352 breast cancer specimens and 83 benign breast tissue specimens including 43 cases of cystic fibrosis and 40 cases of fibroadenoma from 2 independent breast tumor center using immunohistochemical staining for specific peripheral nerve fiber markers.In all, nerve fibers were present in 130 out of 352 breast cancer tissue specimens, while none were detected in normal breast tissue specimens. Among 352 cases, we defined 239 cases from Sun Yat-Sen Memorial Hospital, Guangzhou, China, as the training set, and 113 cases from the First Affiliated Hospital of Shantou University, Guangdong, China, as the validation set. The thickness of tumor-involving nerve fibers is significantly correlated with poor differentiation, lymph node metastasis, high clinical staging, and triple negative subtype in breast cancer. More importantly, Cox multifactor analysis indicates that the thickness of tumor-involving nerve fibers is a previously unappreciated independent prognostic factors associated with shorter disease-free survival of breast cancer patients. Our findings are further validated by online Oncomine database. In conclusion, our results show that nerve fiber involvement in breast cancer is associated with progression of the malignancy and warrant further studies in the future.

A positive feedback loop between mesenchymal-like cancer cells and macrophages is essential to breast cancer metastasis.

The close vicinity of cancer cells undergoing epithelial-mesenchymal transition (EMT) and tumor-associated macrophages (TAMs) at the invasive front of tumors suggests that these two cell type may mutually interact. We show that mesenchymal-like breast cancer cells activate macrophages to a TAM-like phenotype by GM-CSF. Reciprocally, CCL18 from TAMs induces cancer cell EMT, forming a positive feedback loop, in coculture systems and humanized mice. Inhibition of GM-CSF or CCL18 breaks this loop and reduces cancer metastasis. High GM-CSF expression in breast cancer samples is associated with more CCL18(+) macrophages, cancer cell EMT, enhanced metastasis, and reduced patient survival. These findings suggest that a positive feedback loop between GM-CSF and CCL18 is important in breast cancer metastasis.

Breaking the vicious cycle between breast cancer cells and tumor-associated macrophages.

We recently identified a vicious cycle between granulocyte macrophage colony stimulating factor (GM-CSF) arising from breast cancer cells that have undergone epithelial-mesenchymal transition (EMT) and the tumor-associated macrophage (TAM)-derived chemokine CCL18, a signaling loop that promotes tumor metastasis. Tumor-derived lactate skews GM-CSF-activated macrophages to an anti-inflammatory and immunosuppressive M2 phenotype, suggesting that breaking this cycle in combination with glycolysis inhibitors may inhibit tumor development.