Sodium Pentobarbital Suppresses Breast Cancer Cell Growth Partly via Normalizing Microcirculatory Hemodynamics and Oxygenation in Tumors.

Breast cancer remains the leading cause of cancer-related death among women worldwide. Sodium pentobarbital was found to play an inhibitory role in glioma growth in rats. In this study, we aimed to evaluate the effects of sodium pentobarbital on breast cancer growth both in vitro and in vivo, and its impacts on the microcirculatory changes on both skin and tumor surface in mice bearing subcutaneous xenograft. Cell counting assay was used to assess the antiproliferative effect of sodium pentobarbital on MDA-MB-231 breast cancer cells. Subcutaneous xenograft model was established to study the role of sodium pentobarbital on in vivo tumor growth. Speed-resolved blood perfusion, hemoglobin oxygen saturation (SO2, %), total hemoglobin tissue concentration (ctTHb, µM), and red blood cell (RBC) tissue fraction (%) were examined simultaneously by using enhanced perfusion and oxygen saturation system to investigate the effects of sodium pentobarbital on microcirculatory hemodynamics and oxygenation. Sodium pentobarbital suppressed breast tumor growth both in vitro and in vivo. Cutaneous blood flux in nutritive capillaries with low-speed flow was significantly increased in tumor-bearing mice, and high-dose sodium pentobarbital treatment cause a reduction in this low-speed blood flux, whereas sodium pentobarbital therapy caused an elevated blood flux in larger microvessels with mid and high speed in a dose-dependent manner. Different doses of sodium pentobarbital exerted different actions on SO2, ctTHb, and RBC tissue fraction. Collectively, the inhibitory effect of sodium pentobarbital on breast tumor growth was at least partly associated with its ability to normalize microcirculatory hemodynamics and oxygenation in tumors. SIGNIFICANCE STATEMENT: This study is the first to demonstrate the inhibiting effect of sodium pentobarbital on breast cancer growth both in vitro and in vivo, and such an inhibition was at least partly associated with its ability to normalize microcirculatory hemodynamics and oxygenation in tumors.

Roquin2 suppresses breast cancer progression by inhibiting tumor angiogenesis via selectively destabilizing proangiogenic factors mRNA.

Tumor angiogenesis is an essential step in tumor growth and metastasis. The initiation of tumor angiogenesis is dictated by a shift in the balance between proangiogenic and antiangiogenic gene expression programs. Roquin2 is a zinc-finger RNA-binding protein with important roles in mediating the expression of inflammatory genes, such as TNF, IL6 and PTGS2, which are also important angiogenic factors. In this study, we demonstrate that Roquin2 functions as a potent tumor angiogenesis regulator that inhibits breast tumor-induced angiogenesis by selectively destabilizing mRNA of proangiogenic gene transcripts, including endoglin (ENG), endothelin-1 (EDN1), vascular endothelial growth factor B (VEGFB) and platelet derived growth factor C (PDGFC). Roquin2 recognizes and binds the stem-loop structure in the 3'untranslated region (3'UTR) of these mRNAs via its ROQ domain to destabilize mRNA. Moreover, we found that Roquin2 expression was reduced in breast cancer cells and tissues, and associated with poor prognosis in breast cancer patients. Overexpression of Roquin2 inhibited breast tumor-induced angiogenesis in vitro and in vivo, whereas silencing Roquin2 enhanced tumor angiogenesis. In vivo induction of Roquin2 by adenovirus significantly suppressed breast tumor growth, metastasis and angiogenesis. Taken together, our results identify that Roquin2 is a novel breast cancer suppressor that inhibits tumor angiogenesis by selectively downregulating the expression of proangiogenic genes.

RNA-binding protein SAMD4A inhibits breast tumor angiogenesis by modulating the balance of angiogenesis program.

Tumor-induced angiogenesis is important for further progression of solid tumors. The initiation of tumor angiogenesis is dictated by a shift in the balance between proangiogenic and antiangiogenic gene expression programs. However, the potential mechanism controlling the expression of angiogenesis-related genes in the tumor cells, especially the process mediated by RNA-binding protein (RBP) remains unclear. SAMD4A is a conserved RBP across fly to mammals, and is believed to play an important role in controlling gene translation and stability. In this study, we identified the potential role of SAMD4A in modulating angiogenesis-related gene expression and tumor progression in breast cancer. SAMD4A expression was repressed in breast cancer tissues and cells and low SAMD4A expression in human breast tumor samples was strongly associated with poor survival of patients. Overexpression of SAMD4A inhibited breast tumor angiogenesis and caner progression, whereas knockdown of SAMD4A demonstrated a reversed effect. Mechanistically, SAMD4A was found to specifically destabilize the proangiogenic gene transcripts, including C-X-C motif chemokine ligand 5 (CXCL5), endoglin (ENG), interleukin 1ß (IL1ß), and angiopoietin 1 (ANGPT1), by directly interacting with the stem-loop structure in the 3' untranslated region (3'UTR) of these mRNAs through its sterile alpha motif (SAM) domain, resulting in the imbalance of angiogenic genes expression. Collectively, our results suggest that SAMD4A is a novel breast tumor suppressor that inhibits tumor angiogenesis by specifically downregulating the expression of proangiogenic genes, which might be a potential antiangiogenic target for breast cancer therapy.

TARBP2 promotes tumor angiogenesis and metastasis by destabilizing antiangiogenic factor mRNAs.

Tumor angiogenesis is a crucial step in the further growth and metastasis of solid tumors. However, its regulatory mechanism remains unclear. Here, we showed that TARBP2, an RNA-binding protein, played a role in promoting tumor-induced angiogenesis both in vitro and in vivo through degrading the mRNAs of antiangiogenic factors, including thrombospondin1/2 (THBS1/2), tissue inhibitor of metalloproteinases 1 (TIMP1), and serpin family F member 1 (SERPINF1), by targeting their 3'untranslated regions (3'UTRs). Overexpression of TARBP2 promotes tumor cell-induced angiogenesis, while its knockdown inhibits tumor angiogenesis. Clinical cohort analysis revealed that high expression level of TARBP2 was associated with poor survival of lung cancer and breast cancer patients. Mechanistically, TARBP2 physically interacts with the stem-loop structure located in the 3'UTR of antiangiogenic transcripts, leading to mRNA destabilization by the dsRNA-binding domains 1/2 (dsRBDs1/2). Notably, the expression level of TARBP2 in human tumor tissue is negatively correlated with the expression of antiangiogenic factors, including THBS1/2, and brain-specific angiogenesis inhibitor 1 (BAI1). Moreover, TARBP2 expression is strongly associated with tumor angiogenesis in a group of human lung cancer samples. Collectively, our results highlight that TARBP2 is a novel tumor angiogenesis regulator that could promote tumor angiogenesis by selectively downregulating antiangiogenic gene expression.

Roquin1 inhibits the proliferation of breast cancer cells by inducing G1/S cell cycle arrest via selectively destabilizing the mRNAs of cell cycle-promoting genes.

BACKGROUND: Dysregulation of cell cycle progression is a common feature of human cancer cells; however, its mechanism remains unclear. This study aims to clarify the role and the underlying mechanisms of Roquin1 in cell cycle arrest in breast cancer. METHODS: Public cancer databases were analyzed to identify the expression pattern of Roquin1 in human breast cancers and its association with patient survival. Quantitative real-time PCR and Western blots were performed to detect the expression of Roquin1 in breast cancer samples and cell lines. Cell counting, MTT assays, flow cytometry, and in vivo analyses were conducted to investigate the effects of Roquin1 on cell proliferation, cell cycle progression and tumor progression. RNA sequencing was applied to identify the differentially expressed genes regulated by Roquin1. RNA immunoprecipitation assay, luciferase reporter assay, mRNA half-life detection, RNA affinity binding assay, and RIP-ChIP were used to explore the molecular mechanisms of Roquin1. RESULTS: We showed that Roquin1 expression in breast cancer tissues and cell lines was inhibited, and the reduction in Roquin1 expression was associated with poor overall survival and relapse-free survival of patients with breast cancer. Roquin1 overexpression inhibited cell proliferation and induced G1/S cell cycle arrest without causing significant apoptosis. In contrast, knockdown of Roquin1 promoted cell growth and cycle progression. Moreover, in vivo induction of Roquin1 by adenovirus significantly suppressed breast tumor growth and metastasis. Mechanistically, Roquin1 selectively destabilizes cell cycle-promoting genes, including Cyclin D1, Cyclin E1, cyclin dependent kinase 6 (CDK6) and minichromosome maintenance 2 (MCM2), by targeting the stem-loop structure in the 3' untranslated region (3'UTR) of mRNAs via its ROQ domain, leading to the downregulation of cell cycle-promoting mRNAs. CONCLUSIONS: Our findings demonstrated that Roquin1 is a novel breast tumor suppressor and could induce G1/S cell cycle arrest by selectively downregulating the expression of cell cycle-promoting genes, which might be a potential molecular target for breast cancer treatment.

A cluster of metabolism-related genes predict prognosis and progression of clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) has long been considered as a metabolic disease characterized by metabolic reprogramming due to the abnormal accumulation of lipid droplets in the cytoplasm. However, the prognostic value of metabolism-related genes in ccRCC remains unclear. In our study, we investigated the associations between metabolism-related gene profile and prognosis of ccRCC patients in the Cancer Genome Atlas (TCGA) database. Importantly, we first constructed a metabolism-related prognostic model based on ten genes (ALDH6A1, FBP1, HAO2, TYMP, PSAT1, IL4I1, P4HA3, HK3, CPT1B, and CYP26A1) using Lasso cox regression analysis. The Kaplan-Meier analysis revealed that our model efficiently predicts prognosis in TCGA_KIRC Cohort and the clinical proteomic tumor analysis consortium (CPTAC_ccRCC) Cohort. Using time-dependent ROC analysis, we showed the model has optimal performance in predicting long-term survival. Besides, the multivariate Cox regression analysis demonstrated our model is an independent prognostic factor. The risk score calculated for each patient was significantly associated with various clinicopathological parameters. Notably, the gene set enrichment analysis indicated that fatty acid metabolism was enriched considerably in low-risk patients. In contrast, the high-risk patients were more associated with non-metabolic pathways. In summary, our study provides novel insight into metabolism-related genes' roles in ccRCC.

MicroRNA-132 suppresses migration and invasion of renal carcinoma cells.

BACKGROUND: The aim of this study was to explain the effects of microRNA-132 in renal cell carcinoma by regulating FOXM1 expression. METHODS: Thirty patients with renal cell carcinoma admitted to our hospital were enrolled, and their adjacent normal tissues and cancer tissues were taken. The expression of microRNA-132 was measured by in situ hybridization (ISH) and RT-PCR, and the expression of FOXM1 was evaluated by RT-PCR and immunohistochemistry (IHC), and the correlation between microRNA-132 and FOXM1 was analyzed. In the cell experiment, the KETR-3 cells were divided into three groups: Negative control (NC) group were treated with nothing; blank (BL) group were transfected with empty vector; and microRNA-132 (miRNA) group were transfected with microRNA-132. The cell invasion and migration abilities among groups were assessed by transwell and wound healing assays. The expression levels of related proteins (FOXM1, MMP-2, MMP-9, VEGF-alpha, and uPAR) were determined by Western blot. RESULTS: Depending on clinical data, we found that FOXM1 protein expression of renal cell carcinoma tissues was higher than that in adjacent normal tissues. MiRNA-132 was negative correlation with FOXM1. In vitro, the number of invasive cells and wound healing rate in the microRNA group were significantly suppressed than those in the NC group (P < 0.05, respectively). In the Western blot assay, the results showed that the protein expression levels of FOXM1, MMP-2, MMP-9, VEGF-α, and uPAR were significantly inhibited in the miRNA group compared with the NC group (P < 0.05, respectively). CONCLUSION: miRNA-132 had anti-tumor effects in renal cell carcinoma by suppressing FOXM1 expression.

[Qilin Pills improve sperm quality in patients with oligozoospermia].

OBJECTIVE: To observe the therapeutic effect of Qilin Pills (QLP) on oligoasthenospermia. METHODS: This prospective study included 168 patients diagnosed with oligozoospermia in our hospital between September 2015 and September 2017, and all of them failed to achieve pregnancy. We randomly divided them into a QLP group (n = 82) and a placebo control group (n = 86) to receive oral QLPs or placebo pills, respectively, both at 6 g tid for 6 months. We followed up the patients and recorded their routine semen parameters every month during the medication and the rate of pregnancy in their spouses. RESULTS: After 6 months of treatment, the QLP group, as compared with the placebo controls, showed significantly improved sperm concentration (25.13 ×106/ml vs 11.62 ×106/ml, P < 0.01), grade a+b sperm (33.81% vs 17.32%, P < 0.01), grade a sperm (22.84% vs 13.56%, P < 0.01) and sperm motility (56.33% vs 26.23%, P < 0.01); and 26 pregnancies were achieved in the QLP group (32.91%), remarkably more than 13 in the placebo control group (15.85%) (P < 0.01). CONCLUSIONS: Qilin Pills can effectively improve sperm quality and increase the rate of pregnancy.

Gene expression profiles of glucose toxicity-exposed islet microvascular endothelial cells.

OBJECTIVE: Islet microcirculation is mainly composed by IMECs. The aim of the study was to investigate the differences in gene expression profiles of IMECs upon glucose toxicity exposure and insulin treatment. METHODS: IMECs were treated with 5.6 mmol L-1 glucose, 35 mmol L-1 glucose, and 35 mmol L-1 glucose plus 10-8  mol L-1 insulin, respectively. Gene expression profiles were determined by microarray and verified by qPCR. GO terms and KEGG analysis were performed to assess the potential roles of differentially expressed genes. The interaction and expression tendency of differentially expressed genes were analyzed by Path-Net algorithm. RESULTS: Compared with glucose toxicity-exposed IMECs, 1574 mRNAs in control group and 2870 mRNAs in insulin-treated IMECs were identified with differential expression, respectively. GO and KEGG pathway analysis revealed that these genes conferred roles in regulation of apoptosis, proliferation, migration, adhesion, and metabolic process etc. Additionally, MAPK signaling pathway and apoptosis were the dominant nodes in Path-Net. IMECs survival and function pathways were significantly changed, and the expression tendency of genes from euglycemia and glucose toxicity exposure to insulin treatment was revealed and enriched in 7 patterns. CONCLUSIONS: Our study provides a microcirculatory framework for gene expression profiles of glucose toxicity-exposed IMECs.

Monocyte chemotactic protein-induced protein 1 controls allergic airway inflammation by suppressing IL-5-producing TH2 cells through the Notch/Gata3 pathway.

BACKGROUND: Asthmatic and allergic inflammation is mediated by TH2 cytokines (IL-4, IL-5, and IL-13). Although we have learned much about how TH2 cells are differentiated, the TH2 checkpoint mechanisms remain elusive. OBJECTIVES: In this study we investigate how monocyte chemotactic protein-induced protein 1 (MCPIP1; encoded by the Zc3h12a gene) regulates IL-5-producing TH2 cell differentiation and TH2-mediated inflammation. METHODS: The functions of Zc3h12a-/- CD4 T cells were evaluated by checking the expression of TH2 cytokines and transcription factors in vivo and in vitro. Allergic airway inflammation of Zc3h12a-/- mice was examined with murine asthma models. In addition, antigen-specific CD4 T cells deficient in MCPIP1 were transferred to wild-type recipient mice, challenged with ovalbumin (OVA) or house dust mite (HDM), and accessed for TH2 inflammation. RESULTS: Zc3h12a-/- mice have spontaneous severe lung inflammation, with an increase in mainly IL-5- and IL-13-producing but not IL-4-producing TH2 cells in the lung. Mechanistically, differentiation of IL-5-producing Zc3h12a-/- TH2 cells is mediated through Notch signaling and Gata3 independent of IL-4. Gata3 mRNA is stabilized in Zc3h12a-/- TH2 cells. MCPIP1 promotes Gata3 mRNA decay through the RNase domain. Furthermore, deletion of MCPIP1 in OVA- or HDM-specific T cells leads to significantly increased TH2-mediated airway inflammation in OVA or HDM murine models of asthma. CONCLUSIONS: Our study reveals that MCPIP1 regulates the development and function of IL-5-producing TH2 cells through the Notch/Gata3 pathway. MCPIP1 represents a new and promising target for the treatment of asthma and other TH2-mediated diseases.

MCPIP1 Selectively Destabilizes Transcripts Associated with an Antiapoptotic Gene Expression Program in Breast Cancer Cells That Can Elicit Complete Tumor Regression.

The ability of cancer cells to evade apoptosis is dictated by a shift in the balance between proapoptotic and antiapoptotic gene expression programs. Monocyte chemotactic protein-induced protein 1 (MCPIP1) is a zinc-finger RNA binding protein with important roles in mediating inflammatory responses. Overexpression of MCPIP1 in different cancer cell types has been implicated in eliciting an antitumor response, but a direct role of MCPIP1 in apoptosis has not been established. In this study, we demonstrate that MCPIP1 functions as a potent tumor suppressor that induces apoptosis of breast tumor cells by selectively enhancing mRNA decay of antiapoptotic gene transcripts, including Bcl2L1, Bcl2A1, RelB, Birc3, and Bcl3. Mechanistically, MCPIP1 physically interacted with a stem-loop structure in the 3' untranslated region of these transcripts through its PIN domain, causing mRNA destabilization. Furthermore, we found that MCPIP1 expression was repressed in breast tumor cells, and overexpression of MCPIP1 induced apoptosis, whereas its depletion enhanced cancer cell proliferation. Moreover, MCPIP1 induction in vivo resulted in complete regression of established tumors and a significant reduction in metastatic disease. Notably, low MCPIP1 expression in tumor samples from breast cancer patients was strongly associated with poor survival over 13 years of follow-up. Collectively, our results highlight that MCPIP1 is a new tumor suppressor in breast cancer that induces cell death by tipping the balance in favor of proapoptotic gene expression.

Tristetraprolin induces cell cycle arrest in breast tumor cells through targeting AP-1/c-Jun and NF-κB pathway.

The main characteristic of cancers, including breast cancer, is the ability of cancer cells to proliferate uncontrollably. However, the underlying mechanisms of cancer cell proliferation, especially those regulated by the RNA binding protein tristetraprolin (TTP), are not completely understood. In this study, we found that TTP inhibits cell proliferation in vitro and suppresses tumor growth in vivo through inducing cell cycle arrest at the S phase. Our studies demonstrate that TTP inhibits c-Jun expression through the C-terminal Zn finger and therefore increases Wee1 expression, a regulatory molecule which controls cell cycle transition from the S to the G2 phase. In contrast to the well-known function of TTP in regulating mRNA stability, TTP inhibits c-Jun expression at the level of transcription by selectively blocking NF-κB p65 nuclear translocation. Reconstitution of NF-κB p65 completely abolishes the inhibition of c-Jun transcription by TTP. Moreover, reconstitution of c-Jun in TTP-expressing breast tumor cells diminishes Wee1 overexpression and promotes cell proliferation. Our results indicate that TTP suppresses c-Jun expression that results in Wee1 induction which causes cell cycle arrest at the S phase and inhibition of cell proliferation. Our study provides a new pathway for TTP function as a tumor suppressor which could be targeted in tumor treatment.

Melatonin inhibits IL1ß-induced MMP9 expression and activity in human umbilical vein endothelial cells by suppressing NF-κB activation.

Matrix metalloproteinases (MMPs) have been involved in inflammatory and degradative processes in pathologic conditions. The purpose of this study was to investigate the protective effect of melatonin in human umbilical vein endothelial cell (HUVEC) monolayer permeability and the regulation of MMP9 induced by interleukin 1ß (IL1ß (IL1B)) in HUVECs. Protection studies were carried out with melatonin, a well-known antioxidant and antiinflammatory molecule. MMP9 expression was increased with IL1ß induction in HUVECs. Melatonin showed a barrier-protective role by downregulation of MMP9 and upregulation of tissue inhibitor of metalloproteinase-1 expression in HUVECs. Meanwhile, melatonin also decreased sodium fluorescein permeability and counteracted the downregulation of vascular endothelial cadherin and occludin expression in HUVECs. During inflammatory stimulus, nuclear factor-κB (NF-κB) plays a significant role in regulating MMP genes expression, thus the function of NF-κB in HUVECs' barrier disruption was investigated. IL1ß induced nuclear translocation of NF-κB in HUVECs and regulated MMP9 expression. However, NF-κB translocation into the nucleus was inhibited significantly by melatonin. Our results show that melatonin decreases the permeability of monolayer endothelial cell induced by IL1ß. At the same time, melatonin decreased the expression and activity of MMP9 by a NF-κB-dependent pathway in HUVECs induced by IL1ß.