MicroRNA-10b Promotes Migration and Invasion through KLF4 in Human Esophageal Cancer Cell Lines

Recently, microRNAs have emerged as regulators of cancer metastasis through acting on multiple signaling pathways involved in metastasis. In this study, we have analyzed the level of miR-10b and cell motility and invasiveness in several human esophageal squamous cell carcinoma cell lines. Our results reveal a significant correlation of miR-10b level with cell motility and invasiveness. Overexpression of miR-10b in KYSE140 cells increased cell motility and invasiveness, whereas inhibition of miR-10b in EC9706 cells reduced cell invasiveness, although it did not alter cell motility. Additionally, we identified KLF4, a known tumor suppressor gene that has been reported to suppress esophageal cancer cell migration and invasion, as a direct target of miR-10b. Furthermore, overexpression of miR-10b in KYSE140 and KYSE450 cells led to a reduction of endogenous KLF4 protein, whereas silencing of miR-10b in EC9706 cells caused up-regulation of KLF4 protein. Coexpression of miR-10b and KLF4 in KYSE140 cells and coexpression of small interfering RNA for KLF4 mRNA and miR-10b-AS in EC9706 cells partially abrogated the effect of miR-10b on cell migration and invasion. Finally, analyses of the miR-10b level in 40 human esophageal cancer samples and their paired normal adjacent tissues revealed an elevated expression of miR-10b in 95% (38 of 40) of cancer tissues, although no significant correlation of the miR-10b level with clinical metastasis status was observed in these samples.     

Klf4 haploinsufficiency in Sp7+ lineage leads to underdeveloped mandibles and insufficient elongation of mandibular incisor

The mandible is an important component of the craniofacial bones, whose development is regulated by complex molecular networks and involves the well-coordinated development of the bone, cartilage, and teeth. Previously, we demonstrated that Krüppel-like factor 4 (KLF4) promoted dentinogenesis and osteogenesis, but it was enigmatic whether Klf4 participated in the development of the mandible. In this study, the Sp7-Cre; Klf4^f/+ mice exhibited underdeveloped mandibles and insufficient elongation of the mandibular incisor when compared with Klf4^f/+ and Sp7-Cre mice. Moreover, morphological and molecular analysis showed that the alveolar bone mass was significantly decreased in KLF4 deficient mice, accompanied by reduced expression of osteoblast-related genes. Meanwhile, the KLF4 deficient mice had decreased expression of receptor activator of nuclear factor kappa-Β ligand (RANKL) and no significant change of osteoprotegerin (OPG) in the alveolar bone near the mandibular incisor. Simultaneously, the osteoclastogenesis in the alveolar bone of KLF4 deficient mice was attenuated, which was demonstrated by a diminished number of tartrate-resistant acid phosphatase positive (TRAP+), matrix metallopeptidase 9 positive (MMP9+), and cathepsin K positive (CTSK+) multinucleated osteoclasts, respectively. Collectively, our study suggested that Klf4 participated in mandibular development, and Klf4 in Sp7+ lineage affected osteogenesis directly and osteoclastogenesis indirectly.     

Methylseleninic acid activates Keap1/Nrf2 pathway via up-regulating miR-200a in human oesophageal squamous cell carcinoma cells

Oesophageal squamous cell carcinoma (ESCC) occurs at a very high rates in certain regions of China. There are increasing evidences demonstrating that selenium could act as a potential anti-oesophageal cancer agent, but the precise mechanisms involved are still not completely understood. Methylseleninic acid (MSA), as a potent second-generation selenium compound, is a promising chemopreventive agent. Previous studies demonstrated that the kelch-like ECH-associated protein 1 (Keap1)/nuclear factor E2-related factor 2 (Nrf2) system plays a critical role in cancer prevention, but little is known about its association with MSA in ESCC cells. In the present study, we observed that MSA treatment significantly down-regulated Keap1, induced nuclear accumulation of Nrf2 and enhance the antioxidant response element (ARE) promoter activity in ESCC cells. MSA could also significantly induce miR-200a expression and inhibit Keap1 directly. Antagomir-200a could attenuate MSA treatment-induced Keap1 down-regulation in ESCC cells. Moreover, MSA-induced miR-200a expression was dependent on the mediation of Krüpple-like factor 4 (KLF4). These results reaffirm the potential role of MSA as a chemopreventive agent via the regulation of KLF4/miR-200a/Keap1/Nrf2 axis in ESCC cells.     

Antagonistic Interplay between MicroRNA-155 and IL-10 during Lyme Carditis and Arthritis

MicroRNA-155 has been shown to play a role in immune activation and inflammation, and is suppressed by IL-10, an important anti-inflammatory cytokine. The established involvement of IL-10 in the murine model of Borrelia burgdorferi-induced Lyme arthritis and carditis allowed us to assess the interplay between IL-10 and miR-155 in vivo. As reported previously, Mir155 was highly upregulated in joints from infected severely arthritic B6 Il10^-/- mice, but not in mildly arthritic B6 mice. In infected hearts, Mir155 was upregulated in both strains, suggesting a role of miR-155 in Lyme carditis. Using B. burgdorferi-infected B6, Mir155^-/-, Il10^-/-, and Mir155^-/- Il10^-/- double-knockout (DKO) mice, we found that anti-inflammatory IL-10 and pro-inflammatory miR-155 have opposite and somewhat compensatory effects on myeloid cell activity, cytokine production, and antibody response. Both IL-10 and miR-155 were required for suppression of Lyme carditis. Infected Mir155^-/- mice developed moderate/severe carditis, had higher B. burgdorferi numbers, and had reduced Th1 cytokine expression in hearts. In contrast, while Il10^-/- and DKO mice also developed severe carditis, hearts had reduced bacterial numbers and elevated Th1 and innate cytokine expression. Surprisingly, miR-155 had little effect on Lyme arthritis. These results show that antagonistic interplay between IL-10 and miR-155 is required to balance host defense and immune activation in vivo, and this balance is particularly important for suppression of Lyme carditis. These results also highlight tissue-specific differences in Lyme arthritis and carditis pathogenesis, and reveal the importance of IL-10-mediated regulation of miR-155 in maintaining healthy immunity.     

Dysregulation of Protease and Protease Inhibitors in a Mouse Model of Human Pelvic Organ Prolapse

Mice deficient for the fibulin-5 gene (Fbln5^−/−) develop pelvic organ prolapse (POP) due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP)-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5^−/− mice, herein named V1 (25 kDa). V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS) 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5^−/− mice. PRSS3 was (a) localized in epithelial secretions, (b) detected in media of vaginal organ culture from both Fbln5^−/− and wild type mice, and (c) cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin) and Elafin] was dysregulated in Fbln5^−/− epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice.     

Kruppel-Like Factor 2-Mediated Suppression of MicroRNA-155 Reduces the Proinflammatory Activation of Macrophages

Objective

Recent evidence indicates that significant interactions exist between Kruppel-like factor 2 (KLF2) and microRNAs (miRNAs) in endothelial cells. Because KLF2 is known to exert anti-inflammatory effects and inhibit the pro-inflammatory activation of monocytes, we sought to identify how inflammation-associated miR-155 is regulated by KLF2 in macrophages.

Approach and Results

Peritoneal macrophages from wild-type (WT) C57Bl/6 mice were transfected with either recombinant adenovirus vector expressing KLF2 (Ad-KLF2) or siRNA targeting KLF2 (KLF2-siRNA) for 24 h–48 h, then stimulated with oxidized low-density lipoproteins (ox-LDL, 50 μg/mL) for 24 h. Quantitative real-time polymerase chain reaction showed that KLF2 markedly reduced the expression of miR-155 in quiescent/ox-LDL-stimulated macrophages. We also found that the increased expression of miR-155, monocyte chemoattractant protein (MCP-1) and interleukin (IL)-6 and the decreased expression of the suppressor of cytokine signaling (SOCS)-1 and IL-10 in ox-LDL-treated macrophages were significantly suppressed by KLF2. Most importantly, over-expression of miR-155 could partly reverse the suppressive effects of KLF2 on the inflammatory response of macrophages. Conversely, the suppression of miR-155 in KLF2 knockdown macrophages significantly overcame the pro-inflammatory properties associated with KLF2 knockdown. Finally, Ad-KLF2 significantly attenuated the diet-induced formation of atherosclerotic lesions in apolipoprotein E-deficient (apoE^-/-) mice, which was associated with a significantly reduced expression of miR-155 and its relative inflammatory cytokine genes in the aortic arch and in macrophages.

Conclusion

KLF2-mediated suppression of miR-155 reduced the inflammatory response of macrophages.     

MicroRNA-146a Provides Feedback Regulation of Lyme Arthritis but Not Carditis during Infection with Borrelia burgdorferi

MicroRNAs have been shown to be important regulators of inflammatory and immune responses and are implicated in several immune disorders including systemic lupus erythematosus and rheumatoid arthritis, but their role in Lyme borreliosis remains unknown. We performed a microarray screen for expression of miRNAs in joint tissue from three mouse strains infected with Borrelia burgdorferi. This screen identified upregulation of miR-146a, a key negative regulator of NF-κB signaling, in all three strains, suggesting it plays an important role in the in vivo response to B. burgdorferi. Infection of B6 miR-146a^−/− mice with B. burgdorferi revealed a critical nonredundant role of miR-146a in modulating Lyme arthritis without compromising host immune response or heart inflammation. The impact of miR-146a was specifically localized to the joint, and did not impact lesion development or inflammation in the heart. Furthermore, B6 miR-146a^−/− mice had elevated levels of NF-κB-regulated products in joint tissue and serum late in infection. Flow cytometry analysis of various lineages isolated from infected joint tissue of mice showed that myeloid cell infiltration was significantly greater in B6 miR-146a^−/− mice, compared to B6, during B. burgdorferi infection. Using bone marrow-derived macrophages, we found that TRAF6, a known target of miR-146a involved in NF-κB activation, was dysregulated in resting and B. burgdorferi-stimulated B6 miR-146a^−/− macrophages, and corresponded to elevated IL-1β, IL-6 and CXCL1 production. This dysregulated protein production was also observed in macrophages treated with IL-10 prior to B. burgdorferi stimulation. Peritoneal macrophages from B6 miR-146a^−/− mice also showed enhanced phagocytosis of B. burgdorferi. Together, these data show that miR-146a-mediated regulation of TRAF6 and NF-κB, and downstream targets such as IL-1β, IL-6 and CXCL1, are critical for modulation of Lyme arthritis during chronic infection with B. burgdorferi.     

Circ_0004015 silencing represses cisplatin chemoresistance and tumor progression by reducing KLF8 in a miR-198-dependent manner in non-small cell lung cancer

Circular RNA (circRNA) plays vital roles in diverse cancer progression, including non-small cell lung cancer (NSCLC). Herein, the role of circ_0004015 in regulating the sensitivity of NSCLC to cisplatin (DDP) is revealed. The RNA expression of circ_0004015, microRNA-198 (miR-198) and kruppel like factor 8 (KLF8) was detected by quantitative real-time polymerase chain reaction. Protein expression was checked by western blot. The half maximal inhibitory concentration of DDP and cell proliferation were determined by cell counting kit-8 assay. Cell colony formation ability, migration, invasion and apoptosis were investigated by colony-forming assay, transwell assay and flow cytometry analysis, respectively. The effect of circ_0004015 knockdown on DDP sensitivity in vivo was demonstrated by mouse model assay. The interactions among circ_0004015, miR-198 and KLF8 were predicted by bioinformatics methods, and identified by mechanism assays. The expression of circ_0004015 and KLF8 was apparently upregulated, while miR-198 expression was downregulated in DDP-resistant NSCLC tissues and cells compared with control groups. Additionally, circ_0004015 silencing repressed DDP resistance, cell proliferation, migration and invasion, but induced cell apoptosis in DDP-resistant NSCLC cells. Circ_0004015 knockdown promoted the effect of DDP on tumor formation in vivo. Also, miR-198 inhibitors attenuated circ_0004015 depletion-mediated action though associating with circ_0004015. MiR-198 regulated DDP sensitivity and NSCLC progression by targeting KLF8. Furthermore, circ_0004015 modulated KLF8 expression through interaction with miR-198. Circ_0004015 conferred DDP resistance and promoted NSCLC progression by miR-198/KLF8 pathway, proving a potential target for studying DDP-mediated treatment of NSCLC.     

Expression of newly identified secretory CEACAM1 isoforms in the intestinal epithelium

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) regulates intestinal immunological homeostasis. However, precise expression patterns of CEACAM1 isoforms remain poorly understood in the intestinal epithelia. Focusing on the small intestinal epithelium of BALB/c mice, we identified three novel splice variants encoding CEACAM1^a-2, -2C1, and -4C1 by RT-PCR. CEACAM1^a-2, -2C1, and -4C1 demonstrated secretory properties by transfection experiments in vitro. Among them, CEACAM1^a-4C1 was the major secreted isoform in vivo due to the soluble/secreted CEACAM1^a with a frameshift sequence in the C-terminus, specific for CEACAM1^a-2C1 and -4C1. CEACAM1^a-4C1 was capable of binding murine hepatitis virus (MHV) and was detected at approximately 120 kDa in the small intestinal secretions. Neutralizing effects of the soluble CEACAM1^a on MHV infectivity in vitro were demonstrated by using recombinant CEACAM1^a-4C1. Our data suggest an intrinsic mechanism operated by free CEACAM1 for surveillance of pathogens and maintenance of homeostasis in the intestine.     

Transgenic Expression of miR-222 Disrupts Intestinal Epithelial Regeneration by Targeting Multiple Genes Including Frizzled-7

Defects in intestinal epithelial integrity occur commonly in various pathologies. miR-222 is implicated in many aspects of cellular function and plays an important role in several diseases, but its exact biological function in the intestinal epithelium is underexplored. We generated mice with intestinal epithelial tissue-specific overexpression of miR-222 to investigate the function of miR-222 in intestinal physiology and diseases in vivo. Transgenic expression of miR-222 inhibited mucosal growth and increased susceptibility to apoptosis in the small intestine, thus leading to mucosal atrophy. The miR-222–elevated intestinal epithelium was vulnerable to pathological stress, since local overexpression of miR-222 not only delayed mucosal repair after ischemia/reperfusion-induced injury, but also exacerbated gut barrier dysfunction induced by exposure to cecal ligation and puncture. miR-222 overexpression also decreased expression of the Wnt receptor Frizzled-7 (FZD7), cyclin-dependent kinase 4 and tight junctions in the mucosal tissue. Mechanistically, we identified the Fzd7 messenger ribonucleic acid (mRNA) as a novel target of miR-222 and found that [miR-222/Fzd7 mRNA] association repressed Fzd7 mRNA translation. These results implicate miR-222 as a negative regulator of normal intestinal epithelial regeneration and protection by downregulating expression of multiple genes including the Fzd7. Our findings also suggest a novel role of increased miR-222 in the pathogenesis of mucosal growth inhibition, delayed healing and barrier dysfunction.     

Aurka deficiency in the intestinal epithelium promotes age-induced obesity via propionate-mediated AKT activation

Aurora-A kinase, a serine/threonine mitotic kinase involved in mitosis, is overexpressed in several human cancers. A recent study showed that Aurora-A mediates glucose metabolism via SOX8/FOXK1 in ovarian cancer. However, the roles of Aurora-A in metabolic diseases remain unclear. This study found that Aurka loss in the intestinal epithelium promoted age-induced obesity and enlargement of lipid droplets in parallel with an increase in infiltrated macrophages in the white adipocyte tissue (WAT) of male mice. Moreover, loss of Aurka induced the expression of lipid metabolism regulatory genes, including acetyl-coenzyme A carboxylase 1 (Acc1), in association with an increase in the levels of p-AKT in the intestinal epithelium as well as WAT. Blockade of AKT activation reduced the expression of lipid metabolism regulatory genes. In subsequent experiments, we found that the Firmicutes abundance and the levels of short-chain fatty acids (SCFAs) in the gut were dramatically increased in Aurka^f/+;Villin^Cre/+ mice compared with Aurka^f/+ mice. Additionally, propionate increased the phosphorylation of AKT in vitro. These observations indicated that Aurka loss in the intestinal epithelium contributed to gut microbiota dysbiosis and higher levels of SCFAs, especially propionate, leading to AKT activation and lipid metabolism regulatory gene expression, which in turn promoted age-induced obesity.     

Identification of miR-200a as a novel suppressor of connexin 43?in breast cancer cells

Both miRNAs (miRs) and connexin 43 (Cx43) were important regulators of the metastasis of breast cancer, whereas the miRs regulating Cx43 expression in breast cancer cells were still obscure. In the present study, we scanned and found miR-1, miR-206, miR-200a, miR-381, miR-23a/b and miR-186 were functional suppressors of human Cx43 mRNA and protein expression. Specially, we demonstrated that only miR-200a could directly target the 3′-untranslated region (3′-UTR) of human Cx43 gene. Functionally, overexpression of Cx43 in MCF cells potentiated the migration activity, whereas additional miR-200a treatment notably prevented this effect. Finally, we demonstrated that decreased levels of miR-200a and elevated expression of Cx43 in the metastatic breast cancer tissues compared with the primary ones. Thus, we are the first to identify miR-200a as a novel and direct suppressor of human Cx43, indicating that miR200a/Cx43 axis might be a useful diagnostic and therapeutic target of metastatic breast cancer.     

MicroRNA-155 Regulates Cell Survival,Growth, and Chemosensitivity by Targeting FOXO3a in Breast Cancer

Breast cancer is the second leading cause of cancer death in women. Despite improvement in treatment over the past few decades, there is an urgent need for development of targeted therapies. miR-155 (microRNA-155) is frequently up-regulated in breast cancer. In this study, we demonstrate the critical role of miR-155 in regulation of cell survival and chemosensitivity through down-regulation of FOXO3a in breast cancer. Ectopic expression of miR-155 induces cell survival and chemoresistance to multiple agents, whereas knockdown of miR-155 renders cells to apoptosis and enhances chemosensitivity. Further, we identified FOXO3a as a direct target of miR-155. Sustained overexpression of miR-155 resulted in repression of FOXO3a protein without changing mRNA levels, and knockdown of miR-155 increases FOXO3a. Introduction of FOXO3a cDNA lacking the 3′-untranslated region abrogates miR-155-induced cell survival and chemoresistance. Finally, inverse correlation between miR-155 and FOXO3a levels were observed in a panel of breast cancer cell lines and tumors. In conclusion, our study reveals a molecular link between miR-155 and FOXO3a and presents evidence that miR-155 is a critical therapeutic target in breast cancer.