Clcn3 deficiency ameliorates high-fat diet-induced obesity and adipose tissue macrophage inflammation in mice.

Obesity induces accumulation of adipose tissue macrophages (ATMs) and ATM-driven inflammatory responses that promote the development of glucose and lipid metabolism disorders. ClC-3 chloride channel/antiporter, encoded by the Clcn3, is critical for some basic cellular functions. Our previous work has shown significant alleviation of type 2 diabetes in Clcn3 knockout (Clcn3-/-) mice. In the present study we investigated the role of Clcn3 in high-fat diet (HFD)-induced obesity and ATM inflammation. To establish the mouse obesity model, both Clcn3-/- mice and wild-type mice were fed a HFD for 4 or 16 weeks. The metabolic parameters were assessed and the abdominal total adipose tissue was scanned using computed tomography. Their epididymal fat pad tissue and adipose tissue stromal vascular fraction (SVF) cells were isolated for analyses. We found that the HFD-fed Clcn3-/- mice displayed a significant decrease in obesity-induced body weight gain and abdominal visceral fat accumulation as well as an improvement of glucose and lipid metabolism as compared with HFD-fed wild-type mice. Furthermore, the Clcn3 deficiency significantly attenuated HFD-induced ATM accumulation, HFD-increased F4/80+ CD11c+ CD206- SVF cells as well as HFD-activated TLR-4/NF-κB signaling in epididymal fat tissue. In cultured human THP-1 macrophages, adenovirus-mediated transfer of Clcn3 specific shRNA inhibited, whereas adenovirus-mediated cDNA overexpression of Clcn3 enhanced lipopolysaccharide-induced activation of NF-κB and TLR-4. These results demonstrate a novel role for Clcn3 in HFD-induced obesity and ATM inflammation.

MiR-1-3p that correlates with left ventricular function of HCM can serve as a potential target and differentiate HCM from DCM.

BACKGROUND: MicroRNAs (miRNAs) are non-coding RNAs that function as regulators of gene expression and thereby contribute to the complex disease phenotypes. Hypertrophic cardiomyopathy (HCM) and Dilated cardiomyopathy (DCM) can cause sudden cardiac death and eventually develop into heart failure. However, they have different clinical and pathophysiological phenotype and the expressional spectrum of miRNAs in left ventricles of HCM and DCM has never been compared before. METHODS: This study selected 30 human left ventricular heart samples belonged to three diagnostic groups (Control, HCM, DCM). Each group has ten samples. Based on previous findings, the expression of 13 different microRNAs involving heart failure and hypertrophy (miR-1-3p, miR-10b, miR-21, miR-23a, miR-27a, miR-29a, miR-133a-3p, miR-142-3p, miR-155, miR-199a-3p, miR-199a-5p, miR-214, miR-497) was measured. 17 HCM patients were included as second group to validate the associations. RESULTS: We found miR-155, miR-10b and miR-23a were highly expressed in both HCM and DCM compared with control. MiR-214 was downregulated and miR-21 was upregulated in DCM but not in HCM. We also identified miR-1-3p and miR-27a expressed significantly different between HCM and DCM and both miRNAs downregulated in HCM. And only miR-1-3p correlated with left ventricular end diastolic diameter (LVEDD) and left ventricular ejection fraction (LVEF) that reflected the cardiac function in HCM. A second HCM group also confirmed this correlation. We then predicted Chloride voltage-gated channel 3 (Clcn3) as a direct target gene of miR-1-3p using bioinformatics tools and confirmed it by Luciferase reporter assay. CONCLUSION: Our data demonstrated that different cardiomyopathies had unique miRNA expression pattern. And the expression levels of miR-1-3p and miR-27a had disease-specificity and sensitivity in HCM, whereas only miR-1-3p was significantly associated with left ventricular function in HCM identifying it as a potential target to improve the cardiac function in end-stage HCM. We also provide Clcn3 as a direct target of miR-1-3p which sheds light on the mechanism of HCM.

Overexpression of chloride channel-3 is associated with the increased migration and invasion ability of ectopic endometrial cells from patients with endometriosis.

STUDY QUESTION: Is chloride channel-3 (ClC-3) involved in regulating the biological behavior of endometrial stromal cells (ESCs)? SUMMARY ANSWER: ClC-3 promotes endometriotic cell migration and invasion. WHAT IS KNOWN ALREADY: ClC-3 plays a significant role in the migration and invasion of various kinds of cells. STUDY DESIGN, SIZE, DURATION: An ITALIC! in vitro investigation of the effect of ClC-3 on the migration and invasion of ectopic ESCs from patients with endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: The ectopic and eutopic endometrial samples from 43 female patients with endometriosis and the endometrial samples from 39 non-endometriotic female patients were collected. Primary cells from these samples were isolated and cultured. Real-time RT-PCR, immunohistochemistry and western blot were used to detect the expression of ClC-3 and matrix metalloproteinase 9 (MMP-9). Small interfering RNA (siRNA) technology was employed to knock down ClC-3 expression. The migration and invasion ability of ESCs was measured by the transwell assay with uncoated or Matrigel-coated membranes. MAIN RESULTS AND THE ROLE OF CHANCE: The expression of ClC-3 mRNA and proteins was significantly up-regulated in the ectopic tissues from endometriotic patients, while that in the eutopic endometrial tissues of the same patients did not significantly differ from that in non-endometriotic patients. The migration and invasion ability and MMP-9 expression was increased in the ESCs from ectopic endometrial tissues. The knockdown of ClC-3 expression by ClC-3 siRNA inhibited ESC migration and invasion and attenuated the expression of MMP-9. ClC-3 expression level was well-correlated to the clinical characteristics and symptoms of endometriosis patients, including infertility, dysmenorrhea, chronic pelvic pain, dyspareunia and diameter of endometriosis lesion. LIMITATIONS, REASONS FOR CAUTION: Further studies are needed to examine the regulatory mechanism of estrogen on ClC-3 expression of ESCs. WIDER IMPLICATIONS OF THE FINDINGS: ClC-3 is involved in the migration and invasion processes of ESCs and can regulate MMP-9 expression. Up-regulation of ClC-3 expression may contribute to endometriosis development by regulating MMP-9 expression. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the National Natural Science Foundation of China (81173064, 81272223, 81273539), the Ministry of Education of China (20124401110009), the Natural Science Foundation of Guangdong Province (S2011010001589) and the Science and Technology Programs of Guangdong (2013B051000059), Guangzhou (2013J500015) and Dongguan (2011108102006). The authors have no conflict of interest.

Clcn3 deficiency ameliorates high-fat diet-induced obesity and improves metabolism in mice.

Objective: Obesity is defined as excess body fat and is a current health epidemic associated with increased risk for type 2 diabetes and cardiovascular disease. The ClC-3 chloride channel/antiporter, encoded by the Clcn3, is associated with some diseases, like carcinoma, nervous system diseases, and metabolic diseases. To verify the relationship between the Clcn3 and weight including metabolic changes, searching for a new target for metabolic therapy of obesity, we designed the experiment. Methods: The mice were divided into 4 different groups: Clcn3+/+ mice + high-fat diet (HFD), Clcn3-/- mice + HFD, Clcn3+/+ mice + normal diet (ND), Clcn3-/- mice + ND, and fed for 16 weeks. After the glucose tolerance test and insulin tolerance test, peripheral blood and adipose tissues were collected. Moreover, we performed transcriptome sequencing for the epididymal white adipose tissue from Clcn3+/+ and Clcn3-/- mice with the high-fat diet. Western blotting verified the changes in protein levels of relevant metabolic genes. Results: We found that the Clcn3-/- mice had lower body weight and visceral fat, refining glucose and lipid metabolism in HFD-induced mice, but had no effect in normal diet mice. RNA-seq and Western blotting indicated that Clcn3 deficiency may inhibit obesity through the AMPK-UCP1 axis. Conclusion: Modulation of Clcn3 may provide an appealing therapeutic target for obesity and associated metabolic syndrome.

CLCN3 in mediating the proliferation of human ovarian cancer cells.

Background: Chloride channel-3 (CLCN3), a crucial component of the voltage-gated chloride channel family, is implicated in numerous physiological and pathophysiological processes. This study aimed to investigate the characteristics of CLCN3 in pancancer and its influence on the immune response through the use of a range of databases. Concurrently, we assessed the impact of CLCN3 on the proliferation of ovarian cancer (OC) cells and explored its potential mechanisms. Methods: We employed the Tumor Immune Estimation Resource (TIMER) 2.0 and Clinical Proteomic Tumor Analysis Consortium (CPTAC) databases to examine the messenger RNA (mRNA) and the protein expression of CLCN3 across various cancers. The prognostic significance of CLCN3 was evaluated using the Gene Expression Profiling Interactive Analysis 2.0 (GEPIA 2.0) database. The University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) facilitated the analysis of CLCN3 promoter methylation levels. The association between CLCN3 expression and tumor-infiltrating immune cells was investigated using various algorithms. The cBioportal database facilitated the analysis of CLCN3 mutations and mutation sites across various cancers. The Tumor-Immune System Interactions Database (TISIDB) database was employed to explore the correlation between CLCN3 expression and immune or molecular subtypes across a variety of cancer types. We collected ovarian tissue samples, encompassing both normal ovarian and OC tissues. The human OC cell lines, SKOV3 cells and OVCAR433 cells, were cultured. CLCN3 expression was determined via reverse-transcription quantitative polymerase chain reaction (RT-qPCR), while phosphatidylinositol 3-kinase/Akt kinase (PI3K/AKT) expression was detected using Western blot. We utilized small interfering RNA (siRNA) technology to suppress CLCN3 expression. The proliferative capacity of SKOV3 and OVCAR433 cells was assessed using the Cell Counting Kit 8 (CCK-8) assay. Results: CLCN3 demonstrated an aberrant expression in a number of cancer types and was markedly reduced in OC tissues. Poor prognosis in cervical squamous cell cancer and myeloid leukemia was linked to excessive expression of CLCN3. The examination of immune cell infiltration, which included CD8+ T cells, B cells, T regulatory cells, and cancer-associated fibroblast cells, showed a strong association with aberrant CLCN3 expression. Following the use of siRNA technology, the ability of the ovarian carcinoma cell line SKOV3 and OVCAR433 to proliferate as well as the expression of PI3K/AKT both increased. Conclusions: CLCN3 is a possible biomarker for immune-related processes and the prognosis of cancer, and the PI3K/AKT signaling pathway may affect OC cells' ability to proliferate.

HNRNPK/CLCN3 axis facilitates the progression of LUAD through CAF-tumor interaction.

Background: Chloride channel 3 (CLCN3) is regulated by transcription-coactivator, however, it is unclear which core transcription factor regulates CLCN3. The role of CLCN3 in lung adenocarcinoma (LUAD) is unexplored and the relationship between CLCN3 and tumor microenvironment is unknown. Methods: A 5'-biotin-labeled promoter probe of CLCN3 was used to pull down the promoter-binding transcription factor. Further study was investigated using LUAD samples, cell lines, and xenograft mice models, and the mechanism was explored. Results: CLCN3 was upregulated in human LUAD, and CLCN3 knockdown inhibited tumor proliferation and migration in vitro. Next, heterogeneous nuclear ribonucleoprotein K (HNRNPK) was first validated as a CLCN3 promoter-binding transcription factor. Mechanistically, HNRNPK knockdown suppressed the promoter activity of CLCN3, thus regulating CLCN3 expression at the transcriptional level, and the binding motif 'GCGAGG' and binding site '-538/-248 bp' were identified. Subsequently, the RNA-seq data illustrated that the primary functions of HNRNPK were similar to those of CLCN3. The results from in vitro and in vivo trials indicated that the expression and function of CLCN3 were regulated by HNRNPK. By isolating primary cancer-associated fibroblasts (CAFs) from human LUAD, we confirmed that decreased extracellular CLCN3 secretion induced by HNRNPK knockdown inhibited CAFs activation and TGF-ß1 production, thus suppressing nuclear HNRNPK expression and LUAD progression in a feedback way. Furthermore, this phenomenon was rescued after the addition of TGF-ß1, revealing that the HNRNPK/CLCN3 axis facilitated LUAD progression through intercellular interactions. Finally, we identified that CLCN3 and HNRNPK were upregulated and correlated with poor prognosis in LUAD patients. Conclusions: HNRNPK/CLCN3 axis facilitates the progression of LUAD through CAF-tumor interaction.

miR-24 protects against ischemia-induced brain damage in rats via regulating microglia polarization by targeting Clcn3.

Microglia and macrophages play important roles in ischemic brain injury. Changes in their M1/M2 polarization phenotypes significantly impact disease progression. The M2 microglia/macrophages are anti-inflammatory and have a protective effect against ischemic injury. The microRNA 24 (miR-24) promotes M2 macrophage polarization and suppresses inflammation. We tested the hypothesis that miR-24 is protective in ischemic brain injury by regulating microglia polarization. We treated rats with miR-24 inhibitor or mimic and subsequently subjected the rats to middle cerebral artery occlusion (MCAO) to induce ischemic brain injury. Neurological deficit and infarct volume were analyzed. Microglia and macrophages were assessed by fluorescence-activated cell sorting. Microglia polarization was determined by genes specific for M1 and M2 both in vivo and in BV-2 cells. The effect of miR-24 target Clcn3 on microglia polarization was examined. We found that miR-24 inhibition aggravated MCAO induced damage, while miR-24 overexpression alleviated brain injury by suppressing microglia/macrophage infiltration. miR-24 suppressed M1 and promoted M2 microglia polarization both in vivo and in vitro. Finally, we showed that miR-24 targeted Clcn3 to regulate microglia polarization. Our study indicates that miR-24 plays a neuroprotective role by promoting anti-proinflammatory microglia polarization during ischemic brain injury.

MicroRNA-205-5p inhibits three-dimensional spheroid proliferation of ErbB2-overexpressing breast epithelial cells through direct targeting of CLCN3.

We previously reported that microRNA-205-5p (miR-205-5p) is significantly decreased in the ErbB2-overexpressing breast epithelial cell line MCF10A-ErbB2 compared with control cells. In this study, we identified a direct target of miR-205-5p, chloride voltage-gated channel 3 (CLCN3). CLCN3 expression was induced by ErbB2 overexpression; this induced expression was then reduced to control levels by the transfection of the miR-205-5p precursor. In RNA-binding protein immunoprecipitation with Ago1/2/3 antibody, CLCN3 was significantly enriched in 293T embryonic kidney cells with miR-205-5p mimic transfection compared with negative control mimic transfection. In luciferase reporter assays using CLCN3 3'-UTR constructs, the miR-205-5p mimic significantly decreased reporter activity of both wild-type and partial mutant constructs in MCF10A-ErbB2 cells. In contrast, no inhibitory effects of the miR-205-5p mimic were detected using the complete mutant constructs. Since miR-205-5p expression in exosomes derived from MCF10A-neo cells was substantially higher than in exosomes derived from MCF10A-ErbB2 cells, we next investigated whether an exosome-mediated miR-205-5p transfer could control CLCN3 expression. To this end, exosomal miR-205-5p derived from MCF10A-neo cells was functionally transferred to MCF10A-ErbB2 cells, which served to decrease the expression of CLCN3. To assess the roles of CLCN3 in breast cancer, we next performed three-dimensional (3D) spheroid proliferation analyses using MCF10A-ErbB2 cells treated with MCF10A-neo-derived exosomes or CLCN3 shRNA stably expressing SKBR3 and MDA-MB-453 breast cancer cells. Our results showed that both treatment with MCF10A-neo-derived exosome and CLCN3 shRNA expression suppressed 3D spheroid proliferation. Collectively, these novel findings suggest that CLCN3 may be a novel direct target of miR-205-5p and this CLCN3/miR-205-5p interaction may serve a pivotal role in regulating breast cancer cellular proliferation under physiological conditions.

Identification of Chloride Channels CLCN3 and CLCN5 Mediating the Excitatory Cl- Currents Activated by Sphingosine-1-Phosphate in Sensory Neurons.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in numerous physiological and pathophysiological processes. We have previously reported a S1P-induced nocifensive response in mice by excitation of sensory neurons via activation of an excitatory chloride current. The underlying molecular mechanism for the S1P-induced chloride conductance remains elusive. In the present study, we identified two CLCN voltage-gated chloride channels, CLCN3 and CLCN5, which mediated a S1P-induced excitatory Cl- current in sensory neurons by combining RNA-seq, adenovirus-based gene silencing and whole-cell electrophysiological voltage-clamp recordings. Downregulation of CLCN3 and CLCN5 channels by adenovirus-mediated delivery of shRNA dramatically reduced S1P-induced Cl- current and membrane depolarization in sensory neurons. The mechanism of S1P-induced activation of the chloride current involved Rho GTPase but not Rho-associated protein kinase. Although S1P-induced potentiation of TRPV1-mediated ionic currents also involved Rho-dependent process, the lack of correlation of the S1P-activated Cl- current and the potentiation of TRPV1 by S1P suggests that CLCN3 and CLCN5 are necessary components for S1P-induced excitatory Cl- currents but not for the amplification of TRPV1-mediated currents in sensory neurons. This study provides a novel mechanistic insight into the importance of bioactive sphingolipids in nociception.

Genome-wide identification and functional analyses of microRNA signatures associated with cancer pain.

Cancer pain remains a major challenge and there is an urgent demand for the development of specific mechanism-based therapies. Various diseases are associated with unique signatures of expression of microRNAs (miRNAs), which reveal deep insights into disease pathology. Using a comprehensive approach combining genome-wide miRNA screening, molecular and in silico analyses with behavioural approaches in a clinically relevant model of metastatic bone-cancer pain in mice, we now show that tumour-induced conditions are associated with a marked dysregulation of 57 miRNAs in sensory neurons corresponding to tumour-affected areas. By establishing protocols for interference with disease-induced miRNA dysregulation in peripheral sensory neurons in vivo, we functionally validate six dysregulated miRNAs as significant modulators of tumour-associated hypersensitivity. In silico analyses revealed that their predicted targets include key pain-related genes and we identified Clcn3, a gene encoding a chloride channel, as a key miRNA target in sensory neurons, which is functionally important in tumour-induced nociceptive hypersensitivity in vivo. Our results provide new insights into endogenous gene regulatory mechanisms in cancer pain and open up attractive and viable therapeutic options.

Establishment and identification of a ClC-3/MMTV-PyMT hybrid mouse strain / 中国实验动物学报

Objective To establish a CLCN3/MMTV-PyMT double transgenic mouse model of spontaneous breast tumor with simultaneously overexpressing ClC-3.Method CLCN3 transgenic mice were crossed with MMTV-PyMT spon-taneous mammary tumor model mice.The genotype was determined by PCR.The expression of ClC-3 in tissues was detec-ted by immunofluorescence and Western blot.Results CLCN3 and MMTV-PyMT transgenic mice were bred and CLCN3/MMTV-PyMT hybrid mouse model was successfully established.The ClC-3 expression in CLCN3/MMTV-PyMT hybrid mice was higher than that in the MMTV-PyMT mice, assessed by immunofluorescence and Western blot analysis.Conclu-sions Transgenic mouse models of spontaneous breast cancer with simultaneously overexpressing ClC-3 are successfully es-tablished.The double transgenic mice provide a good animal model for further research of ClC-3 in tumor growth and metas-tasis.