Zinc finger BED-type containing 3 promotes hepatic steatosis by interacting with polypyrimidine tract-binding protein 1.

AIMS/HYPOTHESIS: The relationship between metabolic dysfunction-associated steatotic liver disease (MASLD) and type 2 diabetes mellitus, insulin resistance and the metabolic syndrome is well established. While zinc finger BED-type containing 3 (ZBED3) has been linked to type 2 diabetes mellitus and the metabolic syndrome, its role in MASLD remains unclear. In this study, we aimed to investigate the function of ZBED3 in the context of MASLD. METHODS: Expression levels of ZBED3 were assessed in individuals with MASLD, as well as in cellular and animal models of MASLD. In vitro and in vivo analyses were conducted using a cellular model of MASLD induced by NEFA and an animal model of MASLD induced by a high-fat diet (HFD), respectively, to investigate the role of ZBED3 in MASLD. ZBED3 expression was increased by lentiviral infection or tail-vein injection of adeno-associated virus. RNA-seq and bioinformatics analysis were employed to examine the pathways through which ZBED3 modulates lipid accumulation. Findings from these next-generation transcriptome sequencing studies indicated that ZBED3 controls SREBP1c (also known as SREBF1; a gene involved in fatty acid de novo synthesis); thus, co-immunoprecipitation and LC-MS/MS were utilised to investigate the molecular mechanisms by which ZBED3 regulates the sterol regulatory element binding protein 1c (SREBP1c). RESULTS: In this study, we found that ZBED3 was significantly upregulated in the liver of individuals with MASLD and in MASLD animal models. ZBED3 overexpression promoted NEFA-induced triglyceride accumulation in hepatocytes in vitro. Furthermore, the hepatocyte-specific overexpression of Zbed3 promoted hepatic steatosis. Conversely, the hepatocyte-specific knockout of Zbed3 resulted in resistance of HFD-induced hepatic steatosis. Mechanistically, ZBED3 interacts directly with polypyrimidine tract-binding protein 1 (PTBP1) and affects its binding to the SREBP1c mRNA precursor to regulate SREBP1c mRNA stability and alternative splicing. CONCLUSIONS/INTERPRETATION: This study indicates that ZBED3 promotes hepatic steatosis and serves as a critical regulator of the progression of MASLD. DATA AVAILABILITY: RNA-seq data have been deposited in the NCBI Gene Expression Omnibus ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE231875 ). MS proteomics data have been deposited to the ProteomeXchange Consortium via the iProX partner repository ( https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD041743 ).

Zbed3 Is Indispensable for Wnt Signaling Regulation of Cortical Layers Formation in Developing Brain.

Wnt/ß-catenin signaling plays multiple important roles during mammalian brain development, and it regulates the proliferation and differentiation of neural progenitors in a context-dependent manner and affects neocortex layer formation. However, the specific role of Wnt/ß-catenin in neuronal layer fate determination in the neocortex is still unclear. Here, we report that Zbed3, which is a positive regulator of Wnt/ß-catenin signaling, colocalizes with ß-catenin at the endfeet of radial glia in the ventricular zone of embryo mouse neocortex. Overexpression and knockdown of Zbed3 increased and decreased the activity of Wnt/ß-catenin signaling in the neocortex, respectively. Interestingly, knockdown of Zbed3 in vivo could significantly shift neuronal fates from deep layers to upper layers but is not required for the proliferation and differentiation of neural progenitors. Overexpression of Zbed3 led to increased generation of deep-layer neurons without impairing cell cycle exit of neural progenitors. More importantly, knockdown of Zbed3 could effectively block the effects of the ectopic expression of stabilized ß-catenin on neocortex layer formation. Hence, our results demonstrate that Zbed3 is indispensable for Wnt/ß-catenin signaling regulating neuronal layer fates in the developing brain.

STAT3-induced ZBED3-AS1 promotes the malignant phenotypes of melanoma cells by activating PI3K/AKT signaling pathway.

Melanoma is considered as the most frequent primary malignancy occurring in skin. Accumulating studies have suggested that long non-coding RNAs (lncRNAs) play critical parts in multiple cancers. In this study, we explored the molecular mechanism of ZBED3 antisense RNA 1 (ZBED3-AS1) in melanoma. We observed that ZBED3-AS1 expression was remarkably up-regulated in melanoma tissues, and high ZBED3-AS1 level was linked to unsatisfactory survival of melanoma patients. Then, we discovered that ZBED3-AS1 was overexpressed in melanoma cells compared with human epidermal melanocytes. In addition, loss-of-function assays verified that ZBED3-AS1 knockdown restrained cell proliferation, migration, epithelial-mesenchymal transition (EMT), and stemness in melanoma. In addition, signal transducer and activator of transcription 3 (STAT3), which also showed tumour-facilitating functions in melanoma, was confirmed as a transcriptional activator of ZBED3-AS1. Moreover, ZBED3-AS1 enhanced the expression of AT-rich interaction domain 4B (ARID4B) through sequestering miR-381-3p. Importantly, we further confirmed that ZBED3-AS1 promoted the malignant progression of melanoma by regulating miR-381-3p/ARID4B axis to activate the phosphatidylinositol 3-kinase/AKT serine/threonine kinase (PI3K/AKT) signalling pathway. In a word, our research might provide a novel therapeutic target for melanoma.

Subcortical maternal complex (SCMC) expression during folliculogenesis is affected by oocyte donor age in sheep.

PURPOSE: The age-associated decline in female fertility is largely ascribable to the decrease in oocyte quality. The subcortical maternal complex (SCMC) is a multiprotein complex essential for early embryogenesis and female fertility and functionally conserved across mammals. The present work evaluated expression dynamics of its components during folliculogenesis in relation to maternal age in sheep. METHODS: The expression of the SCMC components (KHDC3/FILIA, NLRP2, NLRP5/MATER, OOEP/FLOPED, PADI6, TLE6 and ZBED3) was analyzed by real-time PCR in pools of growing oocytes (GO) of different diameters (70-90 µm (S), 90-110 µm (M), or 110-130 µm (L)) derived from non-hormonally treated adult (Ad; age < 4 years), prepubertal (Pr; age 40 days), or aged ewes (age > 6 years). RESULTS: Specific expression patterns associated with donor age were observed during folliculogenesis for all genes, except ZBED3. In oocytes of adult donors, the synthesis of NLRP2, NLRP5, PADI6, and ZBED3 mRNAs was complete in S GO, while FILIA, TLE6, and OOEP were actively transcribed at this stage. Conversely, Pr GO showed active transcription of all mRNAs, except for ZBED3, during the entire window of oocyte growth. Notably, aged GO showed a completely inverse pattern, with a decrease of NLRP2, TLE6, FILIA, and PADI6 mRNA abundance during the latest stage of oocyte growth (L GO). Interestingly, MATER showed high expression variability, suggesting large inter-oocyte differences. CONCLUSION: Our study describes the SCMC expression dynamics during sheep oogenesis and reports age-specific patterns that are likely involved in the age-related decline of oocyte quality.

Zbed3 promotes proliferation and invasion of lung cancer partly through regulating the function of Axin-Gsk3ß complex.

Our previous work showed that Zbed3 is overexpressed in nonsmall cell lung cancer and that down-regulation of Zbed3 inhibited ß-catenin expression and cancer cell proliferation and invasiveness. Here, we investigated Zbed3's ability to promote lung cancer cell proliferation and invasion and the involvement of the Axin/TPC/glycogen synthase kinase 3ß (Gsk-3ß) complex to the response. Coimmunoprecipitation assays showed that wild-type Zbed3 bound to Axin but a Zbed3 mutant lacking the Axin binding site did not. In A549 and H1299 lung cancer cells, Zbed3 overexpression promoted cancer cell proliferation and invasiveness, as well as Wnt signalling and expression of downstream mediators, including ß-catenin, cyclin D1 and MMP7 (P < 0.05). In contrast, the Zbed3 mutant failed to enhance ß-catenin expression (P > 0.05), and its ability to promote cancer cell proliferation and invasiveness was much less than wild-type Zbed3 (P < 0.05). The ability of Zbed3 to increase ß-catenin levels was abolished by Axin knockdown in A549 cells (P > 0.05). Similarly, treating the cells with a GSK-3ß inhibitor abolished Zbed3's ability to increase ß-catenin levels and Wnt signalling. These results indicate that Zbed3 enhances lung cancer cell proliferation and invasiveness at least in part by inhibiting Axin/adenomatous polyposis coli/GSK-3ß-mediated negative regulation of ß-catenin levels.

Long noncoding RNA ZBED3-AS1 induces the differentiation of mesenchymal stem cells and enhances bone regeneration by repressing IL-1ß via Wnt/ß-catenin signaling pathway.

Bone regeneration, as a physiological process of bone formation, is regulated by multiple cytokines. Long noncoding RNAs are involved in the progress of bone formation. The present study investigated role by which ZBED3-AS1 acts to control the differentiation of mesenchymal stem cells (MSCs) and bone regeneration. Bioinformatics prediction and dual luciferase reporter gene assay identified putative ZBED3-AS1 binding sites on the 3'-untranslated region of interleukin-1ß (IL-1ß). Then, RNA immunoprecipitation and chromatin immunoprecipitation assays confirmed that ZBED3-AS1 could regulate the expression of IL-1ß by binding to the transcription factor CREB. Notably, ZBED3-AS1 was shown to negatively regulate IL-1ß expression. After model establishment in rats simulating bone injury, MSCs were isolated and delivered with ZBED3-AS1, Si-ZBED3-AS1, Si-IL-1ß, or DKK (inhibitor of Wnt/ß-catenin signaling pathway) to identify their roles in osteogenic differentiation by evaluating MSC colony formation and proliferation. Then, number of mineralized nodules, alkaline phosphatase (ALP) activity and osteocalcin (OCN) expression, and expression of osteogenesis-related genes were determined. Overexpression of ZBED3-AS1 or silencing of IL-1ß was shown to accelerate ectopic osteogenesis, as reflected by increasing the number of mineralized nodules, ALP activity, and OCN expression, and promoting MSC colony formation and proliferation. Additionally, ZBED3-AS1 activated the Wnt/ß-catenin signaling pathway by negatively regulating IL-1ß. IL-1ß inhibited osteogenic differentiation by suppressing the Wnt/ß-catenin signaling pathway. Furthermore, the effect of ZBED3-AS1 and IL-1ß on osteogenic differentiation was confirmed in vivo. Taken together, upregulation of ZBED3-AS1 could restore differentiation of MSCs and enhance bone regeneration via activation of Wnt/ß-catenin signaling pathway by repressing IL-1ß.

The function of BED finger domain of Zbed3 in regulating lung cancer cell proliferation.

Zbed3, a BED finger domain-containing protein was found to promote cancer proliferation by regulating ß-catenin expression through interacting with Axin. But whether and how BED finger domain function in regulating cancer proliferation is unknown. We constructed five mutants of Zbed3, which lacks the Axin-Zbed3 binding site, and the 43 to 52, 69 to 77, 87 to 92, and 97 to 104 sequences in BED finger domain, respectively and named them as Z-A, Z1, Z2, Z3, and Z4. Transfection of both wild-type of Zbed3 and the mutants Z1, Z3, and Z4 (P < 0.05), but not Z2 (P > 0.05) significantly upregulated ß-catenin expression in NCI-H1299 cells. Overexpression of both wild-type of Zbed3 and the mutants Z1, Z3, and Z4 (P < 0.05) but not Z2 (P > 0.05) significantly promoted cancer cell proliferation and invasion. The ability of proliferation (P < 0.05) but not invasion (P < 0.05) of cancer cells transfected with Z1 and Z4 was significantly lower than that with wild-type Zbed3 and Z3. Overexpression of wild-type Zbed3 (P < 0.05) but not the mutant Z-A, which lacks the binding site with Axin and Z2 (P > 0.05) significantly upregulated the interaction of Axin and Zbed3, ß-catenin expression and the activity of Wnt signaling. Both overexpression of wild-type Zbed3 and the mutant Z1 and Z4 significantly upregulated the activity of Wnt signaling and promoted cancer cell proliferation (P < 0.05) but only overexpression of wild-type Zbed3 (P < 0.05), but not the mutant Z1, and Z4 (P > 0.05), significantly upregulated the expression of proliferating cell nuclear antigen (PCNA) in NCI-H1299 cells. These results indicate that Zbed3 may promote lung cancer cell proliferation through regulating PCNA expression besides regulating ß-catenin expression and BED finger domain can impact on this function.

The LncRNA ZBED3-AS1 induces chondrogenesis of human synovial fluid mesenchymal stem cells.

Human synovial fluid-derived mesenchymal stem cells (SFMSCs) have great potential for cartilage induction and are promising for cell-based strategies for articular cartilage repair. Many long non-coding RNAs (lncRNAs) regulate chondrogenesis of MSCs. We hypothesized that the divergent lncRNA ZBED3-AS1, which binds locally to chromatin, could promote the expression of zbed3, a novel Axin-interacting protein that activates Wnt/ß-catenin signaling, involved in chondrogenesis. However, the function of ZBED3-AS1 in SFMSCs is unclear. In this study, the expression, biological function, and roles of ZBED3-AS1 in SFMSC chondrogenesis were examined by multilineage differentiation, flow cytometry, and gain-of-function studies. We found that ZBED3-AS1 promotes chondrogenesis. Furthermore, ZBED3-AS1 could directly increase zbed3 expression. Finally, the wnt-inhibitor DKK1 could reverse the stimulatory effect of ZBED3-AS1 on chondrogenesis. These findings demonstrate the role of a new lncRNA, ZBED3-AS1, in SFMSC chondrogenesis and may improve osteoarthritis treatment.

Zbed3 contributes to malignant phenotype of lung cancer via regulating ß-catenin and P120-catenin 1.

Our previous studies indicate that abnormal expression of several Wnt signaling molecules including Axin, Dvl and ß-catenin are involved in proliferation, invasion and metastasis of lung cancer. Zbed3 was found to inhibit function of Axin-GSK3ß complex and thus lead to accumulation of ß-catenin in NIH3T3 and HEK293T cells. However its function in malignant tumors is largely unknown. Here we investigate the clinico-pathological significance of Zbed3 expression and its function in non-small cell lung cancer. We use immunohistochemistry and Western blotting to examine Zbed3 expression in non-small cell lung cancer and lung tissues. Transfection of siRNA and plasmid was used to study the function of Zbed3 in lung cancer cells in vitro. We found Zbed3 expression was elevated in cancer tissues compared to normal lung tissues. Increased Zbed3 expression is significantly associated with lymph node metastasis, advanced TNM stages, higher Ki67 status and patients' poor clinical outcome. Higher Zbed3 expression was also found in lung cancer cell lines compared to bronchial epithelial cell line HBE. Downregulation of Zbed3 by siRNA significantly inhibits cancer cell proliferation and invasion in vitro. Downregulation of Zbed3 also significantly inhibits expression of ß-catenin, downstream molecules of Wnt signaling and P120ctn-1 in lung cancer cells. These results suggest that Zbed3 may contribute to lung cancer cell invasion through regulating ß-catenin and p120ctn-1 and may be a promissing cancer marker in non-small cell lung cancer.

lncRNA Biomarkers of Glioblastoma Multiforme.

Long noncoding RNAs (lncRNAs) are RNA molecules of 200 nucleotides or more in length that are not translated into proteins. Their expression is tissue-specific, with the vast majority involved in the regulation of cellular processes and functions. Many human diseases, including cancer, have been shown to be associated with deregulated lncRNAs, rendering them potential therapeutic targets and biomarkers for differential diagnosis. The expression of lncRNAs in the nervous system varies in different cell types, implicated in mechanisms of neurons and glia, with effects on the development and functioning of the brain. Reports have also shown a link between changes in lncRNA molecules and the etiopathogenesis of brain neoplasia, including glioblastoma multiforme (GBM). GBM is an aggressive variant of brain cancer with an unfavourable prognosis and a median survival of 14-16 months. It is considered a brain-specific disease with the highly invasive malignant cells spreading throughout the neural tissue, impeding the complete resection, and leading to post-surgery recurrences, which are the prime cause of mortality. The early diagnosis of GBM could improve the treatment and extend survival, with the lncRNA profiling of biological fluids promising the detection of neoplastic changes at their initial stages and more effective therapeutic interventions. This review presents a systematic overview of GBM-associated deregulation of lncRNAs with a focus on lncRNA fingerprints in patients' blood.

Implication of lncRNA ZBED3-AS1 downregulation in acquired resistance to Temozolomide and glycolysis in glioblastoma.

Temozolomide (TMZ) is the recommended drug for glioblastoma (GBM) treatment, but its clinical effect is restricted due to drug resistance. This research studies the effects of long non-coding RNA (lncRNA) ZBED3-AS1 and its related molecules on acquired TMZ resistance in glioblastoma (GBM). ZBED3-AS1 was identified to be downregulated in TMZ-resistant GBM cells by analyzing GSE113510 and GSE100736 datasets. ZBED3-AS1 downregulation was detected in TMZ-resistant GBM tissues and cell lines (U251/TMZ and U87/TMZ). ZBED3-AS1 knockdown promoted, whereas its overexpression suppressed TMZ resistance, viability and mobility, and glycolytic activity of TMZ-resistant cells. ZBED3-AS1 bound to Spi-1 proto-oncogene (SPI1) but did not affect its expression. Instead, it blocked SPI1-mediated transcriptional activation of thrombomodulin (THBD). SPI1 and THBD increased TMZ resistance and glycolysis in TMZ-resistant cells. Either ZBED3-AS1 overexpression or SPI1 knockdown in U87/TMZ cells blocked the growth of orthotopic and subcutaneous xenograft tumors in nude mice. In conclusion, this study demonstrates that ZBED3-AS1 downregulation and THBD activation is linked to increased TMZ resistance and glycolysis in GBM cells.

Circular RNA hsa_circ_0119412 contributes to tumorigenesis of gastric cancer via the regulation of the miR-1298-5p/zinc finger BED-type containing 3 (ZBED3) axis.

Circular RNAs (circRNAs) are associated with the progression of gastric cancer (GC). This study investigates the regulation of the circular RNA, hsa_circ_0119412 in GC and its effects on GC cells. The expression of hsa_circ_0119412, microRNA (miR)-1298-5p, and zinc finger BED-type containing 3 (ZBED3) were measured by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting. The cell counting kit-8 (CCK-8) assay, flow cytometry, transwell, and animal assays were performed to identify the roles of hsa_circ_0119412, miR-1298-5p, and ZBED3 in the viability, apoptosis, invasion, and growth of GC cells. The relationship between hsa_circ_0119412, miR-1298-5p, and ZBED3 was confirmed by luciferase, RNA immunoprecipitation (RIP), and RNA pull-down assays. Our data revealed that hsa_circ_0119412 and ZBED3 expression was upregulated in GC, while miR-1298-5p expression was downregulated. Both the knockdown of hsa_circ_0119412/ZBED3 and miR-1298-5p overexpression inhibited GC cell growth and invasion, and enhanced cell apoptosis, while miR-1298-5p interference or ZBED3 overexpression showed the opposite trend. Mechanistically, hsa_circ_0119412 sponges miR-1298-5p, which regulates ZBED3 expression. Silencing hsa_circ_0119412 inhibits the progression of GC, at least in part, by targeting the miR-1298-5p/ZBED3 axis.

Lnc13728 facilitates human mesenchymal stem cell adipogenic differentiation via positive regulation of ZBED3 and downregulation of the WNT/ß-catenin pathway.

BACKGROUND: Obesity has received increasing attention because of its widespread worldwide occurrence and many threats to health. Human adipose-derived mesenchymal stem cells (hADSCs) are a critical source of adipocytes. Long noncoding RNAs (lncRNAs) play pivotal roles in cell fate determination and differentiation. The objective of the present study was to identify and investigate the function and regulatory mechanism of lncRNAs on adipogenic differentiation of hADSCs. METHODS: We used lncRNA arrays to identify the prominent differentially expressed lncRNAs before and after hADSC adipogenic differentiation and verified their biological function through antisense oligonucleotide knockdown or lentivirus overexpression. The adipogenic differentiation of hADSCs was assessed by oil red O staining as well as the mRNA and protein levels of adipogenic marker genes through qRT-PCR and western blot. Bioinformatic tool LncPro and immunofluorescence was performed to uncover the interaction between lnc13728 and ZBED3. WNT/ß-catenin signaling pathway was evaluated by western blot and immunofluorescence. RESULTS: The lncRNA arrays showed that lnc13728 expression was significantly upregulated after hADSC adipogenic differentiation and was correlated positively with the expression of the adipogenesis-related genes in human adipose tissue. Lnc13728 knockdown in hADSCs suppressed the expression of the adipogenesis-related genes at both mRNA and protein level and weakened lipid droplet production. Accordingly, lnc13728 overexpression enhanced hADSC adipogenic differentiation. Beyond that, lnc13728 co-localized with ZBED3 in the cytoplasm and regulated its expression positively. Downregulating ZBED3 had a negative effect on adipogenic differentiation, while the expression of WNT/ß-catenin signaling pathway-related proteins was upregulated. CONCLUSIONS: Lnc13728 promotes hADSC adipogenic differentiation possibly by positively regulating the expression of ZBED3 which plays a role in inhibiting the WNT/ß-catenin pathway.

Long noncoding RNA ZBED3-AS1 restrains breast cancer progression by targeting the microRNA-513a-5p/KLF6 axis.

Breast cancer (BC) is the most commonly occurring malignancy in women. This study aimed to investigate the functions of the long noncoding RNA ZBED3-AS1 (ZBED3-AS1) in BC and its molecular mechanisms. qRT-PCR was conducted to access the expression of ZBED3-AS1, microRNA-513a-5p (miR-513a-5p), and Kruppel like factor 6 (KLF6) in BC. Additionally, BC cell viability and proliferative capacity were measured by MTT and 5-Ethynyl-20-deoxyuridine (EdU) assays. A transwell assay was used for evaluating BC cell migration and invasion. The interactions among ZBED3-AS1, miR-513a-5p, and KLF6 in BC were confirmed by dual-luciferase reporter assay. Furthermore, feedback approaches were performed to determine whether ZBED3-AS1 influences BC cell behaviors by regulating the miR-513a-5p/KLF6 axis. The murine xenograft model was established to assess the effect of ZBED3-AS1 on tumor growth. The expression of ZBED3-AS1 and KLF6 was reduced, while miR-513a-5p expression was elevated in BC. ZBED3-AS1 elevation attenuated the malignant behaviors of BC cells, including viability, proliferative capacity, migration, and invasion. Mechanical experiments revealed that ZBED3-AS1 targeted miR-513a-5p, and miR-513a-5p targeted KLF6 in BC. Feedback approaches validated that miR-513a-5p overexpression or KLF6 depletion reversed the inhibitory effects of ZBED3-AS1 upregulation on viability, proliferative capacity, migration, and invasion of BC cells. Furthermore, ZBED3-AS1 elevation attenuated the tumor growth in the murine xenograft model. ZBED3-AS1 hindered the malignant development of BC cells by regulating the miR-513a-5p/KLF6 axis, providing a novel therapeutic target in BC.

Circulating Zbed3 Levels in Subjects With and Without Metabolic Syndrome.

BACKGROUND: Zinc-finger BED domain-containing 3 (Zbed3) is a member of the zinc-finger domain protein superfamily. Recent studies have shown that Zbed3 is associated with insulin resistance and type 2 diabetes mellitus. However, no report has demonstrated the association of Zbed3 with metabolic syndrome (MetS) in humans. The purpose of this study is to examine the association between Zbed3 and MetS in a cross-sectional study. METHODS: We conducted a cross-sectional study of a Chinese population, including 167 non-MetS subjects and 144 newly diagnosed MetS (nMetS) patients. Circulating Zbed3 levels were examined by enzyme-linked immunosorbent assay. The relationship between circulating Zbed3 levels and the components of MetS was assessed. RESULTS: Circulating Zbed3 levels were significantly higher in nMetS patients than in non-MetS subjects (134.6 ± 32.1 vs. 106.5 ± 26.1 ng/L, P < 0.01). Circulating Zbed3 correlated positively with markers of adiposity (waist circumference, P < 0.01). It also correlated with glucose and lipid parameters (increasing fasting blood glucose and triglycerides and decreasing high-density lipoprotein cholesterol, all P < 0.01) and blood pressure (elevating systolic blood pressure and diastolic blood pressure, both P < 0.01) and inflammatory marker (elevating tumor necrosis factor alpha, P < 0.01). The relative risks for MetS showed significant elevation with an increase in Zbed3 quartiles. Circulating levels of Zbed3 were progressively elevated with an increased number of components of MetS. CONCLUSIONS: These data suggest that Zbed3 may correlate with the pathogenesis of MetS in humans. CLINICAL TRIAL REGISTRATION NUMBER: ChiCTR-OCC-11001422.