Suppression of FAM83D Inhibits Glioma Proliferation,Invasion and Migration by Regulating the AKT/mTOR Signaling Pathway

ObjectiveTo explore the mechanism by which the family with sequence similarity 83, member D (FAM83D)-mediated AKT/mTOR signaling pathway activation affects the proliferation and metastasis of glioma cells.MethodsFAM83D protein expression in glioma cells and tissues was detected by western blotting. Glioma U87 and U251 cells were selected and divided into the Mock, siNC, siFAM83D, FAM83D, MK2206 and FAM83D + MK2206 groups. Cell proliferation was assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) and clone formation assays, while invasion and migration were evaluated by Transwell assays and wound healing tests. The protein expression of members of the AKT/mTOR pathway was determined via western blotting. Xenograft models were also established in nude mice to observe the in vivo effect of FAM83D on the growth of glioma.ResultsFAM83D was upregulated in glioma patients, especially in those with Stage III-IV. In addition, cells treated with siFAM83D had significant downregulation of p-AKT/AKT and p-mTOR/mTOR, with decreased proliferation and colony numbers, as well as decreased invasion and migration compared to the Mock group. However, FAM83D overexpression could activate the Akt/mTOR pathway and promote the proliferation, invasion and migration of glioma cells. Moreover, treatment with MK2206, an inhibitor of AKT, reversed the promoting effect of FAM83D on the growth of glioma cells. The in vivo experiments demonstrated that silencing FAM83D could inhibit the in vivo growth of glioma cellsConclusionFAM83D was upregulated in glioma and silencing FAM83D suppressed the proliferation, invasion and migration of glioma cells via inhibition of the AKT/mTOR pathway.     

DNA demethylation of promoter region orchestrates SPI-1-induced ADAMTS-5 expression in articular cartilage of osteoarthritis mice

Osteoarthritis (OA) is one of the most prevalent joint diseases in aged people and characterized by articular cartilage degeneration, synovial inflammation, and abnormal bone remodeling. Recent advances in OA research have clearly shown that OA development is associated with aberrant DNA methylation status of many OA-related genes. As one of most important cartilage degrading proteases in OA, a disintegrin and metalloproteinase with thrombospondin motifs subtype 5 (ADAMTS-5) is activated to mediate cartilage degradation in human OA and experimental murine OA models. The pathological factors and signaling pathways mediating ADAMTS-5 activation during OA development are not well defined and have been a focus of intense research. ADAMTS-5 promoter is featured by CpG islands. So far there have been no reports concerning the DNA methylation status in ADAMTS-5 promoter during OA development. In this study, we sought to investigate DNA methylation status in ADAMTS-5 promoter, the role of DNA methylation in ADAMTS-5 activation in OA, and the underlying mechanisms. The potential for anti-OA intervention therapy which is based on modulating DNA methylation is also explored. Our results showed that DNA methyltransferases 1 (Dnmt1) downregulation-associated ADAMTS-5 promoter demethylation played an important role in ADAMTS-5 activation in OA, which facilitated SPI-1 binding on ADAMTS-5 promoter to activate ADAMTS-5 expression. More importantly, OA pathological phenotype of mice was alleviated in response to Dnmt1-induced DNA methylation of ADAMTS-5 promoter. Our study will benefit not only for deeper insights into the functional role and regulation mechanisms of ADAMTS-5 in OA, but also for the discovery of disease-modifying OA drugs on the basis of ADAMTS-5 via modulating DNA methylation status.     

Long noncoding RNA FAM83H‐AS1 exerts an oncogenic role in glioma through epigenetically silencing CDKN1A (p21)

Gliomas are the commonest and most aggressive primary malignant tumor in the central nervous system. Long noncoding RNAs (lncRNAs) have been identified to act as crucial regulators in multiple biological processes, including tumorigenesis. FAM83H antisense RNA1 (FAM83H‐AS1) has been uncovered to be dysregulated in several cancers. However, the biological role of FAM83H‐AS1 in glioma still needs to be investigated. Currently, our findings indicated that FAM83H‐AS1 was upregulated in glioma tissues and cell lines and high level of FAM83H‐AS1 was associated with poor prognosis of glioma. Loss‐of‐function assays demonstrated that silenced FAM83H‐AS1 obviously suppressed cell proliferation via regulating the cell‐cycle distribution and cell apoptosis rate, and mechanistic experiments revealed that FAM83H‐AS1 could epidemically silence CDKN1A expression through recruiting EZH2 to the promoter of CDKN1A, thereby influencing the cell cycle and proliferation. Collectively, our findings suggested that FAM83H‐AS1 participated in the progression of glioma and might act as a potential therapeutic target and prognosis biomarker for human glioma.     

Analyses of the genomic methylation status of the human cyclin A1 promoter by a novel real-time PCR-based methodology

The role of CpG methylation in the regulation of tissue-specific gene expression is highly controversial. Cyclin A1 is a tissue-specifically expressed gene that is strongly methylated in non-expressing tumor cell lines. We have established a novel real-time PCR method to quantitate genomic CpG methylation of the cyclin A1 promoter. Genomic DNA samples from different human organs were treated with bisulfite and amplified with methylation-specific primers and with primers amplifying methylated as well as non-methylated DNA. PCR product quantitation was obtained by using a fluorogenic probe labeled with FAM and TAMRA. These analyses demonstrated that the human cyclin A1 promoter was methylated in kidney, colon, spleen, testis, and small intestine, but not in brain, liver, pancreas, or heart. Expression of cyclin A1 was predominantly found in testis. Low level expression of cyclin A1 was present in spleen, prostate, leukocytes, colon, and thymus. Taken together, our data provide evidence that CpG methylation patterns of the human cyclin A1 promoter in human organs do not generally correlate with cyclin A1 gene expression in vivo.     

LncRNA FAM83A-AS1 facilitates tumor proliferation and the migration via the HIF-1α/ glycolysis axis in lung adenocarcinoma

Background: Lung adenocarcinoma (LUAD), the major subtype of lung cancer, is among the leading cause of cancer-related death worldwide. Energy-related metabolic reprogramming metabolism is a hallmark of cancer shared by numerous cancer types, including LUAD. Nevertheless, the functional pathways and molecular mechanism by which FAM83A-AS1 acts in metabolic reprogramming in lung adenocarcinoma have not been fully elucidated.Methods: We used transwell, wound-healing scratch assay, and metabolic assays to explore the effect of FAM83A-AS1 in LUAD cell lines. Western blotting, Co-IP assays, and ubiquitination assays were used to detect the effects of FAM83A-AS1 on HIF-1α expression, degradation, and its binding to VHL. Moreover, an in vivo subcutaneous tumor formation assay was used to detect the effect of FAM83A-AS1 on LUAD.Results: Herein, we identified FAM83A-AS1 as a metabolism-related lncRNA, which was highly correlated with glycolysis, hypoxia, and OXPHOS pathways in LUAD patients using bioinformatics analysis. In addition, we uncovered that FAM83A-AS1 could promote the migration and invasion of LUAD cells, as well as influence the stemness of LUAD cells in vivo and vitro. Moreover, FAM83A-AS1 was shown to promote glycolysis in LUAD cell lines in vitro and in vivo, and was found to influence the expression of genes related to glucose metabolism. Besides, we revealed that FAM83A-AS1 could affect glycolysis by regulating HIF-1α degradation. Finally, we found that FAM83A-AS1 knockdown could inhibit tumor growth and suppress the expression of HIF-1α and glycolysis-related genes in vivo.Conclusion: Our study demonstrates that FAM83A-AS1 contributes to LUAD proliferation and stemness via the HIF-1α/glycolysis axis, making it a potential biomarker and therapeutic target in LUAD patients.     

心脏特异表达人源FAM55A转基因小鼠的建立

目的建立心脏特异表达的人源FAM55A转基因小鼠,为研究该基因在心肌病发病中的作用提供模型。方法 Western blot检测FAM55A在野生型小鼠与cTnTR141W转基因小鼠心脏组织中的表达变化及其在野生小鼠的组织表达谱。克隆人源FAM55A基因入α-MHC启动子下游构建a-MHC-FAM55A表达载体,显微注射法建立FAM55A转基因小鼠。PCR鉴定转基因首建鼠的基因型。Western blot鉴定人源FAM55A在转基因小鼠心脏中的表达,超声检测转基因小鼠心脏的几何构型和功能。HE染色检测转基因小鼠心脏的病理改变。结果 FAM55A在野生型小鼠心脏中有少量表达,在扩张型心肌病小鼠的心脏中表达增加。建立了1个心脏组织特异表达人源FAM55A转基因小鼠品系。与野生型小鼠相比,FAM55A转基因小鼠的心脏收缩期和舒张期左室前壁从1月龄到5月龄持续增厚,3月龄转基因小鼠心脏射血分数和短轴缩短率稍有增强,1月龄和5月龄转基因小鼠心脏功能则与同龄野生型小鼠相比无变化。组织学检测显示,转基因小鼠心脏左室心肌细胞不均匀肥大,但不发生紊乱。结论 FAM55A在扩张型心肌病小鼠的心脏中表达上调,建立了心脏特异表达的人源FAM55A转基因小鼠,为进一步和心肌病小鼠模型杂交,研究该基因在心肌病发病中的作用提供了工具。     

Increased DNA methylation and decreased expression of PDX-1 in pancreatic islets from patients with type 2 diabetes

Mutations in pancreatic duodenal homeobox 1 (PDX-1) can cause a monogenic form of diabetes (maturity onset diabetes of the young 4) in humans, and silencing Pdx-1 in pancreatic β-cells of mice causes diabetes. However, it is not established whether epigenetic alterations of PDX-1 influence type 2 diabetes (T2D) in humans. Here we analyzed mRNA expression and DNA methylation of PDX-1 in human pancreatic islets from 55 nondiabetic donors and nine patients with T2D. We further studied epigenetic regulation of PDX-1 in clonal β-cells. PDX-1 expression was decreased in pancreatic islets from patients with T2D compared with nondiabetic donors (P = 0.0002) and correlated positively with insulin expression (rho = 0.59, P = 0.000001) and glucose-stimulated insulin secretion (rho = 0.41, P = 0.005) in the human islets. Ten CpG sites in the distal PDX-1 promoter and enhancer regions exhibited significantly increased DNA methylation in islets from patients with T2D compared with nondiabetic donors. DNA methylation of PDX-1 correlated negatively with its gene expression in the human islets (rho = -0.64, P = 0.0000029). Moreover, methylation of the human PDX-1 promoter and enhancer regions suppressed reporter gene expression in clonal β-cells (P = 0.04). Our data further indicate that hyperglycemia decreases gene expression and increases DNA methylation of PDX-1 because glycosylated hemoglobin (HbA1c) correlates negatively with mRNA expression (rho = -0.50, P = 0.0004) and positively with DNA methylation (rho = 0.54, P = 0.00024) of PDX-1 in the human islets. Furthermore, while Pdx-1 expression decreased, Pdx-1 methylation and Dnmt1 expression increased in clonal β-cells exposed to high glucose. Overall, epigenetic modifications of PDX-1 may play a role in the development of T2D, given that pancreatic islets from patients with T2D and β-cells exposed to hyperglycemia exhibited increased DNA methylation and decreased expression of PDX-1. The expression levels of PDX-1 were further associated with insulin secretion in the human islets.     

DNMT3B-mediated FAM111B methylation promotes papillary thyroid tumor glycolysis,growth and metastasis

Over the past decades, the incidence of thyroid cancer (TC) rapidly increased all over the world, with the papillary thyroid cancer (PTC) accounting for the vast majority of TC cases. It is crucial to investigate novel diagnostic and therapeutic targets for PTC and explore more detailed molecular mechanisms in the carcinogenesis and progression of PTC. Based on the TCGA and GEO databases, FAM111B is downregulated in PTC tissues and predicts better prognosis in PTC patients. FAM111B suppresses the growth, migration, invasion and glycolysis of PTC both in vitro and in vivo. Furthermore, estrogen inhibits FAM111B expression by DNMT3B methylation via enhancing the recruitment of DNMT3B to FAM111B promoter. DNMT3B-mediated FAM111B methylation accelerates the growth, migration, invasion and glycolysis of PTC cells. In clinical TC patient specimens, the expression of FAM111B is inversely correlated with the expressions of DNMT3B and the glycolytic gene PGK1. Besides, the expression of FAM111B is inversely correlated while DNMT3B is positively correlated with glucose uptake in PTC patients. Our work established E2/DNMT3B/FAM111B as a crucial axis in regulating the growth and progression of PTC. Suppression of DNMT3B or promotion of FAM111B will be potential promising strategies in the estrogen induced PTC.     

FAM83H-AS1 is associated with clinical progression and modulates cell proliferation,migration, and invasion in bladder cancer

FAM83H-AS1, also known as oncogenic long noncoding RNA (lncRNA)-3, is a novel lncRNA that has been suggested to be dysregulated in a variety of human cancers. However, the expression status and function of FAM83H-AS1 in bladder cancer are still unknown. The object of our study is to explore the clinical value of FAM83H-AS1 in patients with bladder cancer and the biological function of FAM83H-AS1 in bladder cancer cells. In our results, the expression of FAM83H-AS1 was obviously elevated in bladder cancer tissue samples and bladder cancer cell lines compared with adjacent normal tissue samples and normal bladder epithelial cell lines, respectively. In addition, high expression of FAM83H-AS1 was associated with advanced clinical stage and the presence of muscularis invasion and served as an independent poor prognostic factor for overall survival in patients with bladder cancer. The loss-of-function study showed that silencing FAM83H-AS1 expression suppressed cell proliferation, migration, and invasion and induced cycle arrest at G0/G1 phase. In conclusion, FAM83H-AS1 is involved in the progression of bladder cancer and serves as a prognostic biomarker and potential therapeutic target for patients with bladder cancer.     

Increased Expression of DNA Methyltransferase 3a in Obese Adipose Tissue: Studies With Transgenic Mice

Epigenetic mechanisms are likely to be involved in the development of obesity. This study was designed to examine the role of a DNA methyltransferase (Dnmt3a), in obese adipose tissue. The gene expression of Dnmts was examined by quantitative real‐time PCR analysis. Transgenic mice overexpressing Dnmt3a in the adipose tissue driven by the aP2 promoter were created (Dnmt3a mice). DNA methylation of downregulated genes was examined using bisulfite DNA methylation analysis. Dnmt3a mice were fed a methyl‐supplemented or high‐fat diet, and subjected to body weight measurement and gene expression analysis of the adipose tissue. Expression of Dnmt3a was markedly upregulated in the adipose tissue of obese mice. The complementary DNA (cDNA) microarray analysis of Dnmt3a mice revealed a slight decrease in the gene expression of secreted frizzled‐related protein 1 (SFRP1) and marked increase in that of interferon responsive factor 9 (IRF9). In the SFRP1 promoter, DNA methylation was not markedly increased in Dnmt3a mice relative to wild‐type mice. In experiments with a high‐fat diet or methyl‐supplemented diet, body weight did not differ significantly with the genotypes. Gene expression levels of inflammatory cytokines such as tumor necrosis factor‐α (TNF‐α) and monocyte chemoattractant protein‐1 (MCP‐1) were higher in Dnmt3a mice than in wild‐type mice on a high‐fat diet. This study suggests that increased expression of Dnmt3a in the adipose tissue may contribute to obesity‐related inflammation. The data highlight the potential role of Dnmt3a in the adult tissue as well as in the developing embryo and cancer.     

Inactivation of a novel FGF23 regulator, FAM20C, leads to hypophosphatemic rickets in mice

Family with sequence similarity 20,-member C (FAM20C) is highly expressed in the mineralized tissues of mammals. Genetic studies showed that the loss-of-function mutations in FAM20C were associated with human lethal osteosclerotic bone dysplasia (Raine Syndrome), implying an inhibitory role of this molecule in bone formation. However, in vitro gain- and loss-of-function studies suggested that FAM20C promotes the differentiation and mineralization of mouse mesenchymal cells and odontoblasts. Recently, we generated Fam20c conditional knockout (cKO) mice in which Fam20c was globally inactivated (by crossbreeding with Sox2-Cre mice) or inactivated specifically in the mineralized tissues (by crossbreeding with 3.6 kb Col 1a1-Cre mice). Fam20c transgenic mice were also generated and crossbred with Fam20c cKO mice to introduce the transgene in the knockout background. In vitro gain- and loss-of-function were examined by adding recombinant FAM20C to MC3T3-E1 cells and by lentiviral shRNA-mediated knockdown of FAM20C in human and mouse osteogenic cell lines. Surprisingly, both the global and mineralized tissue-specific cKO mice developed hypophosphatemic rickets (but not osteosclerosis), along with a significant downregulation of osteoblast differentiation markers and a dramatic elevation of fibroblast growth factor 23 (FGF23) in the serum and bone. The mice expressing the Fam20c transgene in the wild-type background showed no abnormalities, while the expression of the Fam20c transgene fully rescued the skeletal defects in the cKO mice. Recombinant FAM20C promoted the differentiation and mineralization of MC3T3-E1 cells. Knockdown of FAM20C led to a remarkable downregulation of DMP1, along with a significant upregulation of FGF23 in both human and mouse osteogenic cell lines. These results indicate that FAM20C is a bone formation "promoter" but not an "inhibitor" in mouse osteogenesis. We conclude that FAM20C may regulate osteogenesis through its direct role in facilitating osteoblast differentiation and its systemic regulation of phosphate homeostasis via the mediation of FGF23.