Fengtian He

LincRNA-p21 Regulates Neointima Formation, Vascular Smooth Muscle Cell Proliferation, Apoptosis and Atherosclerosis by Enhancing p53 Activity

Background— Long noncoding RNAs (lncRNAs) have recently been implicated in many biological processes and diseases. Atherosclerosis is a major risk factor for cardiovascular disease. However, the functional role of lncRNAs in atherosclerosis is largely unknown. Methods and Results— We identified lincRNA-p21 as a key regulator of cell proliferation and apoptosis during atherosclerosis. The expression of lincRNA-p21 was dramatically downregulated in atherosclerotic plaques of ApoE−/− mice, an animal model for atherosclerosis. Through loss- and gain-of-function approaches, we showed that lincRNA-p21 represses cell proliferation and induces apoptosis in vascular smooth muscle cells and mouse mononuclear macrophage cells in vitro. Moreover, we found that inhibition of lincRNA-p21 results in neointimal hyperplasia in vivo in a carotid artery injury model. Genome-wide analysis revealed that lincRNA-p21 inhibition dysregulated many p53 targets. Furthermore, lincRNA-p21, a transcriptional target of p53, feeds back to enhance p53 transcriptional activity, at least in part, via binding to mouse double minute 2 (MDM2), an E3 ubiquitin-protein ligase. The association of lincRNA-p21 and MDM2 releases MDM2 repression of p53, enabling p53 to interact with p300 and to bind to the promoters/enhancers of its target genes. Finally, we show that lincRNA-p21 expression is decreased in patients with coronary artery disease. Conclusions— Our studies identify lincRNA-p21 as a novel regulator of cell proliferation and apoptosis and suggest that this lncRNA could serve as a therapeutic target to treat atherosclerosis and related cardiovascular disorders.

MicroRNA-613 represses lipogenesis in HepG2 cells by downregulating LXRα

BackgroundMicroRNAs (miRNAs) emerge as new important regulators of lipid homeostasis by regulating corresponding genes. MiR-613 is a newly discovered microRNA, of which the biological function is unknown. A recent report has shown that miR-613 downregulates liver X receptor α (LXRα), a ligand-activated nuclear receptor playing an important role in the regulation of lipid metabolism. The purpose of this study is to explore the effect and the molecular basis of miR-613 on lipogenesis in HepG2 cells.MethodsHepG2 cells were transiently transfected with miR-613 mimic or control microRNA. Real time PCR, Western blot, Luciferase reporter assay and Oil Red O staining were employed to examine the expression of LXRα and its target genes involved in lipogenesis, binding site for miR-613 in 3′-untranslated region (3′-UTR) of LXRα mRNA and lipid droplet accumulation in the cells.ResultsMiR-613 dramatically suppressed the expression of LXRα and its target genes including sterol-regulatory element binding protein 1c (SREBP-1c), fatty acid synthase (FAS), carbohydrate responsive element-binding protein (ChREBP) and acetyl-CoA carboxylase (ACC). Reporter assay showed that miR-613 directly bound to 3′-UTR of LXRα mRNA. Moreover, miR-613 significantly repressed LXRα-induced lipid droplet accumulation in HepG2 cells. Ectopic expression of LXRα without 3′-UTR markedly attenuated the miR-613-mediated downregulation of LXRα’s target genes and LXRα-induced lipid droplet accumulation.ConclusionsMiR-613 suppresses lipogenesis by directly targeting LXRα in HepG2 cells, suggesting that miR-613 may serve as a novel target for regulating lipid homeostasis.

Curcumin Protects Against Collagen-Induced Arthritis via Suppression of BAFF Production

PurposeThe aim of the present study was to evaluate whether the anti-Rheumatoid arthritis (RA) effect of curcumin is associated with the regulation of B cell-activating factor belonging to the TNF family (BAFF) production.MethodsCollagen-induced arthritis (CIA) was induced in DBA/1xa0J mice by immunization with bovine type II collagen. To investigate the anti-arthritic effect of curcumin in the CIA model, mice were injected intraperitoneally with curcumin (50xa0mg/kg) on every other day either from day 1 or from day 28 after the first immunization. The clinical severity of arthritis was monitored. BAFF, interleukin-6 (IL-6) and interferon-γ (IFNγ) production in serum were measured. Furthermore, the effect of curcumin on IFNγ-induced BAFF expression and transcriptional activation in B lymphocytes was determined by qPCR, Western Blot, and luciferase assay. Finally, IFNγ related signal transducers and activators of transcription 1 (STAT1) signaling in B lymphocytes were studied using Western Blot.ResultsCurcumin dramatically attenuated the progression and severity of CIA in DBA/1xa0J mice, accompanied with decrease of BAFF production in serum and spleen cells as well as decrease of serum IFNγ and IL-6. Treatment of B lymphocytes with curcumin suppressed IFNγ-induced BAFF expression, STAT1 phosphorylation and nuclear translocation, suggesting that curcumin may repress IFNγ-induced BAFF expression via negatively interfering with STAT1 signaling.ConclusionThe results of the present study suggest that suppression of BAFF production may be a novel mechanism by which curcumin improves RA.

MicroRNA-363-mediated downregulation of S1PR1 suppresses the proliferation of hepatocellular carcinoma cells

S1PR1 plays a crucial role in promoting proliferation of hepatocellular carcinoma (HCC). Over expression of S1PR1 is observed in HCC cell lines. The mechanisms underlying the aberrant expression of S1PR1 are not known well. MircroRNAs are important regulators of gene expression and disproportionate microRNAs can result in dysregulation of oncogenes in cancer cells. In this study, we found that miR-363, a potential tumor suppressor microRNA, downregulated the expression of S1PR1 and inhibited the proliferation of HCC cells. Bioinformatic analysis predicted a putative binding site of miR-363 within the 3-UTR of S1PR1 mRNA. Luciferase reporter assay showed that miR-363 directly targeted the 3-UTR of S1PR1 mRNA. Transfection of miR-363 mimics suppressed S1PR1 expression in HCC cells, followed by the repression of the activation of ERK and STAT3. Moreover, we found that the expression of downstream genes of ERK and STAT3, including PDGF-A, PDGF-B, MCL-1 and Bcl-xL, was suppressed after miR-363 transfection. Taken together, the present study demonstrated that miR-363 was a negative regulator of S1PR1 expression in HCC cells and inhibited cell proliferation, suggesting that the miR-363/S1PR1 pathway might be a novel target for the treatment of HCC.

The FOXM1-induced resistance to oxaliplatin is partially mediated by its novel target gene Mcl-1 in gastric cancer cells.

Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein that belongs to the Bcl-2 family. The aberrant expression of Mcl-1 is important for sensitivity to chemotherapy drugs in gastric cancer. However, the regulatory mechanism of Mcl-1 in gastric cancer cells remains unclear. In this study, we first found that Forkhead box M1 (FOXM1) and Mcl-1 expression levels were positively correlated in human gastric cancer specimens and that both are associated with poor prognosis of patients treated with oxaliplatin. Second, we demonstrated that the expression level of Mcl-1 was correlated with FOXM1 expression in gastric cancer cells. Third, reporter assays showed that FOXM1 upregulated the promoter activity of the Mcl-1 gene. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) assays further demonstrated that FOXM1 could bind to a particular site (-635acaaacaa-628) in the promoter region of the Mcl-1 gene. Moreover, CCK-8 assays and analyses of apoptosis revealed that the suppression of the FOXM1/Mcl-1 pathway induced apoptosis and thus increased sensitivity to oxaliplatin in gastric cancer cells, whereas the enhancement of the FOXM1/Mcl-1 pathway inhibited apoptosis and decreased sensitivity to oxaliplatin in gastric cancer cells. Taken together, this study is the first to not only show that Mcl-1 is a novel target gene of FOXM1 but also suggest that targeting FOXM1/Mcl-1 may be a novel strategy to enhance sensitivity to oxaliplatin in gastric cancer.

A feedback loop in PPARγ–adenosine A2A receptor signaling inhibits inflammation and attenuates lung damages in a mouse model of LPS-induced acute lung injury

Although peroxisome proliferator-activated receptor-γ (PPARγ) and adenosine A2A receptor (A2AR) are reported to be anti-inflammatory factors in acute lung injury (ALI), their internal link and synergic or antagonistic effect after activation are poorly understood. Here, we found that PPARγ and A2AR could upregulate the mRNA and protein expressions of each other in lung tissues of LPS-induced mouse ALI model and murine macrophages. Further investigation demonstrated that PPARγ upregulated A2AR expression by directly binding to a DR10 response element (-218 to -197) within A2AR gene promoter region. Instead of directly interacting with PPARγ, A2AR stimulated PPARγ expression via protein kinase A (PKA)-cAMP response element binding protein (CREB) signaling by provoking the binding of CREB to a cAMP responsive element (CRE)-like site in PPARγ gene promoter region. In addition, combination of PPARγ and A2AR agonists was found to exert obviously better effect on suppressing neutrophil infiltration and inflammatory cytokine expressions, attenuating lung edema, pathological changes and improving lung function of blood gas exchange than their single application. These findings reveal a novel functional positive feedback loop between PPARγ and A2AR signaling to potentialize their effect on inhibiting inflammation and attenuating lung damages in ALI. It suggests that targeting this PPARγ-A2AR signaling rather than PPARγ or A2AR alone may be a more attractive and efficient potential therapeutic strategy for ALI.

Activation of Adenosine 2A receptor inhibits neutrophil apoptosis in an autophagy-dependent manner in mice with systemic inflammatory response syndrome

Systemic inflammatory response syndrome (SIRS) is an overwhelming whole body inflammation caused by infectious diseases or sterile insults. Neutrophils are the dominant participants during inflammation, and their survival and death determine the initiation as well as resolution of SIRS. Apoptosis and autophagy are two fundamental cellular processes that modulating cell fate, but their correlation and regulators in neutrophils under SIRS condition have not been elucidated. In this study, we demonstrated that high dose of LPS induced both apoptosis and autophagy of neutrophils in a mouse SIRS model and LPS-stimulated neutrophils in vitro. Moreover, we found that the adenosine 2A receptor (A2AR), a known anti-inflammatory G protein-coupled receptor (GPCR), could inhibit LPS-induced neutrophil apoptosis by suppressing the LPS-induced autophagy. Activation of A2AR suppressed LPS-induced autophagy by inhibiting the ROS-JNK pathway as well as promoting GPCR βϒ subunit–AKT signaling. The A2AR-inhibited autophagy suppressed apoptosis of neutrophils by blocking caspase8, caspase3 and PARP signaling. These findings not only increase our understandings of neutrophils’ fate and function in response to systemic inflammation, but also identify a novel anti-inflammatory role of A2AR in modulating neutrophils’ survival during inflammation.

The mutual regulation between miR-214 and A2AR signaling plays an important role in inflammatory response.

Inflammation is a pathological course involved in several diseases. Both adenosine A2A receptor (A2AR) and miR-214 play important roles in regulation of inflammation. However, the internal link between them and their synergic modulation in inflammatory response has not been elucidated. In this study, we found that miR-214 and A2AR activation could downregulate the expressions of each other in murine macrophages. Comparing with the well known anti-inflammatory role of A2AR, miR-214 promoted the release of inflammatory cytokines TNF-α and IL-6. Further investigation demonstrated that miR-214 downregulated A2AR expression by directly targeting the 3-untranslated region of A2AR mRNA. Instead of directly interacting with miR-214, A2AR activation repressed miR-214 expression by stimulating PKA signaling to suppress the nuclear translocation of NF-κB which could enhance the transcript activity of miR-214 gene promoter. Then using an LPS-induced ALI mouse model, in which inflammation is a hallmark, we confirmed their negative relationship and demonstrated that combination of miR-214 antagomir and A2AR agonist CGS21680 exerts more anti-inflammatory effect including alleviating the pathological changes, suppressing the neutrophil infiltration and the expression of inflammatory cytokines than using one of them alone. These findings for the first time uncovered a mutual suppression feedback loop between A2AR signaling and miR-214 in inflammation, which may provide new insight of inflammatory regulation and potential therapeutic significance for some inflammation-associated diseases.

PinX1, a novel target gene of p53, is suppressed by HPV16 E6 in cervical cancer cells.

The aberrant activation of telomerase is critical for the initiation and development of human cervical cancer, which is dependent on the activation of human telomerase reverse transcriptase (hTERT). Recently, Pin2/TRF1-interacting protein X1 (PinX1) has been identified as a suppressor of hTERT. It has been found that the telomerase is activated while the level of PinX1 is decreased in cervical cancer. However, the regulatory mechanism of PinX1 in cervical cancer cells remains unclear. In the present study, we demonstrated that the level of PinX1 is regulated by p53, and p53 functions as a transcriptional factor to directly activate the expression of PinX1 in cervical cancer cells. Moreover, we found that HPV16 E6 suppresses the expression of PinX1 via inhibiting p53 transcriptional activity, resulting in the enhancement of telomerase activity. This study not only for the first time shows that PinX1 is a novel target gene of p53 but also suggests that suppression of p53/PinX1 pathway may be a novel mechanism by which HPV16 E6 enhances the telomerase activity in cervical cancer cells.

Activation of FXR protects against renal fibrosis via suppressing Smad3 expression

Renal fibrosis is the common pathway of most chronic kidney disease progression to end-stage renal failure. The nuclear receptor FXR (farnesoid X receptor), a multiple functional transcription factor, plays an important role in protecting against fibrosis. The TGFβ-Smad signaling has a central role in kidney fibrosis. However, it remains unclear whether FXR plays direct anti-fibrotic effect in renal fibrosis via regulating TGFβ-Smad pathway. In this study, we found that the level of FXR was negatively correlated with that of Smad3 and fibronectin (a marker of fibrosis) in human fibrotic kidneys. Activation of FXR suppressed kidney fibrosis and downregulated Smad3 expression, which was markedly attenuated by FXR antagonist. Moreover, the FXR-mediated repression of fibrosis was significantly alleviated by ectopic expression of Smad3. Luciferase reporter assay revealed that FXR activation inhibited the transcriptional activity of Smad3 gene promoter. The in vivo experiments showed that FXR agonist protected against renal fibrosis and downregulated Smad3 expression in UUO mice. These results suggested that FXR may serve as an important negative regulator for manipulating Smad3 expression, and the FXR/Smad3 pathway may be a novel target for the treatment of renal fibrosis.