Up-regulation of THY1 attenuates interstitial pulmonary fibrosis and promotes lung fibroblast apoptosis during acute interstitial pneumonia by blockade of the WNT signaling pathway

Acute interstitial pneumonia (AIP) is an idiopathic pulmonary disease featuring rapid progressive dyspnea and respiratory failure. These symptoms typically develop within several days or weeks in patients without any pre-existing lung disease or external chest disease. Thymocyte differentiation antigen-1 (THY1) has been reported to have an effect on lung fibroblast proliferation and fibrogenic signaling. In this study, the mechanism of THY1 in AIP in influencing pulmonary fibrosis in terms of lung fibroblast proliferation and apoptosis was examined. An AIP mouse model with the pathological changes of lung tissues observed was established to identify the role of THY1 in the pathogenesis of AIP. The expression of THY1, a key regulator of the WNT pathway β-catenin and fibroblasts markers MMP-2, Occludin, α-SMA and Vimentin were determined. Lung fibroblasts of mice were isolated, in which THY1 expression was altered to identify roles THY1 plays in cell viability and apoptosis. A TOP/TOPflash assay was utilized to determine the activation of WNT pathway. Decrement of pulmonary fibrosis was achieved through THY1 up-regulation. The expression of MMP-2, Occludin, α-SMA, Vimentin and β-catenin, and the extent of β-catenin phosphorylation, significantly decreased, thereby indicating that THY1 overexpression inactivated WNT. Cell proliferation was inhibited and apoptosis was accelerated in lung fibroblasts transfected with vector carrying overexpressed THY1. Altogether, this study defines the potential role of THY1 in remission of AIP, via the upregulation of THY1, which renders the WNT pathway inactive. This inactivation of the WNT signaling pathway could alleviate pulmonary fibrosis by reducing lung fibroblast proliferation in AIP.

Abbreviations: AIP: Acute interstitial pneumonia; ILDs: interstitial lung diseases; DAD: diffuse alveolar damage; SPF: specific-pathogen-free; NC: negative control; HCMV: human cytomegalovirus; HE: Hematoxylin-eosin; RIPA: radio-immunoprecipitation assay; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BSA: bovine serum albumin; HRP: horseradish peroxidase; ECL: electrochemiluminescence; FBS: fetal bovine serum; DMSO: dimethyl sulfoxide; OD: optical density     

Retracted: DLX5 gene regulates the Notch signaling pathway to promote glomerulosclerosis and interstitial fibrosis in uremic rats

Uremia largely results from the accumulation of organic waste products normally cleared by the kidneys, which commonly accompanies kidney failure and chronic kidney disease. However, genetic investigations in a uremia remain largely unclear. This study aimed to determine the expression patterns of distal-less homeobox 5 (DLX5) in uremia rat model and further to study its effects on glomerulosclerosis and interstitial fibrosis. Uremic expression chip was applied to screen differentially expressed genes in uremia. Next, we used small interfering RNA-mediated RNA interference to specifically silence DLX5 in experimental uremic rats to understand the regulatory mechanism of DLX5. To understand effect of Notch1 signaling pathway in uremia, we also treated experimental uremic rats with γ-secretase inhibitor (GSI), an inhibitor of Notch1 signaling pathway. The expression of fibronectin (FN), laminin (LN), transforming growth factor-β1 (TGF-β1), Hes1, Hes5, and Jagged2 was determined. The semiquantitative assessment was applied to verify the effects of DLX5 on glomerulosclerosis. In the uremic expression chip, we found that DLX5 was upregulated in uremia samples, and considered to regulate the Notch signaling pathway. We found that small interfering RNA-mediated DLX5 inhibition or Notch1 signaling pathway inhibitory treatment relieved and delayed the kidney injury and glomerulosclerosis in uremia. Meanwhile, inhibition of DLX5 or Nothch1 signaling pathway reduced expression of FN, LN, Nothch1, TGF-β1, Hes1, Hes5, and Jagged2. Intriguingly, we discovered that Notch1 signaling pathway was inhibited after silencing DLX5. In conclusion, these findings highlight that DLX5 regulates Notch signaling, which may, in turn, promote complications of uremia such as kidney fibrosis, providing a novel therapeutic target for treating uremia.     

The potential signaling pathway between peroxisome proliferator-activated receptor gamma and retinoic acid receptor alpha in renal interstitial fibrosis disease

Abstract

Peroxisome proliferator-activated receptorγ (PPARγ) can regulate the process of cell apoptosis and is related to the progression of renal disorders. Retinoic acid receptor alpha (RARα) is one of the nuclear receptors involved in a variety of kidney diseases. Renal interstitial fibrosis (RIF) is a common denominator of chronic kidney disease (CKD). This study investigated whether a potential signaling pathway existed between PPARγ and RARα in RIF rats with unilateral ureteral obstruction (UUO). The rats were randomly divided into four groups: a model group subjected to UUO (GU), and three other groups treated with rosiglitazone sodium (GRS), GW9662 and dimethyl sulfoxide (DMSO), n?=?40, respectively. Renal tissues were collected two and four weeks after post-surgery. The relevant indicators were detected. In comparison with the GU group, the expressions of PPARγ and RARα (protein and mRNA) were increased in the GRS group, and decreased in the GW9662 group (all p?<?0.01). The RIF index, mRNA and protein expression of transforming growth factor-β1 (TGF-β1), and the protein expressions of collagen-IV (Col-IV) and fibronectin (FN) in the GRS group were more markedly reduced than those in the GU group; their levels in the GW9662 group were elevated (all p?<?0.01). PPARγ or RARα was negatively correlated to the RIF index, TGF-β1, Col-IV and FN. PPARγ was positively correlated with RARα (all p?<?0.01). In conclusion, PPARγ agonist can elevate the expression of PPARγ or RARα in RIF rats. There might be a potential signaling pathway between PPARγ and RARα in RIF disease.     

microRNA-665 silencing improves cardiac function in rats with heart failure through activation of the cAMP signaling pathway

Heart failure (HF) is a disease with high mortality and morbidity rate. Previous studies have shown that microRNAs (miRNAs) may be implicated in the pathogenesis of HF, potentially being able to improve the cardiac function in an HF rat model. The present study was designed to define the role of miR-665 in the cardiac function of the HF rats. Following the establishm;ent of the rat models of HF, the functional role miR-665 in HF was determined using an ectopic expression and knockdown experiments. The cardiac function was evaluated with the determination of ventricular mass index and hemodynamic parameters. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining was performed, with the apoptosis of cardiac cells detected in the process. The expression of miR-665, glucagon-like peptide 1 receptor (GLP1R), cyclic adenosine monophosphate (cAMP) signaling pathway-related, and apoptosis-related genes was examined. Enzyme-linked immunosorbent assay was conducted to determine the levels of inflammation-related genes. Initially, the upregulation of miR-665, downregulation of GLP1R, and inactivation of cAMP signaling pathway were observed in HF rats. GLP1R was a target of miR-665. Forced expression of miR-665 promoted cell apoptosis and inhibited GLP1R and the cAMP signaling pathway. In addition, miR-665 overexpression has been known to impair cardiac function, promote inflammatory response while elevating malondialdehyde and superoxide dismutase levels, and decreasing mitochondrial respiratory chain enzyme activities. Furthermore, we also observed that the effects of miR-665 inhibition had been reversed when the cAMP signaling pathway was also inhibited. This study demonstrates that miR-665 inhibition can stabilize the cardiac function of HF rats via the cAMP signaling pathway via upregulation of the GLP1R.     

Retracted: Silencing of keratin 1 inactivates the Notch signaling pathway to inhibit renal interstitial fibrosis and glomerular sclerosis in uremia

Renal interstitial fibrosis is a key factor in the development of chronic renal diseases, possibly leading to uremia. The present study conducted aimed to assess the hypothesis whether keratin 1 (KRT1) silencing could suppress kidney interstitial fibrosis and glomerular sclerosis via the Notch pathway to alleviate uremic symptoms. Differentially expressed genes associated with uremia were identified using the gene expression omnibus (GEO) database. Uremic rat models were established, in which short hairpin-RNA against KRT1, activators, and inhibitors of the Notch pathway were transfected. To further validate the mechanism of KRT1 in uremia, KRT1 expression, cell apoptosis, glomerular area (GA), and glomerular capillary volume (GV), the score of glomerular sclerosis, and tubulointerstitial injury were assayed and investigated. GEO database revealed that KRT1 was upregulated in uremia and regulated the Notch pathway. GA, GV, cell apoptosis, glomerular sclerosis, and tubulointerstitial injury were typically located in more elevated levels of uremia in rats. KRT1 silencing and Notch pathway inhibition decreased the expression of Jagged1, Notch1, NICD1, Hey1, Hes1, α-SMA, and FN, which further resulted in decreased cell apoptosis, GA, GV, the score of glomerular sclerosis, and tubulointerstitial injury. Subsequently, the effect of KRT1 silencing on uremia was no longer evident once the Notch pathway was activated. The co-localization of high expression KRT1 and Notch1 was found in uremia. In summary, the results identified KRT1 as a key regulator in uremia progression, and KRT1 silencing can suppress glomerular sclerosis and tubulointerstitial injury via inactivation of the Notch pathway in uremic rats.     

microRNA-206 overexpression inhibits epithelial-mesenchymal transition and glomerulosclerosis in rats with chronic kidney disease by inhibiting JAK/STAT signaling pathway

Chronic kidney disease (CKD) is a traumatic disease with significant psychic consequences to the patient's overall physical condition. microRNA-206 (miR-206) has been reported to play an essential role in the development of various diseases. The purpose of the present study is to investigate the effect of miR-206 through the JAK/STAT signaling pathway on epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells and glomerulosclerosis in rats with CKD. The targeting relationship between miR-206 and ANXA1 was verified. To explore the role of miR-206 in CKD, the model of CKD rats was established to detect glomerular sclerosis index (GSI), contents of interleukin-6 (IL-6) and transforming growth factor-beta1 (TGF-β1), and expression of type IV collagen. Moreover, to further determine the roles of both miR-206 and the JAK/STAT signaling pathway in CKD, the gain- and loss-of function approaches were performed with the expression of ANXA1, α-SMA, E-cadherin, vimentin, N-cadherin, and the JAK/STAT signaling pathway-related genes detected. miR-206 negatively targeted ANXA1. Overexpressed miR-206 inhibited the degeneration and interstitial fibrosis of renal tubular epithelial cells, decreased GSI of rats, and the expression of type IV collagen, TGF-β1 and IL-6. Overexpressed miR-206 inhibited the degeneration of renal tubular epithelial cells, the expression of ANXA1, α-SMA, TGF-β1, p-STAT3, STAT3, p-STAT1, STAT1, p-JAK2, and JAK2, while promoted the expression of E-cadherin. Taken together the results, miR-206 inhibits EMT of renal tubular epithelial cells and glomerulosclerosis by inactivating the JAK/STAT signaling pathway via ANXA1 in CKD.     

Overexpression of microRNA-186 inhibits angiogenesis in retinoblastoma via the Hedgehog signaling pathway by targeting ATAD2

Retinoblastoma (RB) represents an aggressive malignancy in the eye during the period of infancy and childhood. We delineated the ability of microRNA-186 (miR-186) to influence viability, invasion, migration, angiogenesis, and apoptosis of RB via the Hedgehog signaling pathway by targeting AAA domain-containing protein 2 (ATAD2). The microarray-based analysis was adopted to identify differentially expressed genes (DEGs) related to RB. Subsequently, RB cells were treated with miR-186 mimic, miR-186 inhibitor, or si-ATAD2. The expression of miR-186, ATAD2, Hedgehog signaling pathway-related genes were evaluated, and the target relationship between miR-186 and ATAD2 was verified. Finally, cell proliferation, invasion, migration, apoptosis, and angiogenesis were assessed. ATAD2 was identified as a DEG and modulated by miR-186. Moreover, we revealed that ATAD2 was highly expressed, whereas miR-186 was lowly expressed, and the Hedgehog signaling pathway was activated in RB. Then, ATAD2 as a putative target of miR-186 was validated using a luciferase assay. miR-186 mimic or siRNA-ATAD2 in RB cells reduced cell viability, invasion, and migration coordinating with elevated apoptosis via impairing the Hedgehog signaling pathway, where repressed angiogenesis was observed. Overexpression of miR-186 attenuates RB via the inactivation of the Hedgehog signaling pathway by downregulating ATAD2.     

Downregulation of microRNA-23b protects against ischemia-reperfusion injury via p53 signaling pathway by upregulating MDM4 in rats

Total knee arthroplasty is a commonly performed safe procedure and typically executed in severe knee arthritis, but it also triggers ischemia-reperfusion injury (IRI). More recently, microRNAs (miRs) have been reported to play a contributory role in IRI through the key signaling pathway. Hence, the current study aimed to investigate the effect and specific mechanism of microRNA-23b (miR-23b), murine double minute 4 (MDM4), and the p53 signaling pathway in IRI rat models. First, the IRI model was established, and the expression pattern of miR-23b, MDM4, and the p53 signaling pathway-related genes was characterized in cartilaginous tissues. Then, miR-23b mimics or inhibitors were applied for the elevation or the depletion of the miR-23b expression and siRNA-MDM4 for the depletion of the MDM4 expression in the articular chondrocytes. By means of immunohistochemistry, quantitative real-time polymerase chain reaction, and Western blot analysis, IRI rats exhibited increased miR-23b expression, activated p53 signaling pathway, and decreased MDM4 expression. MDM4 was verified as a target gene of miR-23b through. Downregulated miR-23b increased the expression of MDM4, AKT, and Bcl-2, but decreased the expression of p53, p21, and Bax. In addition, a series of cell experiments demonstrated that downregulated miR-23b promoted articular chondrocyte proliferation and cell cycle entry, but inhibited articular chondrocyte apoptosis. The absence of the effects of miR-23b was observed after MDM4 knocked down. Our results indicate that silencing miR-23b could act to attenuate IRI and reduce the apoptosis of articular chondrocytes through inactivation of the p53 signaling pathway by upregulating MDM4, which provide basic therapeutic considerations for a novel target against IRI.     

Umbilical cord-derived mesenchymal stem cells alleviated inflammation and inhibited apoptosis in interstitial cystitis via AKT/mTOR signaling pathway

Interstitial cystitis (IC) is a bladder syndrome characterized by pelvic pain and urinary frequency without infection or other identifiable pathology. There are no effective treatments to cure IC. This study investigated the effects of human umbilical cord-derived mesenchymal stem cells (UC-MSCs) injection on IC rat model. Furthermore, we used a coculture system to find the possible molecular mechanism on the human uroepithelial cells (SV-HUC-1), which was the cell model of IC. A rat model of IC was established via systemic injection with cyclophosphamide (CYP) and a cell model of IC was induced by being exposed to tumor necrosis factor (TNF)-α (10 ng/ml). After one week, UC-MSCs injection significantly ameliorated the bladder voiding function in IC rat model. And the Histo- and immunohistochemical analyses showed that UC-MSCs can repair impaired bladder, reduce mast cell infiltration and inhibit apoptosis of urothelium. ELISA results showed that UC-MSCs can decrease IL-1β, IL-6 and TNF-α in bladder. In the coculture system, UC-MSCs can promote proliferation of impaired SV-HUC-1 cells, and inhibit apoptosis. However, while knocked down EGF secreted by UC-MSCs with siRNA, the effects would be weaken. Western blot showed that UC-MSCs increase protein expression levels of p-AKT and p-mTOR in SV-HUC-1 cells, and decrease the levels of cleaved caspase-3. Taken together, we provide evidence that UC-MSCs therapy can successfully alleviate IC in a preclinical animal Model and cell model by alleviating inflammation, promoting proliferation and inhibiting apoptosis. In addition, we demonstrate that the AKT/mTOR signaling pathway was activated.     

Protective effect of microRNA-224 on acute lower extremity ischemia through activation of the mTOR signaling pathway via CHOP in mice

Acute lower extremity ischemia (ALEXI) is known worldwide as an urgent condition, occurring when there is an abrupt interruption in blood flow into an extremity. This study aims to investigate whether microRNA-224 (miR-224) affects the ALEXI mice and the underlying mechanism. The miR-224 expression and C/EBP homologous protein (CHOP), mammalian target of rapamycin (mTOR), translation initiation factor 4E-binding protein 1 (4E-BP1), and phosphoprotein 70 ribosomal protein S6 kinase (p70S6K) messenger RNA (mRNA), as well as protein expressions, were determined. The target gene of miR-224 was also verified by using a luciferase reporter gene assay. The vascular endothelial cells from the ALEXI mice were transfected with miR-224 mimics, miR-224 inhibitors, or small-interfering RNA against CHOP. Cell proliferation was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cell cycle distribution along with the cell apoptosis were both evaluated by using a flow cytometry. The muscle fibers of the lower extremities found in the ALEXI mice were evidently swollen and rounded, presenting with a remarkably narrowed gap. The positive CHOP expression increased in ALEXI mice than normal mice, while the miR-224 expression and mTOR, 4E-BP1, and p70S6K mRNA, as well as the protein expression, decreased. Luciferase reporter gene assay validated that the miR-224 gene directly targeted CHOP. MiR-224 facilitated cell proliferation but inhibited cell apoptosis; by contrast, CHOP increased cell apoptosis. Moreover, the cells transfected along with miR-224 mimic exhibited a lower CHOP expression as well as increased mTOR, 4E-BP1, and p70S6K expression. Our study provided evidence that miR-224 could alleviate the occurrence and development of ALEXI in mice through activation of the mTOR signaling pathway by downregulating CHOP.     

Curcumin induces growth-arrest and apoptosis in association with the inhibition of constitutively active JAK-STAT pathway in T cell leukemia

Adult T cell leukemia is an aggressive and frequently fatal malignancy that expressess constitutively activated growth-signaling pathways in association with deregulated growth and resistance to apoptosis. Curcumin (diferuloylmethane) is a naturally occurring yellow pigment, isolated from the rhizomes of the plant Curcuma longa that has traditionally been used in the treatment of injury and inflammation. But the effect and mechanism of action of curcumin on T cell leukemia is not known. To investigate the antitumor activity of curcumin in T cell leukemia, we examined its effect on constitutive phosphorylation of JAK and STAT proteins, proliferation, and apoptosis in HTLV-I-transformed T cell lines. HTLV-I-transformed T cell leukemia lines, MT-2, HuT-102, and SLB-1, express constitutively phosphorylated JAK3, TYK2, STAT3, and STAT5 signaling proteins. In vitro treatment with curcumin induced a dose-dependent decrease in JAK and STAT phosphorylation resulting in the induction of growth-arrest and apoptosis in T cell leukemia. The induction of growth-arrest and apoptosis in association with the blockade of constitutively active JAK-STAT pathway suggests this be a mechanism by which curcumin induces antitumor activity in T cell leukemia.     

microRNA-448 inhibits stemness maintenance and self-renewal of hepatocellular carcinoma stem cells through the MAGEA6-mediated AMPK signaling pathway

Hepatocellular carcinoma (HCC) occurs mainly in patients with chronic liver disease and cirrhosis. Increasing evidence has identified the involvement of microRNAs (miRNAs) acting as essential regulators in the progression of HCC. As predicted by microarray analysis, miR-448 might potentially affect HCC progression by regulating the melanoma-associated antigen (MAGEA). Therefore, the present investigation focused on exploring whether or not miR-448 and MAGEA6 were involved in the self-renewal and stemness maintenance of HCC stem cells. The interaction among miR-448, MAGEA6, and the AMPK signaling pathway was evaluated. It was noted that miR-448 targeted and downregulated MAGEA6, thus activating the AMP-activated protein kinase (AMPK) signaling pathway in HCC. Furthermore, for the purpose of exploring the functional relevance of MAGEA6 and miR-448 on the sphere formation, colony formation, and invasion and migration of HCC stem cells, the CD133⁺CD44 ⁺ HCC stem cells were sorted and treated with the mimic or inhibitor of miR-448, small interfering RNA (siRNA) against MAGEA6 or an AMPK activator AICAR. MAGEA6 silencing or miR-448 overexpression was demonstrated to inhibit the abilities of sphere formation, colony formation, cell migration, and invasion of HCC cells. Afterwards, a rescue experiment was conducted and revealed that MAGEA6 silencing reversed the effects of miR-448 inhibitor on stemness maintenance and self-renewal of HCC stem cells. Finally, after the in vivo experiment was carried out, miR-448 was observed to restrain the tumor formation and stemness in vivo. Altogether, miR-448 activates the AMPK signaling pathway by downregulating MAGEA6, thus inhibiting the stemness maintenance and self-renewal of HCC stem cells, which identifies miR-448 as a new therapeutic strategy for HCC.     

microRNA-96 promotes osteoblast differentiation and bone formation in ankylosing spondylitis mice through activating the Wnt signaling pathway by binding to SOST

Ankylosing spondylitis (AS) refers to a type of arthritis manifested with chronic inflammation of spine joints. microRNAs (MiRNAs) have been identified as new therapeutic targets for inflammatory diseases. In this study, we evaluated the influence of microRNA-96 (miR-96) on osteoblast differentiation together with bone formation in a murine model of AS. The speculated relationship that miR-96 could bind to sclerostin (SOST) was verified by dual luciferase reporter assay. After successful model establishment, the mice with AS and osteoblasts isolated from mice with AS were treated with mimics or inhibitors of miR-96, or DKK-1 (a Wnt signaling inhibitor). The effects of gain- or loss-of-function of miR-96 on the inflammatory cytokine release (IL-6, IL-10, and TNF-α), alkaline phosphatase (ALP) activity, calcium nodule formation, along with the viability of osteoblasts were determined. It was observed that miR-96 might target and regulate SOST. Besides, miR-96 was expressed at a high level in AS mice while SOST expressed at a low level. TOP/FOP-Flash luciferase reporter assay confirmed that miR-96 activated the Wnt signaling pathway. Moreover, AS mice overexpressing miR-96 exhibited increased contents of IL-6, IL-10 and TNF-α, ALP activity, calcium nodule numbers, and viability of osteoblasts. In contrast, inhibition of miR-96 resulted in suppression of the osteoblast differentiation and bone formation. In conclusion, the study implicates that overexpressing miR-96 could improve osteoblast differentiation and bone formation in AS mice via Wnt signaling pathway activation, highlighting a potential new target for AS treatment.