LncRNA MALAT1-deficiency restrains lipopolysaccharide (LPS)-induced pyroptotic cell death and inflammation in HK-2 cells by releasing microRNA-135b-5p

Long non-coding RNAs (LncRNAs) participate in the regulation of chronic kidney disease (CKD), and acute kidney injury (AKI) is identified as an important risk factor for CKD. This study investigated the involvement of a novel LncRNA MALAT1 in regulating lipopolysaccharide (LPS)-induced cell pyroptosis and inflammation in the human renal tubular epithelial HK-2 cells. Here, the HK-2 cells were subjected to LPS (2 μg/mL) treatment to establish cellular AKI models in vitro, and we validated that LPS triggered NLRP3-mediated pyroptotic cell death, promoted cell apoptosis and inflammation-associated cytokines secretion to induce HK-2 cell injury. Then, a novel LncRNA MALAT1/miRNA (miRNA)-135b-5p axis was verified to rescue cell viability in LPS treated HK-2 cells by targeting NLRP3. Mechanistically, miRNA-135b-5p bound to LncRNA MALAT1, and LncRNA MALAT1 positively regulated NLRP3 through acting as RNA sponger for miRNA-135b-5p. Further gain- and loss-of-function experiments evidenced that both LncRNA MALAT1 ablation and miRNA-135b-5p overexpression reversed LPS-induced cell pyroptosis, apoptosis, and inflammation in the HK-2 cells, and the protective effects of LncRNA MALAT1 knock-down on LPS-treated HK-2 cells were abrogated by silencing miRNA-135b-5p. In general, our study firstly investigated the role of the LncRNA MALAT1/ miRNA-135b-5p/NLRP3 signaling cascade in regulating LPS-induced inflammatory death in HK-2 cells.     

LncRNA MALAT1 Promotes Lung Cancer Proliferation and Gefitinib Resistance by Acting as a miR-200a Sponge

IntroductionLung cancer is a major public health problem, as the second causes of cancer related death worldwide, with relatively low survival rates, and accessible drug resistance. Long non-coding RNAs (LncRNAs) have been identified as activator in lung cancer with elusive mechanisms. We aimed to detect the regulation of LncRNA MALAT1 in the proliferation and gefitinib resistance in lung cancer cells.MethodsMALAT1 in A549 and HCC 1299 human lung adenocarcinoma cell lines was silenced by shRNA or overexpressed using plasmid, and the cell viability and cell proliferation were evaluated by MTT assay and soft agar colony formation assay. RNA levels were detected by RT-PCR, and the protein expression was measured by western blot. The binding between MALAT1 and miR-200a was validated by luciferase reporter assays using pSi-Chech 2 vectors.ResultsThe cell viability and proliferation of A549 cells transfected with MALAT1 shRNA were significantly lower than the control. The MALAT1 expression in gefitinib resistant A549 cells was upregulated. miR-200a significantly inhibited the fluorescence of pSi-Check 2 vector with MALAT1 gene, suggesting the direct binding between MALAT1 and miR-200a. In addition, LncRNA MALAT1 promotes ZEB1 expression in A549 cells.ConclusionOur study showed that MALAT1 promoted the proliferation and gefitinib resistance of lung cancer cells by sponging miR-200a, which regulates expression of ZEB1 in the A549 cells. This MALAT1/miR-200a axis could serve as new therapeutic target for lung cancer treatment.     

An Essential Role of NRF2 in Diabetic Wound Healing

The high mortality and disability of diabetic nonhealing skin ulcers create an urgent need for the development of more efficacious strategies targeting diabetic wound healing. In the current study, using human clinical specimens, we show that perilesional skin tissues from patients with diabetes are under more severe oxidative stress and display higher activation of the nuclear factor-E2–related factor 2 (NRF2)–mediated antioxidant response than perilesional skin tissues from normoglycemic patients. In a streptozotocin-induced diabetes mouse model, Nrf2^−/− mice have delayed wound closure rates compared with Nrf2^+/+ mice, which is, at least partially, due to greater oxidative DNA damage, low transforming growth factor-β1 (TGF-β1) and high matrix metalloproteinase 9 (MMP9) expression, and increased apoptosis. More importantly, pharmacological activation of the NRF2 pathway significantly improves diabetic wound healing. In vitro experiments in human immortalized keratinocyte cells confirm that NRF2 contributes to wound healing by alleviating oxidative stress, increasing proliferation and migration, decreasing apoptosis, and increasing the expression of TGF-β1 and lowering MMP9 under high-glucose conditions. This study indicates an essential role for NRF2 in diabetic wound healing and the therapeutic benefits of activating NRF2 in this disease, laying the foundation for future clinical trials using NRF2 activators in treating diabetic skin ulcers.     

Catalase Overexpression Prevents Nuclear Factor Erythroid 2–Related Factor 2 Stimulation of Renal Angiotensinogen Gene Expression,Hypertension, and Kidney Injury in Diabetic Mice

This study investigated the impact of catalase (Cat) overexpression in renal proximal tubule cells (RPTCs) on nuclear factor erythroid 2–related factor 2 (Nrf2) stimulation of angiotensinogen (Agt) gene expression and the development of hypertension and renal injury in diabetic Akita transgenic mice. Additionally, adult male mice were treated with the Nrf2 activator oltipraz with or without the inhibitor trigonelline. Rat RPTCs, stably transfected with plasmid containing either rat Agt or Nrf2 gene promoter, were also studied. Cat overexpression normalized systolic BP, attenuated renal injury, and inhibited RPTC Nrf2, Agt, and heme oxygenase-1 (HO-1) gene expression in Akita Cat transgenic mice compared with Akita mice. In vitro, high glucose level, hydrogen peroxide, and oltipraz stimulated Nrf2 and Agt gene expression; these changes were blocked by trigonelline, small interfering RNAs of Nrf2, antioxidants, or pharmacological inhibitors of nuclear factor-κB and p38 mitogen-activated protein kinase. The deletion of Nrf2-responsive elements in the rat Agt gene promoter abolished the stimulatory effect of oltipraz. Oltipraz administration also augmented Agt, HO-1, and Nrf2 gene expression in mouse RPTCs and was reversed by trigonelline. These data identify a novel mechanism, Nrf2-mediated stimulation of intrarenal Agt gene expression and activation of the renin-angiotensin system, by which hyperglycemia induces hypertension and renal injury in diabetic mice.     

Absence of Dipeptidyl Peptidase 3 Increases Oxidative Stress and Causes Bone Loss

Controlling oxidative stress through the activation of antioxidant pathways is crucial in bone homeostasis, and impairments of the cellular defense systems involved contribute to the pathogenesis of common skeletal diseases. In this work we focused on the dipeptidyl peptidase 3 (DPP3), a poorly investigated ubiquitous zinc-dependent exopeptidase activating the Keap1-Nrf2 antioxidant pathway. We showed Dpp3 expression in bone and, to understand its role in this compartment, we generated a Dpp3 knockout (KO) mouse model and specifically investigated the skeletal phenotype. Adult Dpp3 KO mice showed a mild growth defect, a significant increase in bone marrow cellularity, and bone loss mainly caused by increased osteoclast activity. Overall, in the mouse model, lack of DPP3 resulted in sustained oxidative stress and in alterations of bone microenvironment favoring the osteoclast compared to the osteoblast lineage. Accordingly, in vitro studies revealed that Dpp3 KO osteoclasts had an inherent increased resorptive activity and ROS production, which on the other hand made them prone to apoptosis. Moreover, absence of DPP3 augmented bone loss after estrogen withdrawal in female mice, further supporting its relevance in the framework of bone pathophysiology. Overall, we show a nonredundant role for DPP3 in the maintenance of bone homeostasis and propose that DPP3 might represent a possible new osteoimmunological player and a marker of human bone loss pathology. © 2019 American Society for Bone and Mineral Research.     

Abrogation of MMP-9 gene protects against the development of retinopathy in diabetic mice by preventing mitochondrial damage

OBJECTIVE

In the development of diabetic retinopathy, mitochondrial dysfunction is considered to play an important role in the apoptosis of retinal capillary cells. Diabetes activates matrix metalloproteinase-9 (MMP-9) in the retina and its capillary cells, and activated MMP-9 becomes proapoptotic. The objective of this study is to elucidate the plausible mechanism by which active MMP-9 contributes to the mitochondrial dysfunction in the retina.

RESEARCH DESIGN AND METHODS

Using MMP-9 gene knockout (MMP-KO) mice, we investigated the effect of MMP-9 regulation on diabetes-induced increased retinal capillary cell apoptosis, development of retinopathy, mitochondrial dysfunction and ultrastructure, and mitochondrial DNA (mtDNA) damage. To understand how diabetes increases mitochondrial accumulation of MMP-9, interactions between MMP-9 and chaperone proteins (heat shock protein [Hsp] 70 and Hsp60) were evaluated. The results were confirmed in the retinal mitochondria from human donors with diabetic retinopathy, and in isolated retinal endothelial cells transfected with MMP-9 small interfering RNA (siRNA).

RESULTS

Retinal microvasculature of MMP-KO mice, diabetic for ∼7 months, did not show increased apoptosis and pathology characteristic of retinopathy. In the same MMP-KO diabetic mice, activation of MMP-9 and dysfunction of the mitochondria were prevented, and electron microscopy of the retinal microvasculature region revealed normal mitochondrial matrix and packed lamellar cristae. Damage to mtDNA was protected, and the binding of MMP-9 with Hsp70 or Hsp60 was also normal. As in the retina from wild-type diabetic mice, activation of mitochondrial MMP-9 and alterations in the binding of MMP-9 with chaperone proteins were also observed in the retina from donors with diabetic retinopathy. In endothelial cells transfected with MMP-9 siRNA, high glucose–induced damage to the mitochondria and the chaperone machinery was ameliorated.

CONCLUSIONS

Regulation of activated MMP-9 prevents retinal capillary cells from undergoing apoptosis by protecting mitochondrial ultrastructure and function and preventing mtDNA damage. Thus, MMP-9 inhibitors could have potential therapeutic value in preventing the development of diabetic retinopathy by preventing the continuation of the vicious cycle of mitochondrial damage.Diabetic retinopathy is one of the major causes of acquired blindness in working adults, but despite extensive research in the field, the molecular mechanism of its development remains elusive. In the pathogenesis of this slow progressing disease, capillary cells and other retinal cells are lost by apoptosis before histopathology characteristic of diabetic retinopathy can be seen in the retina (1–4). However, how a diabetic environment accelerates retinal cell apoptosis is unclear.Matrix metalloproteinases (MMPs), a class of approximately 25 zinc-dependent proteinases, regulate a variety of cellular functions, including apoptosis, proliferation, differentiation, and angiogenesis. In diabetes, MMPs are elevated in the retina and other tissues, and MMP-9, the largest member of the MMP family (5), is associated with many diabetes complications, including nephropathy, cardiomyopathy, and retinopathy (6). Our previous work has shown that in a diabetic environment, the activation of MMP-9 in the retina and its capillary cells is mediated by the Ras/Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase pathway, and activated MMP-9 induces the apoptosis of retinal capillary cells (7,8). The mechanism by which MMP-9 induces apoptosis in the pathogenesis of diabetic retinopathy remains to be explored.Damage to the retinal mitochondria is considered to play a major role in the development of diabetic retinopathy. Retinal mitochondria become dysfunctional, superoxide levels are elevated, and mitochondrial permeability is significantly increased, which leads to leakage of cytochrome c into the cytosol and acceleration of the apoptosis of retinal capillary cells (9,10). Emerging work has shown that MMPs are not present in the matrix alone, they are also found in the mitochondria, and induction of MMP-9 in the myocyte mitochondria is considered to act as a negative regulator of mitochondrial function (11). Our recent work has shown that MMP-2, another important member of MMP family, is present in the retinal mitochondria, and in diabetes, MMP-2 damages retinal mitochondria by modulating connexin43 (12). As diabetes activates both MMP-2 and MMP-9 in the retina, accelerating apoptosis of retinal capillary cells (7,8,12), the role of MMP-9 in retinal mitochondrial dysfunction in diabetes, leading to retinal capillary cell loss, needs further investigation.The aim of this study is to elucidate the plausible mechanism by which active MMP-9 contributes to the mitochondrial dysfunction in the retina, accelerating the apoptosis of capillary cells and ultimately resulting in retinopathy. Using MMP-9 gene knockout (MMP-KO) mice, we have investigated the effect of MMP-9 regulation on diabetes-induced increased retinal capillary cell apoptosis and the development of retinopathy. To understand the mechanism, the effect of regulation of MMP-9 on retinal mitochondrial dysfunction, mitochondrial DNA (mtDNA) damage and ultrastructure was evaluated in MMP-KO mice. As mitochondrial function is largely controlled by their membrane structure, and translocases in the outer membrane (the TOM complex) and translocases in the mitochondrial inner membrane (the TIM complex) help facilitate import of proteins (13,14), we have also investigated the mechanism by which diabetes increases mitochondrial accumulation of MMP-9. The results are confirmed in the retina from human donors with diabetic retinopathy and also in isolated retinal endothelial cells exposed to high glucose with the MMP-9 gene silenced.