scholarly journals Lnc027912 Reduces Lipid Accumulation Through Akt/MTOR/SREBP1C Axis in Hepatic Cells

2022 ◽  
Author(s):  
Kaifei Chu ◽  
Niannian Zhao ◽  
Rong Feng ◽  
Li Zhang ◽  
Xudong Hu ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Western Blot ◽  
Lipid Accumulation ◽  
Cholesterol Metabolism ◽  
Cell Model ◽  
Luciferase Reporter ◽  
Hepatic Cells ◽  
Gene Assay ◽  
Signaling Axis

Abstract Background: Various metabolism diseases are closely related to lipid metabolism disorder, but long noncoding-RNAs (lncRNA) involve in regulating function of lipid was limited elucidated. Previous our work have found that lnc027912 involve in cholesterol metabolism. Here, we further explore the role of lipid metabolism-associated lncRNA-lnc027912 in oleic acid- (OA) and palmitic acid (PA)-induced hepatic cells. Methods: The overexpression of lnc027912 cell model was constructed by using virus particles transfection, and the level of lnc027912 in AML12 cells were detected by RT-qPCR. High fat cell model was established by treating AML12 cells with OA and PA, and the level of lipid drops was detected by Oil red O staining and triglyceride analyze Kit. The lipid metabolism related-genes, such as SREBP1C, FAS, PPARγ, MTTP, ApoE and ApoC3 level, was detected using RT-qPCR and Western blot. The role of SREBP1C in lipid metabolism was further analyzed using double luciferase reporter gene assay and Immunofluorescence. The Akt/mTOR signal pathway related genes was detected by Western blot. Results: We found that TG level was inhibited in overexpression of lnc027912 cell. Upregulated lnc027912 of AML12 cells treated with OA and PA showed a significant decrease in lipid accumulation and TG levels. Furthermore, overexpression of lnc027912, the lipid biosynthesis genes of SREBP1C, FAS and PPARγ was significantly decreased and a significant increase in expression of MTTP and ApoE. Interestingly, lnc027912 inhibited Akt/mTOR signaling axis and decreased SREBP1C transit into nucleus and the promoter activity of SREBP1C and regulated expression of its targets. Conclusions: Our study revealed a new insights into the molecular function of lnc027912 in lipid metabolism by Akt/mTOR/SREBP1C signaling axis and highlights the potential of lnc027912 as a new therapeutic target for lipid disorder diseases (such as, NAFLD).

scholarly journals HOXA10 mediates epithelial-mesenchymal transition to promote gastric cancer metastasis partly via modulation of TGFB2/Smad/METTL3 signaling axis

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Chenlong Song ◽  
Chongzhi Zhou
Keyword(s):  
Gastric Cancer ◽  
Western Blot ◽  
Lung Metastasis ◽  
Essential Role ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Signaling Axis

Abstract Background Homeobox A10 (HOXA10) belongs to the HOX gene family, which plays an essential role in embryonic development and tumor progression. We previously demonstrated that HOXA10 was significantly upregulated in gastric cancer (GC) and promoted GC cell proliferation. This study was designed to investigate the role of HOXA10 in GC metastasis and explore the underlying mechanism. Methods Immunohistochemistry (IHC) was used to evaluate the expression of HOXA10 in GC. In vitro cell migration and invasion assays as well as in vivo mice metastatic models were utilized to investigate the effects of HOXA10 on GC metastasis. GSEA, western blot, qRT-PCR and confocal immunofluorescence experiments preliminarily analyzed the relationship between HOXA10 and EMT. ChIP-qPCR, dual-luciferase reporter (DLR), co-immunoprecipitation (CoIP), colorimetric m6A assay and mice lung metastasis rescue models were performed to explore the mechanism by which HOXA10 accelerated the EMT process in GC. Results In this study, we demonstrated HOXA10 was upregulated in GC patients and the difference was even more pronounced in patients with lymph node metastasis (LNM) than without. Functionally, HOXA10 promoted migration and invasion of GC cells in vitro and accelerated lung metastasis in vivo. EMT was an important mechanism responsible for HOXA10-involved metastasis. Mechanistically, we revealed HOXA10 enriched in the TGFB2 promoter region, promoted transcription, increased secretion, thus triggered the activation of TGFβ/Smad signaling with subsequent enhancement of Smad2/3 nuclear expression. Moreover, HOXA10 upregulation elevated m6A level and METTL3 expression in GC cells possible by regulating the TGFB2/Smad pathway. CoIP and ChIP-qPCR experiments demonstrated that Smad proteins played an important role in mediating METTL3 expression. Furthermore, we found HOXA10 and METTL3 were clinically relevant, and METTL3 was responsible for the HOXA10-mediated EMT process by performing rescue experiments with western blot and in vivo mice lung metastatic models. Conclusions Our findings indicated the essential role of the HOXA10/TGFB2/Smad/METTL3 signaling axis in GC progression and metastasis.

scholarly journals MiR-429 Inhibits the Angiogenesis of Human Brain Microvascular Endothelial Cells through SNAI2-Mediated GSK-3β/β-Catenin Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Yameng Sun ◽  
Shenghao Ding ◽  
Yiling Fan ◽  
Fei Shen ◽  
Qing Dong ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Model ◽  
Glucose Deprivation ◽  
Tube Formation ◽  
Luciferase Reporter ◽  
Regulation Mechanism ◽  
Oxygen And Glucose Deprivation ◽  
Downstream Target ◽  
Gsk 3Β

MicroRNA (miRNA) dysfunction has been confirmed as a key event of ischemic stroke appearance. This study is aimed at revealing the role of miR-429 in the angiogenesis of HBMECs. The HBMECs were treated with oxygen and glucose deprivation (OGD) to establish the ischemic cell model. The qRT-PCR was used to measure the expression levels of the miR-429 in the serums of the patients or cells, and CCK-8, wound healing assay, and tube formation assay were used to observe the effects of miR-429 on the phenotype of HBMECs. Moreover, the Targetscan, dual-luciferase reporter assay, and Western blot were used to reveal the downstream target and regulation mechanism of miR-429 in OGD-induced HBMECs. The results showed that miR-429 was significantly upregulated in the serums of the patients, and overexpressed miR-429 could extremely inhibit the viability, migration, and tube formation of OGD-induced HBMECs. Furthermore, it was found that SNAI2 was a downstream factor of miR-429, and SNAI2 could rescue the effects of miR-429 on OGD-induced HBMECs. Besides, the Western blot showed that miR-429 could affect the activity of GSK-3β/β-catenin pathway via inhibiting the expression of SNAI2. In conclusion, this study suggests that miR-429 inhibits the angiogenesis of HBMECs through SNAI2-mediated GSK-3β/β-catenin pathway.

scholarly journals miR-129-5p Promotes Osteogenic Differentiation of BMSCs and Bone Regeneration via Repressing Dkk3

2021 ◽  
Vol 2021 ◽  
pp. 1-18
Author(s):  
Changming Zhao ◽  
Yulin Gu ◽  
Yan Wang ◽  
Qiaozhen Qin ◽  
Ting Wang ◽  
...  
Keyword(s):  
Western Blot ◽  
Bone Regeneration ◽  
Osteogenic Differentiation ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Alizarin Red ◽  
Gene Assay

Objective. Accumulating evidence indicates that microRNAs (miRNAs) play crucial roles in osteogenic differentiation. However, the associated mechanisms remain elusive. This paper is aimed at exploring the role of miR-129-5p in regulating bone marrow mesenchymal stem cell (BMSC) differentiation and bone regeneration in vivo and in vitro. Methods. BMSCs were transduced by miR-129-5p mimic, miR-129-5p inhibitor, and negative control lentivirus. The ability of BMSC differentiation to osteoblast was tested by alkaline phosphatase (ALP) and alizarin red staining (ARS). The expression of osteogenic genes (Runx2, Bmp2, and OCN) was examined via quantitative RT-PCR and western blot. A mouse model of calvaria defect was investigated by Micro-CT, immunohistochemistry, and histological examination. The luciferase reporter gene assay was performed to confirm the binding between Dkk3 and miR-129-5p. For the transfection experiments, lipofectamine 3000 was used to transfect pcDNA-Dkk3 into BMSCs to overexpress Dkk3. Coimmunoprecipitation and immunofluorescent localization assay were included for exploring the role of Dkk3 and β-catenin. Results. miR-129-5p was induced in BMSCs and MSC cell line C3H10T1/2 cells under osteogenic medium. Overexpression of miR-129-5p significantly promoted osteogenic differentiation of BMSCs in vitro. Moreover, BMSCs transduced with miR-129-5p mimic exhibited better bone regeneration compared with BMSCs transduced with control counterpart in vivo. Luciferase and western blot data showed that Dickkopf3 (Dkk3) is a target gene of miR-129-5p and the expression of Dkk3 was inhibited in BMSCs transduced with miR-129-5p mimic but enhanced in BMSCs transduced with miR-129-5p inhibitor. In addition, Dkk3 interacted with β-catenin directly. Conclusions. miR-129-5p promotes osteogenic differentiation of BMSCs and bone regeneration, and miR-129-5p/Dkk3 axis may be new potential targets for the treatment of bone defect and bone loss.

scholarly journals The Role of Lipids, Lipid Metabolism and Ectopic Lipid Accumulation in Axon Growth, Regeneration and Repair after CNS Injury and Disease

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1078
Author(s):  
Debasish Roy ◽  
Andrea Tedeschi
Keyword(s):  
Nervous System ◽  
Lipid Metabolism ◽  
Lipid Accumulation ◽  
Axon Regeneration ◽  
Axon Growth ◽  
The Body ◽  
Cns Repair ◽  
Cord Injury ◽  
Cns Trauma

Axons in the adult mammalian nervous system can extend over formidable distances, up to one meter or more in humans. During development, axonal and dendritic growth requires continuous addition of new membrane. Of the three major kinds of membrane lipids, phospholipids are the most abundant in all cell membranes, including neurons. Not only immature axons, but also severed axons in the adult require large amounts of lipids for axon regeneration to occur. Lipids also serve as energy storage, signaling molecules and they contribute to tissue physiology, as demonstrated by a variety of metabolic disorders in which harmful amounts of lipids accumulate in various tissues through the body. Detrimental changes in lipid metabolism and excess accumulation of lipids contribute to a lack of axon regeneration, poor neurological outcome and complications after a variety of central nervous system (CNS) trauma including brain and spinal cord injury. Recent evidence indicates that rewiring lipid metabolism can be manipulated for therapeutic gain, as it favors conditions for axon regeneration and CNS repair. Here, we review the role of lipids, lipid metabolism and ectopic lipid accumulation in axon growth, regeneration and CNS repair. In addition, we outline molecular and pharmacological strategies to fine-tune lipid composition and energy metabolism in neurons and non-neuronal cells that can be exploited to improve neurological recovery after CNS trauma and disease.

scholarly journals Knockdown of lncRNA X inactive specific transcript (XIST) radiosensitizes non-small cell lung cancer (NSCLC) cells through regulation of miR-16-5p/WEE1 G2 checkpoint kinase (WEE1) axis

2021 ◽  
Vol 35 ◽  
pp. 205873842096608
Author(s):  
Ran Du ◽  
Feng Jiang ◽  
Yanhua Yin ◽  
Jinfen Xu ◽  
Xia Li ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Western Blot ◽  
Small Cell ◽  
Luciferase Reporter ◽  
Small Cell Lung ◽  
Luciferase Reporter Gene ◽  
G2 Checkpoint ◽  
Qrt Pcr ◽  
Specific Transcript

Long non-coding RNA (lncRNA) X inactive specific transcript (XIST) is reported to play an oncogenic role in non-small cell lung cancer (NSCLC). However, the role of XIST in regulating the radiosensitivity of NSCLC cells remains unclear. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expressions of XIST and miR-16-5p in NSCLC in tissues and cells, and Western blot was used to assess the expression of WEE1 G2 checkpoint kinase (WEE1). Cell counting kit-8 (CCK-8), colony formation and flow cytometry assays were used to determine cell viability and apoptosis after NSCLC cells were exposed to different doses of X-rays. The interaction between XIST and miR-16-5p was confirmed by StarBase database, qRT-PCR and dual-luciferase reporter gene assays. TargetScan database was used to predict WEE1 as a target of miR-16-5p, and their targeting relationship was further validated by Western blot, qRT-PCR and dual-luciferase reporter gene assays. XIST was highly expressed in both NSCLC tissue and cell lines, and knockdown of XIST repressed NSCLC cell viability and cell survival, and facilitated apoptosis under the irradiation. MiR-16-5p was a target of XIST, and rescue experiments demonstrated that miR-16-5p inhibitors could reverse the role of XIST knockdown on radiosensitivity in NSCLC cells. WEE1 was validated as a target gene of miR-16-5p, and WEE1 could be negatively regulated by XIST. XIST promotes the radioresistance of NSCLC cells by regulating the expressions of miR-16-5p and WEE1, which can be a novel target for NSCLC therapy.

microRNA-4458 Regulates Insulin Resistance in Hepatic Cells by Targeting the Insulin-Like Growth Factor 1 Receptor

2021 ◽  
Vol 11 (9) ◽  
pp. 1812-1817
Author(s):  
Jingjing Zhou ◽  
Wenjuan Zhu ◽  
Zheng Mao ◽  
Zhen Li ◽  
Xiaoqin Li ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Mrna Expression ◽  
Glucose Uptake ◽  
Hepg2 Cell ◽  
Molecular Mechanisms ◽  
Cell Model ◽  
Serum Insulin ◽  
Luciferase Reporter ◽  
Control Group ◽  
Hepatic Cells

Background: The objective of the research was to investigate the roles of miR-4458 in the regulation of insulin resistance in hepatic cells and to explore the underlying molecular mechanisms. Methods: The blood samples were collected from the T2D patients and the health controls, and the liver tissues were collected from the DM and control rats. The relationship between IGF1R and miR-4458 was predicted by TargetScan and verified by the dual luciferase reporter gene system. qRT-PCR was used to measure the mRNA expression of miR-4458, IGF1R, G6Pase and PEPCK. The protein expression of IGF1R, p-AKT and AKT were measured by Western blot analysis. The rat insulin ELISA Kit and glucose Uptake Colorimteric Assay Kit were used to determine the level of serum insulin and the glucose uptake. Results: miR-4458 was high expressed in T2D patients. We predicted and verified that IGF1R was a direct target of miR-4458, and the mRNA expression of IGF1R was reduced in type 2 diabetes patients. We established the diabetes model (DM) and IR HepG2 cell model, and found that the blood glucose and serum insulin levels were significantly elevated in the DM group. miR-4458 expression was up-regulated, while the expression of IGF1R and p-AKT, and p-AKT/AKT ratio were reduced in the DM group and IR HepG2 cell model. miR-4458 inhibitor and IGF1R-siRNA significantly decreased the expression of miR-4458 and IGF1R respectively. In comparison with IR+inhibitor control group, miR-4458 inhibitor increased 2-DG6P content, IGF1R expression, p-AKT expression and p-AKT/AKT ratio, reduced the expression of G6Pase and PEPCK, and all the effects were reversed by down-regulating IGF1R. Conclusion: miR-4458 regulated the insulin resistance in hepatic cells by regulating the IGF1R/PI3K/AKT signal pathway, which will be a potential target for the treatment of diabetes.

Effects of pomegranate peel polyphenols on lipid accumulation and cholesterol metabolic transformation in L-02 human hepatic cells via the PPARγ-ABCA1/CYP7A1 pathway

2016 ◽  
Vol 7 (12) ◽  
pp. 4976-4983 ◽  
Author(s):  
Ou Lv ◽  
Lifang Wang ◽  
Jianke Li ◽  
Qianqian Ma ◽  
Wei Zhao
Keyword(s):  
Bile Acid ◽  
Lipid Accumulation ◽  
Cell Model ◽  
Hepatic Cell ◽  
Total Bile Acid ◽  
Lipid Lowering ◽  
Pomegranate Peel ◽  
Hepatic Cells ◽  
Metabolic Transformation ◽  
Lipid Lowering Effect

PPPs, PC and PEA in different concentrations were found to decrease the total cholesterol (TC) content and increase the total bile acid (TBA) content of a human hepatic cell model, and so possess a lipid-lowering effect.

scholarly journals Kaempferol and Kaempferide Attenuate Oleic Acid-Induced Lipid Accumulation and Oxidative Stress in HepG2 Cells

2021 ◽  
Vol 22 (16) ◽  
pp. 8847
Author(s):  
Fangfang Tie ◽  
Jin Ding ◽  
Na Hu ◽  
Qi Dong ◽  
Zhi Chen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Oleic Acid ◽  
Lipid Metabolism ◽  
Western Blot ◽  
Blot Analysis ◽  
Lipid Accumulation ◽  
Hepg2 Cells ◽  
Western Blot Analysis ◽  
Heme Oxygenase 1 ◽  
And Oxidative Stress

Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases which lacks ideal treatment options. Kaempferol and kaempferide, two natural flavonol compounds isolated from Hippophae rhamnoides L., were reported to exhibit a strong regulatory effect on lipid metabolism, for which the mechanism is largely unknown. In the present study, we investigated the effects of kaempferol and kaempferide on oleic acid (OA)-treated HepG2 cells, a widely used in vitro model of NAFLD. The results indicated an increased accumulation of lipid droplets and triacylglycerol (TG) by OA, which was attenuated by kaempferol and kaempferide (5, 10 and 20 μM). Western blot analysis demonstrated that kaempferol and kaempferide reduced expression of lipogenesis-related proteins, including sterol regulatory element-binding protein 1 (SREBP1), fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD-1). Expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT enhancer binding proteins β (C/EBPβ), two adipogenic transcription factors, was also decreased by kaempferol and kaempferide treatment. In addition, western blot analysis also demonstrated that kaempferol and kaempferide reduced expression of heme oxygenase-1 (HO-1) and nuclear transcription factor-erythroid 2-related factor 2 (Nrf2). Molecular docking was performed to identify the direct molecular targets of kaempferol and kaempferide, and their binding to SCD-1, a critical regulator in lipid metabolism, was revealed. Taken together, our findings demonstrate that kaempferol and kaempferide could attenuate OA-induced lipid accumulation and oxidative stress in HepG2 cells, which might benefit the treatment of NAFLD.

Bone Marrow Stromal Cells (BMSCs) Inhibit Retinal Ganglion Cell Apoptosis and Alleviate Inflammation Through Up-Regulation of MicroRNA-43 and Brain-Derived Neurotrophic Factor in Glaucoma

2021 ◽  
Vol 11 (12) ◽  
pp. 2478-2483
Author(s):  
Xiang Ji ◽  
Kai-Wen Zhou
Keyword(s):  
Neurotrophic Factor ◽  
Vision Loss ◽  
Luciferase Reporter ◽  
Retinal Ganglion ◽  
Luciferase Reporter Gene ◽  
Down Regulation ◽  
Related Factors ◽  
Gene Assay

Glaucoma is a leading cause of vision loss mainly due to retinal ganglion cells (RGC) loss. MicroRNAs (miRNAs) are highlighted as potential biomarkers in diseases. This study aims to investigate the role of miR-43 and BMSCs in the RGC apoptosis and glaucoma.RGCs were transfected with miR-43 inhibitors and mimics, and then co-cultured with BMSCs. RT-qPCR analysis was conducted to determine miR-43 expression, whilst Western blot, and flow cytometry were carried out to assess the role of miR-43 in apoptosis and inflammation. The interaction between miR-43 and BDNF, a neurotrophic factor, was detected by dual-luciferase reporter gene assay. Overexpression of miR-43 promoted RGC proliferation and decreased apoptosis. Furthermore, miR-43 overexpression diminished the contents of apoptosis- and inflammatory-related factors, and elevated the expression of BDNF. Down-regulation of BDNF exerted similar effect as down-regulation of miR-43, enhancing apoptosis and aggravating inflammation. Importantly, BMSC treatment reversed the in vitro inhibitory effect of si-BDNF on RGC with enhancement of miR-43 expression. Mechanically, miR-43 was indicated to target BDNF in glaucoma. Collectively, miR-43 delivered by BMSCs plays an important role in the inflammatory injury and abnormal apoptosis of RGC by regulating the expression of BDNF. These findings might help development of new treatment for glaucoma and provide a promising biomarker for diagnosis and treatment.

scholarly journals The Differential Expression of miRNAs and a Preliminary Study on the Mechanism of miR-194-3p in Keloids

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Zhishan Xu ◽  
Bingyu Guo ◽  
Peng Chang ◽  
Qiang Hui ◽  
Wei Li ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Differential Expression ◽  
Real Time Pcr ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Cultured Fibroblasts ◽  
Gene Assay ◽  
And Migration ◽  
Related Proteins

The aim of this study was to detect abnormally expressed microRNA (miRNA) in keloids and to study their functions. The differential expression of miRNAs in keloids and normal tissue was detected by gene microarray. MiRNA expression was verified by real-time PCR. A luciferase reporter gene assay, western blot, and real-time PCR were used to detect the effect of miR-194-3p on RUNX2. An MTT assay and a transwell assay were used to detect the effect of miR-194-3p in both primary cultured fibroblasts and HKF cells. Related proteins were analysed by western blot and real-time PCR. The expression of miR-194-3p was lower in keloids, and MiR-194-3p was shown to target RUNX2 directly. MiR-194-3p inhibited the proliferation and migration of fibroblasts through the inhibition of CDK4 and MMP2. MiR-194-3p and RUNX2 may become new targets for the prevention and treatment of keloids.

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