scholarly journals LncRNA-KCNQ1OT1: a potential target in exosomes derived from adipose-derived stem cells for the treatment of osteoporosis

2021 ◽  
Author(s):  
Shanzheng Wang ◽  
Jun Jia ◽  
Chang-hong Chen
Keyword(s):  
Stem Cells ◽  
Caspase 3 ◽  
Osteoporosis Treatment ◽  
Luciferase Reporter Assay ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Adipose Derived Stem Cells ◽  
Reporter Assay ◽  
Tnf Α ◽  
Primary Osteoblasts

Abstract Background. Osteoporosis is a worldwide medical and socioeconomic threat characterized by systemic impairment of bone strength and microstructure. Exosomes derived from adipose-derived stem cells (ADSCs-Exos) have been confirmed to play effective roles in the repair of various tissues and organs. This study aimed to investigate the role of ADSCs-Exos and a noval long none coding RNA KCNQ1OT1 (lnc-KCNQ1OT1) played in osteoporosis as well as the mechanism. Methods. Primary osteoblasts were treated with different doses of TNF-α (0, 1, 2.5, 5, 10 ng/ml) and then co-cultured with ADSCs-Exos or exosomes-derived from lnc-KCNQ1OT1-modified ADSCs (KCNQ1OT1-Exos). The expression of miRNA-141-5p (miR-141-5p) and lnc-KCNQ1OT1 was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression of cleaved-caspase-3, caspase-3 and Bax was determined by Western blot. Cell viability and apoptosis were assessed by cell counting kit 8 (CCK-8) and flow cytometry analysis, respectively. The binding sites between KCNQ1OT1 and miR-141-5p were validated by dual-luciferase reporter assay. Results. Tumor necrosis factor-α (TNF-α) dose dependently increased miR-141-5p expression, inhibited viability and promoted apoptosis of osteoblasts. However, miR-141-5p silencing or co-culture with ADSCs-Exos attenuated these effects. In addition, KCNQ1OT1-Exos could more significantly attenuate the induced cytotoxicity and apoptosis compared to ADSCs-Exos. Moreover, miR-141-5p was confirmed as the target of lnc-KCNQ1OT1 by luciferase reporter assay. Conclusions. ADSCs-Exos attenuated cytotoxicity and apoptosis of TNF-α-induced primary osteoblasts. KCNQ1OT1-Exos had a more significant inhibitory effect compared to ADSCs-Exos by the function of sponging miR-141-5p, suggesting that KCNQ1OT1-Exos could be promising agents in osteoporosis treatment.

scholarly journals lncRNA-KCNQ1OT1: A Potential Target in Exosomes Derived from Adipose-Derived Stem Cells for the Treatment of Osteoporosis

2021 ◽  
Vol 2021 ◽  
pp. 1-17
Author(s):  
Shan-zheng Wang ◽  
Jun Jia ◽  
Chang-hong Chen
Keyword(s):  
Stem Cells ◽  
Noncoding Rna ◽  
Caspase 3 ◽  
Luciferase Reporter Assay ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Adipose Derived Stem Cells ◽  
Reporter Assay ◽  
Tnf Α ◽  
Primary Osteoblasts

Background. Osteoporosis is a worldwide medical and socioeconomic burden characterized by systemic impairment of bone strength and microstructure. Exosomes derived from adipose-derived stem cells (ADSCs-Exos) have been confirmed to play effective roles in the repair of various tissues and organs. This study was aimed at investigating the role of ADSCs-Exos and a novel long noncoding RNA KCNQ1OT1 played in osteoporosis as well as the underlying mechanism. Methods. Primary osteoblasts were treated with different doses of tumor necrosis factor-α (TNF-α) (0, 1, 2.5, 5, and 10 ng/ml) and then cocultured with ADSCs-Exos or exosome-derived from lnc-KCNQ1OT1-modified ADSCs (KCNQ1OT1-Exos). The expression of miRNA-141-5p (miR-141-5p) and lnc-KCNQ1OT1 was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression of cleaved-caspase-3, caspase-3, and Bax was determined by Western blot. Cell viability and apoptosis were assessed by Cell Counting Kit-8 (CCK-8) and flow cytometry analysis, respectively. The binding sites between KCNQ1OT1 and miR-141-5p were validated by dual-luciferase reporter assay. Results. TNF-α dose-dependently increased miR-141-5p expression, inhibited viability, and promoted apoptosis of osteoblasts. However, miR-141-5p silencing or cocultured with ADSCs-Exos attenuated these effects. In addition, KCNQ1OT1-Exos could more significantly attenuate the induced cytotoxicity and apoptosis compared to ADSCs-Exos. Moreover, miR-141-5p was confirmed as the target of KCNQ1OT1 by luciferase reporter assay. Conclusions. ADSCs-Exos can attenuate cytotoxicity and apoptosis of TNF-α-induced primary osteoblasts. KCNQ1OT1-Exos have a more significant inhibitory effect compared to ADSCs-Exos by the function of sponging miR-141-5p, suggesting that KCNQ1OT1-Exos can be promising agents in osteoporosis treatment.

scholarly journals LncRNA-KCNQ1OT1: A Potential Target in Exosomes Derived From ADSCs for The Treatment of Osteoporosis

2021 ◽  
Author(s):  
Shanzheng Wang ◽  
Jun Jia ◽  
Chang-hong Chen
Keyword(s):  
Caspase 3 ◽  
Osteoporosis Treatment ◽  
Underlying Mechanism ◽  
Inhibitory Effect ◽  
Luciferase Reporter Assay ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Tnf Α ◽  
Factor Α

Abstract Background Osteoporosis is a worldwide medical and socioeconomic threat characterized by systemic impairment to bone strength and microstructure. Exosomes derived from adipose-derived stem cells (ADSCs-Exos) have been confirmed to play effective roles in the repair of various tissues and organs. This study aimed to investigate the role of ADSCs-Exos and a noval long none coding RNAKCNQ1OT1 (lnc-KCNQ1OT1) played in osteoporosis as well as the underlying mechanism. Methods MC3T3-E1 cells were treated with different doses of TNF-α (0, 1, 2.5, 5, 10 ng/ml) and then co-cultured with ADSCs-Exos or exosomes-derived from lnc-KCNQ1OT1-modified ADSCs (KCNQ1OT1-Exos). The expression of microRNA (miRNA)-141-5p (miR-141-5p) and lnc-KCNQ1OT1 was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression of cleaved-caspase-3, caspase-3 and Bax was determined by Western blot. Cell viability and apoptosis were assessed by cell counting kit 8 (CCK-8) and flow cytometry analysis, respectively. The binding sites between KCNQ1OT1 and miR-141-5p were validated by dual-luciferase reporter assay. Results Tumor necrosis factor-α (TNF-α) dose dependently increased miR-141-5p expression, inhibited viability and promoted apoptosis of MC3T3-E1 cells. However, miR-141-5p silencing or co-culture with ADSCs-Exos attenuated these effects. In addition, KCNQ1OT1-Exos could more significantly attenuate the induced cytotoxicity and apoptosis compared to ADSCs-Exos. Moreover, miR-141-5p was confirmed as the target of lnc-KCNQ1OT1 by luciferase reporter assay. Conclusions ADSCs-Exos attenuated cytotoxicity and apoptosis of TNF-α-induced MC3T3-E1 cells. KCNQ1OT1-Exos had a more significant inhibitory effect compared to ADSCs-Exos by the function of sponging miR-141-5p, suggesting that KCNQ1OT1-Exos could be promising agents in osteoporosis treatment.

scholarly journals MicroRNA-513b-5p targets COL1A1 and COL1A2 associated with the formation and rupture of intracranial aneurysm

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zheng Zheng ◽  
Yan Chen ◽  
Yinzhou Wang ◽  
Yongkun Li ◽  
Qiong Cheng
Keyword(s):  
Cell Death ◽  
Intracranial Aneurysm ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Control Group ◽  
Type I ◽  
Mrna Levels ◽  
Reporter Assay ◽  
Over Expression ◽  
Tnf Α

AbstractCollagen-type I alpha 1 chain (COL1A1) and COL1A2 are abnormally expressed in intracranial aneurysm (IA), but their mechanism of action remains unclear. This study was performed to investigate the mechanism of COL1A1 and COL1A2 affecting the occurrence and rupture of IA. Quantitative real-time polymerase chain reaction was used to measure the expression of hsa-miR-513b-5p, COL1A1, COL1A2, TNF-α, IL-6, MMP2, MMP3, MMP9 and TIMP4 in patients with ruptured IA (RA) (n = 100), patients with un-ruptured IA (UA) (n = 100), and controls (n = 100). Then, human vascular smooth muscle cells (HASMCs) were cultured, and dual luciferase reporter assay was performed to analyse the targeting relationship between miR-513b-5p and COL1A1 or COL1A2. The effects of the miR-513b-5p mimic and inhibitor on the proliferation, apoptosis, and death of HASMC and the RIP1-RIP3-MLKL and matrix metalloproteinase pathways were also explored. The effect of silencing and over-expression of COL1A1 and COL1A2 on the role of miR-513b-5p were also evaluated. Finally, the effects of TNF-α on miR-513b-5p targeting COL1A1 and COL1A2 were tested. Compared with those in the control group, the serum mRNA levels of miR-513b-5p, IL-6 and TIMP4 were significantly decreased in the RA and UA groups, but COL1A1, COL1A2, TNF-α, IL-1β, MMP2, MMP3 and MMP9 were significantly increased (p < 0.05). Compared with those in the UA group, the expression of COL1A1, COL1A2, TNF-α, IL-1β and MMP9 was significantly up-regulated in the RA group (p < 0.05). Results from the luciferase reporter assay showed that COL1A1 and COL1A were the direct targets of miR-513b-5p. Further studies demonstrated that miR-513b-5p targeted COL1A1/2 to regulate the RIP1-RIP3-MLKL and MMP pathways, thereby enhancing cell death and apoptosis. Over-expression of COL1A1 or COL1A2, rather than silencing COL1A1/2, could improve the inhibitory effect of miR-513b-5p on cell activity by regulating the RIP1-RIP3-MLKL and MMP pathways. Furthermore, over-expression of miR-513b-5p and/or silencing COL1A1/2 inhibited the TNF-α-induced cell proliferation and enhanced the TNF-α-induced cell death and apoptosis. The mechanism may be related to the inhibition of collagen I and TIMP4 expression and promotion of the expression of RIP1, p-RIP1, p-RIP3, p-MLKL, MMP2 and MMP9. MiR-513b-5p targeted the inhibition of COL1A1/2 expression and affected HASMC viability and extracellular mechanism remodelling by regulating the RIP1-RIP3-MLKL and MMP pathways. This process might be involved in the formation and rupture of IA.

Association study of a functional variant of TNF-α gene and serum TNF-α level with the susceptibility of congenital heart disease in a Chinese population

2019 ◽  
Vol 95 (1128) ◽  
pp. 547-551
Author(s):  
Jun Pan ◽  
Jiang Hu ◽  
Xusheng Qi ◽  
Liqin Xu
Keyword(s):  
Congenital Heart Disease ◽  
Heart Disease ◽  
Congenital Heart ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Control Group ◽  
Reporter Assay ◽  
Tnf Α ◽  
Α Level ◽  
Dual Luciferase Reporter Assay

BackgroundCongenital heart disease (CHD) is among the leading causes of infant death worldwide. Although shortage of folate has been found potentially to contribute to CHD in the embryo, the aetiology of CHD was not completely understood. Inflammation and altered immune processes are involved in all forms of cardiac malformation, including CHD. Tumour necrosis factor-α (TNF-α), was involved in the pathogenesis of multiple kinds of heart diseases. However, no studies have systematically evaluated the associations of genetic variants of TNF-α with susceptibility of CHD.MethodsA case-control study was conducted to evaluate the associations between tagSNPs of TNF-α and CHD susceptibility. Serum level of TNF-α was assessed using ELISA. The dual luciferase reporter assay was used to evaluate the functional significance of variant rs1800629 on TNF-α transcriptional activity.ResultsWe found rs1800629 was significantly correlated with increased CHD susceptibility (OR: 1.72, 95% CI 1.26 to 2.36, p=0.001). Serum levels of TNF-α were significantly higher in CHD group (9.09±1.90 pg/mL) than that in control group (6.12±1.56 pg/mL, p<0.001). The AA genotype and AG genotype of rs1800629 was associated with higher serum TNF-α level, compared with GG genotype. The dual luciferase reporter assay showed that promoter activity was significantly increased by 57% and 76% for plasmids containing the minor A allele compared with the major G allele in H9c2 and HEK 293T, respectively.ConclusionThese results indicate that higher level of serum TNF-α increases risk of CHD, while TNF-α rs1800629 A allele might contribute to higher risk for CHD due to the increase in TNF-α expression.

Exosomes derived from mmu_circ_0000250-modified adipose-derived mesenchymal stem cells promote wound healing in diabetic mice by inducing miR-128-3p/SIRT1-mediated autophagy

2020 ◽  
Vol 318 (5) ◽  
pp. C848-C856 ◽  
Author(s):  
Rongfeng Shi ◽  
Yinpeng Jin ◽  
Weiwei Hu ◽  
Weishuai Lian ◽  
Chuanwu Cao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wound Healing ◽  
Mesenchymal Stem Cells ◽  
Therapeutic Effect ◽  
Diabetic Rats ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
High Concentration ◽  
Promote Wound Healing

More and more evidence advises that circular RNAs (circRNAs) function critically in regulating different disease microenvironments. Our previous study found that autotransplantation of adipose-derived mesenchymal stem cells (ADSCs) promotes diabetes wound healing. Exosomes derived in ADSCs play an important regulatory role. This study aimed to characterize if mmu_circ_0000250 played a role in ADSC-exosome-mediated full-thickness skin wound repair in diabetic rats. Endothelial progenitor cells (EPCs) were selected to study the therapeutic mechanism of exosomes in high-glucose (HG)-induced cell damage and dysfunction. Analysis and luciferase reporter assay were utilized to explore the interaction among mmu_circ_0000250, miRNA (miR)-128-3p, and sirtuin (SIRT)1. The diabetic rats were used to confirm the therapeutic effect of mmu_circ_0000250 against exosome-mediated wound healing. Exosomes containing a high concentration of mmu_circ_0000250 had a greater therapeutic effect on restoration of the function of EPCs by promotion autophagy activation under HG conditions. Expression of mmu_circ_0000250 promoted SIRT1 expression by miR-128-3p adsorption, which was confirmed via luciferase reporter assay and bioinformatics analysis. In vivo, exosomes containing a high concentration of mmu_circ_0000250 had a more therapeutic effect on wound healing when compared with wild-type exosomes from ADSCs. Immunohistochemistry and immunofluorescence detection showed that mmu_circ_0000250 increased angiopoiesis with exosome treatment in wound skin and suppressed apoptosis by autophagy activation. In conclusion, we verified that mmu_circ_0000250 enhanced the therapeutic effect of ADSC-exosomes to promote wound healing in diabetes by absorption of miR-128-3p and upregulation of SIRT1. Therefore, these findings advocate targeting the mmu_circ_0000250/miR-128-3p/SIRT1 axis as a candidate therapeutic option for diabetic ulcers.

scholarly journals MiR-214 Attenuates Osteogenic Differentiation of Mesenchymal Stem Cells via Targeting FGFR1

2016 ◽  
Vol 38 (2) ◽  
pp. 809-820 ◽  
Author(s):  
Lei Yang ◽  
Dawei Ge ◽  
Xiaojian Cao ◽  
Yingbin Ge ◽  
Hongtao Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Postmenopausal Osteoporosis ◽  
Osteoblast Differentiation ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Matrix Mineralization ◽  
Direct Target

Background/Aims: Postmenopausal osteoporosis is closely associated with reduction in the differentiation of mesenchymal stem cells (MSCs) into osteoblasts. Previous studies have demonstrated that miR-214 plays an important role in the genesis and development of postmenopausal osteoporosis. Here, we performed this study to investigate the potential mechanism by which miR-214 regulates osteoblast differentiation of MSCs. Methods: First, we explored the expression of miR-214 in MSCs of osteoporotic mice. Next, we examined the change of miR-214 during osteoblast differentiation of MSCs. Then, MSCs were infected with lentiviral vectors expressing miR-214 or miR-214 sponge to investigate the effect of miR-214 on osteoblast differentiation of MSCs. Further, bioinformatics analysis and luciferase reporter assay were performed to identify and validate the target gene of miR-214. Results: MiR-214 was up-regulated in MSCs of osteoporotic mice and down-regulated during osteoblast differentiation of MSCs. Furthermore, overexpression of miR-214 inhibited osteoblast differentiation of MSCs in vitro, whereas inhibition of miR-214 function promoted this process, evidenced by increased expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Bioinformatics, Western blot analysis and luciferase reporter assay demonstrated that FGFR1 is a direct target of miR-214. Conclusions: MiR-214 attenuates osteogenesis by inhibiting the FGFR1/FGF signaling pathway. Our findings suggest that targeting miR-214 promises to be a potential therapy in treatment of postmenopausal osteoporosis.

scholarly journals MiR-3064 in Epicardial Adipose-Derived Exosomes Targets Neuronatin to Regulate Adipogenic Differentiation of Epicardial Adipose Stem Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Wenkai Yang ◽  
Hanjian Tu ◽  
Kai Tang ◽  
Haozhong Huang ◽  
Shi Ou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Target Gene ◽  
Adipogenic Differentiation ◽  
Adipose Stem Cells ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Sequencing Analysis ◽  
Reporter Assay ◽  
Significant Difference

Backgroud: The metabolism of epicardial adipose tissue (EAT) is closely related to coronary atherosclerotic heart disease (CAHD), but the specific mechanism is not fully understood. In this study, we investigated the effects of EAT microenvironment on adipose metabolism from the viewpoint of EAT-derived exosomes and epicardial adipose stem cells (EASCs).Methods: EAT samples from CAHD patients and non-CAHD patients were collected to obtain exosomes via tissue culture. MiRNA sequencing was performed to analyze differences in miRNA expression in exosomes between groups. Luciferase reporter assay was then performed to verify the miRNA target gene. EAT was digested by collagenase to obtain EASCs, which were induced to mature adipocytes in vitro. Immunochemical staining and western blotting were performed to detect protein expression levels.Results: The results showed that CAHD patients had higher levels of EASCs in EAT, and no significant difference in the adipogenic differentiation ability of EASCs was observed between CAHD and non-CAHD patients in vitro. This indicates that the EAT microenvironment is a key factor affecting the adipogenic differentiation of EASCs. The EAT-derived exosomes from CAHD patients inhibited adipogenic differentiation of EASCs in vitro. Sequencing analysis showed that miR-3064-5p was highly expressed in EAT-derived exosomes in CAHD patients, and its inhibitor could improve the adipogenic differentiation of EASCs. Luciferase reporter assay results showed that the target gene of miR-3064-5p is neuronatin (Nnat). Nnat remained silent in EASCs and was less expressed in EAT of CAHD patients.Conclusion: Abovementioned results suggest that Nnat is the key to regulating the adipogenic differentiation of EASCs, and miR-3064-5p in EAT-derived exosomes can inhibit the expression of Nnat by targeting its mRNA, thereby affecting the adipogenic differentiation of EASCs.

scholarly journals miRNA-1290 Promotes Aggressiveness in Pancreatic Ductal Adenocarcinoma by Targeting IKK1

2018 ◽  
Vol 51 (2) ◽  
pp. 711-728 ◽  
Author(s):  
Na Ta ◽  
Xiaoyi Huang ◽  
Kailian Zheng ◽  
Yunshuo Zhang ◽  
Yisha Gao ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Western Blotting ◽  
Luciferase Reporter Assay ◽  
Cell Counting ◽  
Ductal Adenocarcinoma ◽  
Luciferase Reporter ◽  
Low Grade ◽  
Reporter Assay ◽  
Dual Luciferase Reporter Assay

Background/Aims: MicroRNAs (miRNAs) are a group of non-coding RNAs that play diverse roles in pancreatic carcinogenesis. In pancreatic ductal adenocarcinoma (PDAC), NF-kB is constitutively activated in most patients and is linked to a mutation in KRAS via IkB kinase complex 1 (IKK1, also known as IKKa). We investigated the link between PDAC aggressiveness and miR-1290. Methods: We used miRCURYTM LNA Array and in situ hybridization to investigate candidate miRNAs and validated the findings with PCR. The malignant behavior of cell lines was assessed with Cell Counting Kit-8, colony formation, and Transwell assays. A dual-luciferase reporter assay was used to evaluate the interaction between miR-1290 and IKK1. Protein expression was observed by western blotting. Results: In this study, 36 miRNAs were dysregulated in high-grade pancreatic intraepithelial neoplasia (PanIN) and PDAC tissues compared with low-grade PanIN tissues. The area under the curve values of miR-1290 and miR-31-5p were 0.829 and 0.848, respectively (95% confidence interval, 0.722–0.936 and 0.749–0.948, both P < 0.001). There was a significant correlation between miR-1290 and histological differentiation (P = 0.029), pT stage (P = 0.006), and lymph node metastasis (P = 0.001). In addition, the in vitro work showed that miR-1290 promoted PDAC cell proliferation, invasion, and migration. Western blotting and the dual-luciferase reporter assay showed that miR-1290 promoted cancer aggressiveness by directly targeting IKK1. The synergist effect of miR-1290 on the proliferation and metastasis of PDAC cells was attenuated and enhanced by IKK1 overexpression and knockdown, respectively. Consistent with the in vitro results, a subcutaneous tumor mouse model showed that miR-1290 functioned as a potent promoter of PDAC in vivo. Conclusion: MiR-1290 may act as an oncogene by directly targeting the 3’-untranslated region of IKK1, and the miR-1290/IKK1 pathway may prove to be a novel diagnostic and therapeutic target for PDAC.

scholarly journals CDK6 and miR-320c Co-Regulate Chondrocyte Catabolism Through NF-κB Signaling Pathways

2018 ◽  
Vol 51 (2) ◽  
pp. 909-923 ◽  
Author(s):  
Hao Sun ◽  
Zhiyu Huang ◽  
Peihui Wu ◽  
Zongkun Chang ◽  
Weiming  Liao ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Late Stage ◽  
Chondrogenic Differentiation ◽  
Luciferase Reporter Assay ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Inflammatory Factor ◽  
Reporter Assay ◽  
Middle Stage ◽  
Qrt Pcr

Background/Aims: Cyclin-dependent kinase 6 (CDK6) regulates inflammatory response and cell differentiation. This study sought to determine whether CDK6 and miR-320c co-regulate chondrogenesis and inflammation. Methods: Utilizing quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC), CDK6 and miR-320c expression were assessed in a micromass culture of human bone mesenchymal stem cells that underwent chondrogenesis in vitro as well as in chondrocytes from E16.5 mouse forelimbs. Normal chondrocytes were transfected with miR-320c mimic, miR-320c inhibitor, or CDK6-siRNA. Luciferase reporter assay results confirmed that miR-320c directly targets CDK6 by interacting with the 3′-untranslated region (3′-UTR) of its mRNA. qRT-PCR, Western blotting, and Cell Counting Kit-8 were subsequently used to evaluate the effects of miR-320c overexpression and CDK6 inhibition on inflammatory factor expression, as well as to investigate the effects of NF-kB and MAPK signaling pathway activation on IL-1β-induced chondrocyte inflammation. Results: Our results show that miR-320c expression increased during the middle stage and decreased during the late stage of hBMSC chondrogenic differentiation. In contrast, CDK6 expression decreased during the middle stage and increased during the late stage of hBMSC chondrogenic differentiation. Moreover, CDK6 expression increased in severe OA cartilage and in hypertrophic chondrocytes of mouse forelimbs at E16.5. Results of the luciferase reporter assay showed that miR-320c modulated CDK6 expression by binding to the 3′-UTR of its mRNA. miR-320c overexpression and CDK6 inhibition repressed IL-1β-induced expression of inflammatory factors and regulated the NF-kB signaling pathway. Conclusion: CDK6 and miR-320c co-regulate hBMSC chondrogenesis and IL-1β-induced chondrocyte inflammation through the NF-kB signaling pathway, suggesting that miR-320c and CDK6 inhibitors can be used to repress catabolism in human chondrocytes.

scholarly journals MiR-490 alleviates sepsis-induced acute lung injury by targeting MRP4 in new-born mice

2021 ◽  
Author(s):  
Jie Lin ◽  
Zongze Lin ◽  
Liqun Lin
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Total Protein ◽  
Protein Concentration ◽  
Caspase 3 ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Total Protein Concentration ◽  
Tnf Α ◽  
Dual Luciferase Reporter Assay

The aim of this study was to investigate whether the effects of miR-490 on acute lung injury (ALI) induced by sepsis in vitro and in vivo were through targeting multi-drug resistance-associated protein 4 (MRP4). MiR-490 agomir/NC agomir was injected into mice before cecal ligation and puncture (CLP). Pulmonary microvascular endothelial cells (PMVECs) were transfected with or without miR-490 agomir/NC agomir/MRP4/empty vector before lipopolysaccharide (LPS) stimulation. Histopathology, injure score, and Wet/Dry (W/D) of lung tissues were assessed. The number of neutrophils, macrophages and total cells, total protein concentration, TNF-α and IL-1β level in bronchoalveolar lavage fluid (BALF) were measured. The levels of caspase-3, Bcl-2, TNF-α, and IL-1β were measured in MPVECs. Dual-luciferase reporter assay was used to analyze the relationship between MRP4 and miR-490. When compared to the sham group, in CLP mice, the alveolar lung tissue showed significantly hyperemic, alveolar collapse, the W/D ratio was increased, and the injury index was increased. The number of neutrophils, macrophages and total cells, total protein concentration, TNF-α and IL-1β levels were significantly increased in BALF from CLP mice. The levels of TNF-α and IL-1β were significantly increased in lung tissue from CLP mice. Overexpression of miR-490 alleviated lung injury caused by CLP and inhibited inflammation in mice. The levels of TNF-α, IL-1β and caspase-3 were significantly increased, but the level of Bcl-2 was significantly decreased in MPVECs treated with LPS compared to the control group. Overexpression of miR-490 also reversed the increase of TNF-α, IL-1β, cleaved caspase-3 and Bcl-2 caused by LPS in MPVECs. Dual-luciferase reporter assay confirmed that the target gene of miR-490 was MRP4. Besides, overexpression of MRP4 upregulated TNF-α, IL-1β, and cleaved caspase-3, but downregulated the increase of Bcl-2 induced by miR-490 agomir transfection. These data suggested that miR-490 could relieve sepsis-induced acute lung injury in neonatal mice via targeting MRP4.

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