scholarly journals miR-140-3p enhanced the osteo/odontogenic differentiation of DPSCs via inhibiting KMT5B under hypoxia condition

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Han Zheng ◽  
Ning Wang ◽  
Le Li ◽  
Lihua Ge ◽  
Haichao Jia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Tissue Regeneration ◽  
Molecular Mechanisms ◽  
Bone Tissue Regeneration ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Alizarin Red ◽  
Odontogenic Differentiation ◽  
Gene Assay ◽  
Dentinogenic Differentiation

AbstractHuman dental pulp stem cells (DPSCs) have emerged as an important source of stem cells in the tissue engineering, and hypoxia will change various innate characteristics of DPSCs and then affect dental tissue regeneration. Nevertheless, little is known about the complicated molecular mechanisms. In this study, we aimed to investigate the influence and mechanism of miR-140-3p on DPSCs under hypoxia condition. Hypoxia was induced in DPSCs by Cobalt chloride (CoCl2) treatment. The osteo/dentinogenic differentiation capacity of DPSCs was assessed by alkaline phosphatase (ALP) activity, Alizarin Red S staining and main osteo/dentinogenic markers. A luciferase reporter gene assay was performed to verify the downstream target gene of miR-140-3p. This research exhibited that miR-140-3p promoted osteo/dentinogenic differentiation of DPSCs under normoxia environment. Furthermore, miR-140-3p rescued the CoCl2-induced decreased osteo/odontogenic differentiation potentials in DPSCs. Besides, we investigated that miR-140-3p directly targeted lysine methyltransferase 5B (KMT5B). Surprisingly, we found inhibition of KMT5B obviously enhanced osteo/dentinogenic differentiation of DPSCs both under normoxia and hypoxia conditions. In conclusion, our study revealed the role and mechanism of miR-140-3p for regulating osteo/dentinogenic differentiation of DPSCs under hypoxia, and discovered that miR-140-3p and KMT5B might be important targets for DPSC-mediated tooth or bone tissue regeneration.

scholarly journals Hsa-miR-105-1 Regulates Cisplatin-Resistance in Ovarian Carcinoma Cells by Targeting ANXA9

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Xinxin Kou ◽  
Hui Ding ◽  
Lei Li ◽  
Hongtu Chao
Keyword(s):  
Ovarian Carcinoma ◽  
Cell Lines ◽  
Target Gene ◽  
Molecular Mechanisms ◽  
Cisplatin Resistance ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Qrt Pcr ◽  
Gene Assay ◽  
Increased Sensitivity

Purpose. Cisplatin is one of the most effective drugs for treating ovarian carcinoma (OC), which is among the most lethal types of carcinoma. However, the chemoresistance to cisplatin that develops over time leads to a poor clinical outcome for many OC patients. Therefore, it is necessary to clearly understand the molecular mechanisms of chemoresistance. In this study, we examined how Hsa-miR-105-1 functions in cisplatin-resistant OC cells. Methods. The levels of Hsa-miR-105-1 expression in cisplatin-sensitive and resistant OC cell lines were detected by qRT-PCR. The target gene of Hsa-miR-105-1 was predicted by using the TargetScan and Starbase databases and verified by the double luciferase reporter gene assay. The target gene of Hsa-miR-105-1 was identified as ANXA9, and ANXA9 expression was evaluated by qRT-PCR, western blotting, and immunofluorescence. To validate the function of Hsa-miR-105-1 in OC cells, we silenced or overexpressed Hsa-miR-105-1 in cisplatin-sensitive or resistant OC cell lines, respectively. Furthermore, the expression levels of several apoptosis-related proteins, including P53, P21, E2F1, Bcl-2, Bax, and caspase-3, were examined by western blot analysis. Results. The levels of Hsa-miR-105-1 expression were abnormally downregulated in cisplatin-resistant OC cells, while ANXA9 expression was significantly upregulated in those cells. Treatment with an Hsa-miR-105-1 inhibitor promoted the expression of ANXA9 mRNA and protein, enhanced the resistance to cisplatin, and attenuated the cell apoptosis induced by cisplatin in cisplatin-sensitive OC cells. Moreover, treatment with Hsa-miR-105-1 mimics inhibited ANXA9 expression, which further increased the levels of P53, P21, and Bax expression and decreased the levels of E2F1 and Bcl-2 expression, finally resulting in an increased sensitivity to cisplatin in cisplatin-resistant OC cells. Conclusion. We found that a downregulation of Hsa-miR-105-1 expression enhanced cisplatin-resistance, while an upregulation of Hsa-miR-105-1 restored the sensitivity of OC cells to cisplatin. The Hsa-miR-105-1/ANXA9 axis plays an important role in the cisplatin-resistance of OC cells.

scholarly journals PITPNA-AS1 Activated by H3K27ac Sponged miR-98-5p to Regulate Cisplatin Resistance in Gastric Cancer

2021 ◽  
Author(s):  
Yaping Liu ◽  
Xu Zhao ◽  
Yinnan Chen ◽  
Gang Guo ◽  
Jiansheng Wang ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Proliferation ◽  
Molecular Mechanisms ◽  
Luciferase Reporter ◽  
Platinum Resistance ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Proliferation And Apoptosis ◽  
Gastric Cancer Cell Lines ◽  
Cancer Tissues

Abstract To evaluate the expression of PITPNA-AS1 and miR-98-5p in gastric cancer tissues as well as their association with progression of gastric cancer, and investigate the role of PITPNA-AS1 and miR-98-5p in developing platinum resistance. RNA sequencing was used to identify candidate lncRNAs and microRNAs related to local recurrence of gastric cancer. qRT-PCR was used to investigate the expression of PITPNA-AS1 and miR-98-5p. CCK-8 and caspase3/7 activity were used to evaluate the cell proliferation and apoptosis rate. Dual luciferase reporter gene assay and RNA pull down were used to evaluate the cross talk between PITPNA-AS1 and miR-98-5p. PITPNA-AS1 and miR-98-5p could regulate cell proliferation and inhibit apoptosis in gastric cancer cell lines. Cisplatin and lobaplatin could significantly suppress the expression of PITPNA-AS1, which interacted with negatively regulated miR-98-5p expression. PITPNA-AS1 overexpression impaired the effect of platinum, which was partially reversed by downregulation of miR-98-5p knock down. In gastric cancer, PITPNA-AS1 and miR-98-5p could regulat cell growth, apoptosis and platinum resistance. They have the potential to be biomarkers and curative therapeutic targets. However, further research on molecular mechanisms are needed.

Abstract 13141: Cellular Microrna 449b Upregulation Sensitizes Ischemia/reperfusion Injury via Inhibition of Nrf-1, Increasing Ros Production and Apoptosis in the Diabetic Heart

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Hang Wang ◽  
Wayne Lau ◽  
Erhe Gao ◽  
Walter Koch ◽  
Xin Ma ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Molecular Mechanisms ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Experimental Studies ◽  
Diabetic Heart ◽  
Luciferase Reporter ◽  
Proteomics Data ◽  
Luciferase Reporter Gene ◽  
Gene Assay

Myocardial ischemic/reperfusion (MI/R) injury is significantly enhanced in diabetes by incompletely understood mechanisms. Recent clinical and experimental studies demonstrate that hypoadiponectinemia during diabetes enhances oxidative stress and exaggerates MI/R injury. However, molecular mechanisms responsible for hypoadiponectinemia-induced oxidative stress remain unknown. In a discovery-driven fashion, we determined the role of cardiac microRNAs in the MI/R response in adiponectin knockout (APNKO) mice. From 68 total miRNAs differentially expressed between APNKO and wild type (WT) mice, miRNA 449b was identified as the microRNA most relevant to oxidative stress and apoptosis. In cultured neonatal cardiomyocytes, miRNA 449b silencing inhibited hypoxia/reoxygenation-induced apoptosis, whereas miR-449b overexpression significantly increased oxidative stress and cardiomyocyte apoptosis. In APNKO mice, administration of anti-miR-449b decreased oxidative stress (-17.2±3.8%, p<0.05), reduced caspase-3 activity (-21.3±4.2%, p<0.05), attenuated myocardial apoptosis (-16.3±4.1%, p<0.05), and improved myocardial function (1.4±0.3 fold). To identify the downstream molecule regulated by miRNA 449b, we integrated transcriptomics and proteomics data with computational annotation data, and identified Nrf-1 as a miRNA 449b target. A luciferase reporter gene assay demonstrated that miRNA 449b inhibited Nrf-1 expression via Nrf-1 mRNA 3’UTR region binding. Finally, we demonstrated that miRNA 449b was significantly upregulated, Nrf-1 expression was significantly decreased, and the anti-oxidative molecule metallothionein (MT) was significantly inhibited in the diabetic heart subjected to MI/R. Administration of anti-miR-449b in diabetic animals upregulated Nrf-1 and MT expression, reduced oxidative stress, and improved cardiac function (P<0.01) after MI/R. Taken together, this study provides the first evidence that hypoadiponectinemia during diabetes causes cardiac miRNA-449b upregulation and subsequent downregulation of Nrf-1 and MT, thus enhancing oxidative stress and MI/R injury. MicroRNA 449b may represent a potential therapeutic target against diabetic heart disease.

scholarly journals MicroRNA-346-5p Regulates Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells by Inhibiting Transmembrane Protein 9

2020 ◽  
Vol 2020 ◽  
pp. 1-7
Author(s):  
Yicai Zhang ◽  
Yi Sun ◽  
Jinlong Liu ◽  
Yu Han ◽  
Jinglong Yan
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Protein Level ◽  
Molecular Mechanisms ◽  
Transmembrane Protein ◽  
Luciferase Reporter ◽  
Alizarin Red ◽  
Protein Levels

The molecular mechanisms how bone marrow-derived mesenchymal stem cells (BMSCs) differentiate into osteoblast need to be investigated. MicroRNAs (miRNAs) contribute to the osteogenic differentiation of BMSCs. However, the effect of miR-346-5p on osteogenic differentiation of BMSCs is not clear. This study is aimed at elucidating the underlying mechanism by which miR-346-5p regulates osteogenic differentiation of human BMSCs. Results of alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining indicated that upregulation of miR-346-5p suppressed osteogenic differentiation of BMSCs, whereas downregulation of miR-346-5p enhanced this process. The protein levels of the osteoblastic markers Osterix and Runt-related transcription factor 2 (Runx2) were decreased in cells treated with miR-346-5p mimic at day 7 and day 14 after being differentiated. By contrast, downregulation of miR-346-5p elevated the protein levels of Osterix and Runx2. Moreover, a dual-luciferase reporter assay revealed that Transmembrane Protein 9 (TMEM9) was a target of miR-346-5p. In addition, the Western Blot results demonstrated that the TMEM9 protein level was significantly reduced by the miR-346-5p mimic whereas downregulation of miR-346-5p improved the protein level of TMEM9. These results together demonstrated that miR-346-5p served a key role in BMSC osteogenic differentiation of through targeting TMEM9, which may provide a novel target for clinical treatments of bone injury.

scholarly journals MicroRNA-409-3p promotes osteoblastic differentiation via activation of Wnt/β-catenin signaling pathway by targeting SCAI

2020 ◽  
Author(s):  
Nan Chen ◽  
Hao Yang ◽  
Lijun Song ◽  
Hua Li ◽  
Yi Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Quantitative Polymerase Chain Reaction ◽  
Osteoblastic Differentiation ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Complementary Sequence ◽  
Alizarin Red ◽  
Gene Assay

Osteogenic differentiation is an important process of new bone formation, miR-409-3p has been reported to be upregulated in osteogenic differentiation of human bone marrow mesenchymal stem cells (MSCs). To investigate the regulatory effect of miR-409-3p on osteogenic differentiation of MSCs and its molecular mechanism, the expression of miR-409-3p in osteoblast (HCO) and bone marrow-derived MSCs (MSC-A, MSC-B, MSC-U) were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The binding of miR-409-3p to SCAI in MSC-B was investigated by dual-luciferase reporter gene assay. MSC-B were selected to transfect with miR-409-3p analog/complementary sequence (cs), miR-409-3p analog + SCAI and miR-409-3p cs + small interfering (si)-SCAI, as well as control, respectively. The alkaline phosphatase activity, alizarin red staining, and the expression of osteogenic markers in MSC-B during osteoblastic differentiation were tested by RT-qPCR and Western blotting, respectively. The Wnt/β-catenin pathway was inhibited by dickkopf-related protein 1 to get the roles of miR-409-3p during the osteoblastic differentiation of MSC-B when transfected with miR-409-3p analog. The expression of miR-409-3p in HCO was higher than that in these three MSCs, showing an increasing time-dependent trend on the 0 and 21th day of osteoblastic differentiation. MiR-409-3p directly regulated SCAI by targeting SCAI 3′UTR. Further, miR-409-3p suppressed SCAI expression, but SCAI upregulation suppressed the osteoblastic differentiation, as well as reduced the relative mRNA/protein expression of Wnt/β-catenin signaling pathway-related genes. Importantly, disruption of Wnt signaling also blocked miR-409-3p induced osteoblastic differentiation of MSCs. Therefore, miR-409-3p promotes osteoblastic differentiation through the activation of the Wnt/β-catenin pathway by downregulating SCAI expression.

scholarly journals IFN-γ Enhances the Efficacy of Exosomes Derived from Mesenchymal Stem Cells in Myocardial Infarction Rats via miR-21 Stimulated by STAT1

2021 ◽  
Author(s):  
Jian Zhang ◽  
Yao Lu ◽  
Yangming Mao ◽  
Yue Yu ◽  
Tianyu Wu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Umbilical Vein ◽  
Luciferase Reporter ◽  
H9c2 Cells ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Ifn Γ

Abstract Background: Mesenchymal stem cells (MSCs) activated with IFN-γ elicit more powerful physical effects. Exosomes (Exos) secreted from MSCs have protective against myocardial injury. The aim of this study was to investigate whether Exsos derived from IFN-γ-pretreated MSCs exhibit more potent cardioprotective function and the underlying mechanisms. Methods: Exos were isolated from MSCs (Ctrl-Exo) and IFN-γ-primed MSCs (IFN-γ-Exo) and were then delivered to H9c2 cells or human umbilical vein endothelial cells (HUVECs) in vitro under oxygen and glucose deprivation (OGD) condition or in vivo in an infarcted rat heart. RNA sequencing was to identify the different expressed functional transcription factor (TF). Quantitative reverse transcription-PCR (qPCR) was to confirm the upregulated TF and miRNA in IFN-γ-primed MSCs. Dual-luciferase reporter gene assay were to analyze the transcriptional regulation of miRNAs by STAT1. The target of miR-21-5p (miR-21) was disclosed by luciferase reporter assays and qPCR. The function of BTG2 was verified in vitro under OGD condition.Result: IFN-γ-Exo accelerated migration, tube-like structure formation, and prevented H9c2 from OGD-induced apoptosis. Similarly, IFN-γ-Exo leaded to further reduction in fibrosis size, reduced cardiomyocyte apoptosis and improved cardiac function compared to Ctrl-Exo. miR-21 was significantly upregulated in both IFN-γ-primed MSCs and IFN-γ-Exo. STAT1 transcriptionally induced miR-21 expression. Up-regulated miR-21 can inhibit the expression of BTG2. BTG2 promoted H9c2 cells apoptosis and reversed the protective effect of miR-21 under OGD environment.Conclusion: IFN-γ-Exo have enhanced therapeutic efficacy against acute MI possibly through promoting angiogenesis and anti-apoptotic effect through increasing the level of miR-21, which directly targeted on BTG2.

scholarly journals miR-6807-5p Inhibited the Odontogenic Differentiation of Human Dental Pulp Stem Cells Through Directly Targeting METTL7A

2021 ◽  
Vol 9 ◽  
Author(s):  
Ning Wang ◽  
Xiao Han ◽  
Haoqing Yang ◽  
Dengsheng Xia ◽  
Zhipeng Fan
Keyword(s):  
Stem Cells ◽  
Dental Pulp ◽  
Binding Protein ◽  
Dental Pulp Stem Cells ◽  
Luciferase Reporter ◽  
Control Group ◽  
Alizarin Red ◽  
Odontogenic Differentiation ◽  
Human Dental Pulp ◽  
Tooth Tissue

Background: Tooth tissue regeneration mediated by mesenchymal stem cells (MSCs) has become the most ideal treatment. Although the known regulatory mechanism and some achievements have been discovered, directional differentiation cannot effectively induce regeneration of tooth tissue. In this study, we intended to explore the function and mechanism of miR-6807-5p and its target gene METTL7A in odontogenic differentiation.Methods: In this study, human dental pulp stem cells (DPSCs) were used. Alkaline phosphatase (ALP), Alizarin red staining (ARS), and calcium ion quantification were used to detect the odontogenic differentiation of miR-6807-5p and METTL7A. Real-time RT-PCR, western blot, dual-luciferase reporter assay, and pull-down assay with biotinylated miRNA were used to confirm that METTL7A was the downstream gene of miR-6807-5p. Protein mass spectrometry and co-immunoprecipitation (Co-IP) were used to detect that SNRNP200 was the co-binding protein of METTL7A.Results: After mineralized induction, the odontogenic differentiation was enhanced in the miR-6807-5p-knockdown group and weakened in the miR-6807-5p-overexpressed group compared with the control group. METTL7A was the downstream target of miR-6807-5p. After mineralized induction, the odontogenic differentiation was weakened in the METTL7A-knockdown group and enhanced in the METTL7A-overexpressed group compared with the control group. SNRNP200 was the co-binding protein of METTL7A. The knockdown of SNRNP200 inhibited the odontogenic differentiation of DPSCs.Conclusion: This study verified that miR-6807-5p inhibited the odontogenic differentiation of DPSCs. The binding site of miR-6807-5p was the 3′UTR region of METTL7A, which was silenced by miR-6807-5p. METTL7A promoted the odontogenic differentiation of DPSCs. SNRNP200, a co-binding protein of METTL7A, promoted the odontogenic differentiation of DPSCs.

scholarly journals MiR-128-3p Post-Transcriptionally Inhibits WISP1 to Suppress Apoptosis and Inflammation in Human Articular Chondrocytes via the PI3K/AKT/NF-κB Signaling Pathway

2020 ◽  
Vol 29 ◽  
pp. 096368972093913
Author(s):  
Shujun Chen ◽  
Bo Li
Keyword(s):  
Signaling Pathway ◽  
Proinflammatory Cytokines ◽  
Molecular Mechanisms ◽  
Articular Chondrocytes ◽  
Negative Regulator ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Induced Apoptosis

In osteoarthritis (OA), the synthesis and decomposition of the extracellular matrix (ECM) are imbalanced. High expression levels of Wnt1-inducible signaling pathway protein 1 (WISP1) promote the synthesis of matrix metalloproteinases and induce the degradation of cartilage, which aggravates the OA. The aim of this study was to explore the role of miR-128-3p in the development of OA. In the present study, the expression of WISP1 and miR-128-3p in osteoarthritic tissues and chondrocytes was detected using quantitative reverse transcription PCR (RT-qPCR) and Western blotting. Then we predicted that WISP1 might be a potential target gene of miR-128-3p by TargetScan and verified using luciferase reporter gene assay. The effect of miR-128-3p or WISP1 on chondrocytes was evaluated by cell proliferation assay, apoptosis, and caspase-3 activity assay. To further reveal the molecular mechanisms of miR-128-3p in osteoarthritic development, the degradation of chondrocyte matrix and production of proinflammatory cytokines in osteoarthritic chondrocyte model were detected by ELISA. To mimic the osteoarthritic microenvironment in vitro studies, chondrocytes were stimulated with interleukin (IL)-1β, and then we found that the expression of miR-128-3p was downregulated. Overexpression of WISP1 inhibited the proliferation of chondrocytes, which induced apoptosis, degradation of chondrocyte matrix, production of proinflammatory cytokines, and activated the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. Then, we identified that miR-128-3p was a negative regulator of WISP1 by directly targeting its 3′-untranslated region (UTR). Moreover, the PI3K allosteric activator 740 Y-P abolished the inhibition of miR-128-3p in apoptosis, degradation of chondrocyte matrix, and inflammation. Our results showed that miR-128-3p targets WISP1 to regulate chondrocyte proliferation, apoptosis, degradation of chondrocyte matrix, and production of proinflammatory cytokines via the PI3K/Akt/NF-κB pathway, which plays a suppressed role in OA.

Bone Marrow Mesenchymal Stem Cells (BMSC)-Originated miR-133 Impedes Migration and Invasiveness of Glioma Cells While Enhancing Apoptosis via Targeting the Receptor for Activated C Kinase 1 (RACK1)

2021 ◽  
Vol 11 (9) ◽  
pp. 1818-1824
Author(s):  
Jiangbo Xiong ◽  
Sheng Liu ◽  
Bin Xiang ◽  
Weibo Zhang ◽  
Jun Du ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Glioma Cells ◽  
Luciferase Reporter ◽  
Mechanistic Study ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
Marrow Mesenchymal Stem Cells

This study aims to dissect the effects of bone marrow mesenchymal stem cells (BMSC) on the in vitro activity of glioma cells and the underlying mechanisms. The glioma cells were transfected with miR-133 mimics, RACK1-Vector, negative control (NC) and miR-133 mimic+RACK1-Vector, respectively, and then co-cultured with BMSC followed by analysis of miR-133 expression via PCR, apoptosis via flow cytometry, proliferation via CCK-8, invasion and migration via Transwell assay, the expression of proteins involved in apoptosis, anti-apoptosis, invasiveness and RACK1 by western blot, and the targeting relationship between miR-133 and RACK1 by dual-luciferase reporter gene assay. In comparison with normal glial cells, glioma cells exhibited a significantly diminished miR-133 level. miR-133 was upregulated in glioma cells after co-culture with BMSC, along with significantly restrained proliferation rate, migration and invasion activities as well as reduced protein levels (MMP-2, Vimentin, N-cadherin and MMP-9). Mechanistic study showed that miR-133 can retard the expression of RACK1, thereby impeding the invasion, migration and proliferation activities of cells while triggering cell apoptosis. In conclusion, BMSC-originated miR-133 can impede the migration and invasion while enhancing the apoptosis of glioma cells via targeting RACK1.

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