Downregulation of lncRNA ANRIL Inhibits Osteogenic Differentiation of Periodontal Ligament Cells via Sponging miR-7 through NF-κB Pathway

Periodontal Ligament ◽

Molecular Mechanisms ◽

Luciferase Reporter ◽

Periodontal Ligament Cells ◽

Protein Levels ◽

Alp Activity ◽

Non Coding Rnas ◽

Abstract Background Long non-coding RNAs (lncRNAs) are dysregulation in periodontitis development and involved in osteogenesis. The current study aimed was to investigate the function of lncRNA ANRIL in periodontal ligament cells (PDLCs) and potential molecular mechanisms.Methods Firstly, the level of ANRIL was tested by qPCR. Then PDLCs were treated with a mineralizing solution to induce the osteogenic differentiation. ALP activity was measured and protein levels of BMP2, Osterix, and OCN were measured by western blot. A target of ANRIL was verified using dual-luciferase reporter assay. MiR-7 level was measured by qPCR and the signalings of NF-κB pathway were tested by western blot.Results ANRIL expression was downregulated in PDL tissues. Next, ALP activity and protein levels of BMP2, Osterix, and OCN were reduced to show PDLCs were differentiated. ANRIL level was increased in differential PDLCs, and which knockdown inhibited osteogenic differentiation. Then, miR-7 was found as a target of ANRIL. The miR-7 level was upregulated in PDL tissues and reduced in differential PDLCs. Inhibition of miR-7 suppressed ALP activity and BMP2, Osterix, and OCN expression. Moreover, inhibition of miR-7 reversed the effects on the osteogenic differentiation induced by knockdown of ANRIL. Besides, the levels of p-P65 and p-IκBα were elevated by ANRIL downregulation and were rescued by suppressing miR-7.Conclusions Knockdown of ANRIL inhibited osteogenic differentiation via sponging miR-7 through the NF-κB pathway, suggesting that ANRIL might be a therapeutic target for periodontitis.

miRNA-125b Regulates Osteogenic Differentiation of Periodontal Ligament Cells Through NKIRAS2/NF-κB Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000492350 ◽

2018 ◽

Vol 48 (4) ◽

pp. 1771-1781 ◽

Cited By ~ 9

Author(s):

Nan Xue ◽

Lin Qi ◽

Guorong Zhang ◽

Yang Zhang

Keyword(s):

Western Blot ◽

Periodontal Ligament ◽

Osteoblast Differentiation ◽

Bone Repair ◽

Alveolar Bone ◽

Osteoblastic Differentiation ◽

Luciferase Reporter ◽

Periodontal Ligament Cells ◽

Glycerol Phosphate

Background/Aims: Osteogenesis of periodontal ligament cells (PDLCS) is essential for alveolar bone repair. Varieties of factors have been found involved in the regulation of PDLCs osteoblast differentiation. Aim of this study was to identify microRNA as a regulator of the os-teogenic differentiation of PDLCs. Methods: The CD markers were analyzed by flow cytometry analysis. Osteoblast differentiation of PDLCs was induced by treatment with dexamethasone, β-glycerol phosphate and α-ascorbic acid. The expression of osteoblastic phenotype was evaluated after the induction by simultaneous monitoring of alkaline phosphatase activity, the expression of genes involved in osteoblastic differentiation by RT-qPCR and Western Blot, and mineralization at the same time. MicroRNA and NKIRAS2 expression was determined by RT-qPCR. Luciferase reporter assays were performed to test whether miR-125b is capable of interacting with the 3’UTR sequence of NKIRAS2. The possible signaling pathway was determined by Western Blot. Results: In this study, we found that the expression of miR-125b was down regulated during the process of ostoblast differentiation of PDLCs. When the expression of miR-125b was up regulated, the osteogenic differentiation of PDLCs was inhibited. During this process, the over-expressed miR-125b led to the activation of NF-κB. NF-κB inhibitor interacting RAS-like 2 (NKIRAS2) is one of target gene of miR-125b, and it is a regulator of NF-κB signaling that plays various roles in osteoblastic differentiation. We demonstrate thatmiR-125b is involved in osteogenic differentiation of PDLCs. Conclusion: Our data support the hypothesis that that miR-125b attenuates PDLCs osteoblastic differentiation by targeting NKIRAS2 and enhancing NF-κB signaling.

Knockdown of Long Non-Coding RNA XIST Inhibited Doxorubicin Resistance in Colorectal Cancer by Upregulation of miR-124 and Downregulation of SGK1

Cellular Physiology and Biochemistry ◽

10.1159/000495168 ◽

2018 ◽

Vol 51 (1) ◽

pp. 113-128 ◽

Cited By ~ 35

Author(s):

Jia Zhu ◽

Rui Zhang ◽

Dongxiang Yang ◽

Jibin Li ◽

Xiaofei Yan ◽

...

Keyword(s):

Colorectal Cancer ◽

Western Blot ◽

Molecular Mechanisms ◽

Luciferase Reporter ◽

Regulatory Function ◽

Glutathione S Transferase ◽

Protein Levels ◽

Qrt Pcr ◽

Ic50 Value

Background/Aims: Doxorubicin (DOX) is a widely used chemotherapeutic agent for colorectal cancer (CRC). However, the acquirement of DOX resistance limits its clinical application for cancer therapy. Mounting evidence has suggested that aberrantly expressed lncRNAs contribute to drug resistance of various tumors. Our study aimed to explore the role and molecular mechanisms of lncRNA X-inactive specific transcript (XIST) in chemoresistance of CRC to DOX. Methods: The expressions of XIST, miR-124, serum and glucocorticoid-inducible kinase 1 (SGK1) mRNA in DOX-resistant CRC tissues and cells were detected by qRT-PCR or western blot analysis. DOX sensitivity was assessed by detecting IC50 value of DOX, the protein levels of P-glycoprotein (P-gp) and glutathione S-transferase-π (GST-π) and apoptosis. The interactions between XIST, miR-124 and SGK1 were confirmed by luciferase reporter assay, qRT-PCR and western blot. Xenograft tumor assay was used to verify the role of XIST in DOX resistance in CRC in vivo. Results: XIST expression was upregulated and miR-124 expression was downregulated in DOX-resistant CRC tissues and cells. Knockdown of XIST inhibited DOX resistance of CRC cells, as evidenced by the reduced IC50 value of DOX, decreased P-gp and GST-π levels and enhanced apoptosis in XIST-silenced DOX-resistant CRC cells. Additionally, XIST positively regulated SGK1 expression by interacting with miR-124 in DOX-resistant CRC cells. miR-124 suppression strikingly reversed XIST-knockdown-mediated repression on DOX resistance in DOX-resistant CRC cells. Moreover, SGK1-depletion-elicited decrease of DOX resistance was greatly restored by XIST overexpression or miR-124 inhibition in DOX-resistant CRC cells. Furthermore, XIST knockdown enhanced the anti-tumor effect of DOX in CRC in vivo. Conclusion: XIST exerted regulatory function in resistance of DOX possibly through miR-124/SGK1 axis, shedding new light on developing promising therapeutic strategy to overcome chemoresistance in CRC patients.

lncRNA CRNDE promotes the proliferation and metastasis by acting as sponge miR-539-p to regulate POU2F1 expression in HCC

10.21203/rs.3.rs-15899/v2 ◽

2020 ◽

Author(s):

Zhixi Li ◽

Gang Wu ◽

Jie Li ◽

Youyu Wang ◽

Xueming Ju ◽

...

Keyword(s):

Western Blot ◽

Luciferase Reporter ◽

Akt Pathway ◽

Migration And Invasion ◽

Reporter Assay ◽

Normal Cells ◽

Normal Tissues ◽

Qrt Pcr ◽

Hcc Cells ◽

Abstract Background This article focuses on the roles and mechanism of lncRNA CRNDE on the progression of HCC. Methods We used qRT-PCR to detect the expression of lncRNA CRNDE in HCC cells, normal cells and clinical tissues. MTT assay, FCM analysis, Transwell migration and invasion assay were used to detect the effects of lncRNA CRNDE on cell viability, apoptosis, migration and invasion of HCC cells. The expression of apoptosis-related proteins Bcl-2, Bax, Cleaved Caspase 3, Cleaved Caspase 9, EMT epithelial marker E-cadherin and mesothelial marker Vimentin were analyzed by Western blot. Online prediction software was used to predict the binding sites between lncRNA CRNDE and miR-539-5p, or miR-539-5p and POU2F1 3’UTR. Dual luciferase reporter assay, qRT-PCR and RNA pulldown were used to detect target-relationship between lncRNA CRNDE and miR-539-5p. Dual luciferase reporter assay, qRT-PCR, Western blot and Immunofluorescence were used to detect target-relationship between miR-539-5p and POU2F1. qRT-PCR was used to detect the expression of miR-539-5p and POU2F1 in clinical tissues. Rescue experiments was used to evaluate the association among lncRNA CRNDE, miR-539-5p and POU2F1. Finally, we used Western blot to detect the effects of lncRNA CRNDE, miR-539-5p and POU2F1 on NF-κB and AKT pathway. Results lncRNA CRNDE was highly expressed in HCC cells and HCC tissues compared with normal cells and the corresponding adjacent normal tissues. lncRNA CRNDE promoted the cell viability, migration and invasion of HCC cells, while inhibited the apoptosis and promoted the EMT process of HCC cells. lncRNA CRNDE adsorbed miR-539-5p acts as a competitive endogenous RNA to regulate POU2F1 expression indirectly. In HCC clinical tissues, miR-539-5p expression decreased and POU2F1 increased compared with the corresponding adjacent normal tissues. lncRNA CRNDE/miR-539-5p/POU2- F1 participated the NF-κB and AKT pathway in HCC. Conclusion lncRNA CRNDE promotes the expression of POU2F1 by adsorbing miR-539-5p, thus promoting the progression of HCC. Keywords: HCC, lncRNA CRNDE, miR-539-5p, POU2F1, ceRNA

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

BioMed Research International ◽

10.1155/2020/8822232 ◽

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 ◽

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.

Osteocyte-derived exosomes induced by mechanical strain promote human periodontal ligament stem cell proliferation and osteogenic differentiation in an inflammatory environment via the miR-181b-5p/PTEN/AKT signaling pathway

10.21203/rs.3.rs-21278/v1 ◽

2020 ◽

Author(s):

Peiying Lv ◽

Pengfei Gao ◽

Yanyan Yang ◽

Zihui Wang ◽

Lu Sun ◽

...

Keyword(s):

Stem Cell ◽

Periodontal Ligament ◽

Mechanical Strain ◽

Akt Signaling ◽

Luciferase Reporter ◽

Periodontal Tissue ◽

Reporter Assay ◽

Akt Signaling Pathway ◽

Periodontal Ligament Stem Cell

Abstract Background: The oral cavity is a complex environment in which periodontal tissue is constantly stimulated by external microorganisms and mechanical forces. Proper mechanical force helps maintain periodontal tissue homeostasis and improper inflammatory response can break the balance. Periodontal ligament (PDL) cells plays crucial roles in responding these challenges and maintaining the homeostasis of periodontal tissue. However, the mechanisms underlying PDL cell property changes induced by inflammatory and mechanical force microenvironments are still unclear. Recent studies have shown that exosomes function as a mean of cell-cell and cell-matrix communication in biological processes. Methods: Human periodontal ligament stem cells (HPDLSCs) were tested by the CCK8 assay, EdU, alizarin red and ALP staining to evaluate the functions of exosomes induced by mechanical strain. MicroRNA sequencing was used to find the discrepancy miRNA in exosomes. In addition, RT-PCR, FISH, luciferase reporter assay and western blotting assay were used to investigated the mechanism of miR-181b-5p regulating proliferation and osteogenic differentiation through the PTEN/AKT pathway. Results: In this study, the exosomes secreted by MLO-Y4 cells exposed to mechanical strain (Exosome-MS) contributed to human periodontal ligament stem cell (HPDLSC) proliferation and osteogenic differentiation. High-throughput miRNA sequencing showed that miR181b-5p was upregulated in Exosome-MS compared to the exosomes derived from MLO-Y4 cells lacking MS. The luciferase reporter assay demonstrated that miR-181b-5p may target Phosphatase tension homolog deletion (PTEN). In addition, PTEN was negatively regulated by overexpressing miR-181b-5p. PCR and western blot analyses verified that miR‐181b-5p enhanced the protein kinase B (AKT) activity and improved downstream factor transcription. Furthermore, miR-181b-5p effectively ameliorated the inhibition of HPDLSC proliferation and osteogenesis induced by inflammation. Conclusions: This study concluded that exosomes induced by mechanical strain promote HPDLSC proliferation and osteogenic differentiation via the miR-181b-5p/PTEN/AKT signaling pathway, suggesting a potential mechanism for maintaining periodontal homeostasis.

Knockdown of LincRNA PADNA Promotes Bupivacaine-Induced Neurotoxicity by miR-194/FBXW7 Axis

10.21203/rs.3.rs-30999/v1 ◽

2020 ◽

Author(s):

Fan Yuning ◽

Chen Liang ◽

Wang Tenghuan ◽

Nan Zhenhua ◽

Shengkai Gong

Keyword(s):

Functional Analysis ◽

Western Blot ◽

Cell Viability ◽

Luciferase Reporter Assay ◽

Luciferase Reporter ◽

Reporter Assay ◽

Drg Neurons ◽

Abstract The aim of the study was to explore the function and mechanism of lincRNA PADNA in bupivacaine-induced neurotoxicity. Mouse DRG neurons were cultured in vitro and treated with bupivacaine to establish the neurotoxicity model. Caspase3 activity, cell viability, tunel assay were analyzed to assess the role of lincRNA PADNA. Dual-luciferase reporter assay was used to determine the binding target of lincRNA PANDA. The expression of lincRNA PADNA was significantly increased with the increasing concentration of bupivacaine. Functional analysis revealed that knockdown of lincRNA PADNA accelerated the caspase3 activity and inhibited the cell viability. Western blot showed that knockdown of lincRNA PADNA promoted the occurrence of cleaved-caspase3. We also proved that lincRNA PADNA may bind with miR-194. Overexpression of miR-194 could rescued the function of lincRNA PADNA, suggesting that lincRNA PADNA may sponge miR-194. In addition, we provided new evidences that lincRNA PADNA/miR-194/FBXW7 axis play an important role in the neurotoxicity process. We performed comprehensive experiments to verify the function and mechanism of lincRNA PADNA in bupivacaine-induced neurotoxicity. Our study provided new evidences and clues for prevention of neurotoxicity.

MicroRNA Hsa-Let-7b Regulates the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Targeting CTHRC1

Stem Cells International ◽

10.1155/2021/5791181 ◽

2021 ◽

Vol 2021 ◽

pp. 1-15

Author(s):

Lin Fu ◽

Na Li ◽

Yu Ye ◽

Xiaying Ye ◽

Tong Xiao ◽

...

Keyword(s):

Stem Cells ◽

Periodontal Ligament ◽

Cell Counting ◽

Luciferase Reporter ◽

Periodontal Ligament Stem Cells ◽

Alizarin Red ◽

Proliferation Ability ◽

Polymerase Chain ◽

Let-7 miRNA family has been proved as a key regulator of mesenchymal stem cells’ (MSCs’) biological features. However, whether let-7b could affect the differentiation or proliferation of periodontal ligament stem cells (PDLSCs) is still unknown. Here, we found that the expression of hsa-let-7b was visibly downregulated after mineralization induction of PDLSCs. After transfected with hsa-let-7b mimics or inhibitor reagent, the proliferation ability of PDLSCs was detected by cell counting kit-8 (CCK-8), flow cytometry, and 5-ethynyl-2-deoxyuridine (EdU) assay. On the other hand, the osteogenic differentiation capacity was detected by alkaline phosphatase (ALP) staining and activity, alizarin red staining, Western blot, and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). We verified that hsa-let-7b did not significantly impact the proliferation ability of PDLSCs, but it could curb the osteogenic differentiation of PDLSCs. Besides, we predicted CTHRC1 acts as the downstream gene of hsa-let-7b to affect this process. Moreover, the combination of CTHRC1 and hsa-let-7b was verified by dual luciferase reporter assay. Our results demonstrated that the osteogenic differentiation of PDLSCs was enhanced after inhibiting hsa-let-7b, while was weakened after cotransfection with Si-CTHRC1. Collectively, hsa-let-7b can repress the osteogenic differentiation of PDLSCs by regulating CTHRC1.

Osteocyte-derived exosomes induced by mechanical strain promote human periodontal ligament stem cell proliferation and osteogenic differentiation via the miR-181b-5p/PTEN/AKT signaling pathway

10.21203/rs.3.rs-21278/v2 ◽

2020 ◽

Author(s):

Peiying Lv ◽

Pengfei Gao ◽

Guangjie Tian ◽

Yanyan Yang ◽

Feifei Mo ◽

...

Keyword(s):

Stem Cell ◽

Periodontal Ligament ◽

Mechanical Strain ◽

Akt Signaling ◽

Luciferase Reporter ◽

Periodontal Tissue ◽

Reporter Assay ◽

Akt Signaling Pathway ◽

Periodontal Ligament Stem Cell

Abstract Background: The oral cavity is a complex environment in which periodontal tissue is constantly stimulated by external microorganisms and mechanical forces. Proper mechanical force helps maintain periodontal tissue homeostasis and improper inflammatory response can break the balance. Periodontal ligament (PDL) cells plays crucial roles in responding these challenges and maintaining the homeostasis of periodontal tissue. However, the mechanisms underlying PDL cell property changes induced by inflammatory and mechanical force microenvironments are still unclear. Recent studies have shown that exosomes function as a mean of cell-cell and cell-matrix communication in biological processes. Methods: Human periodontal ligament stem cells (HPDLSCs) were tested by the CCK8 assay, EdU, alizarin red and ALP staining to evaluate the functions of exosomes induced by mechanical strain. MicroRNA sequencing was used to find the discrepancy miRNA in exosomes. In addition, RT-PCR, FISH, luciferase reporter assay and western blotting assay were used to investigated the mechanism of miR-181b-5p regulating proliferation and osteogenic differentiation through the PTEN/AKT pathway. Results: In this study, the exosomes secreted by MLO-Y4 cells exposed to mechanical strain (Exosome-MS) contributed to human periodontal ligament stem cell (HPDLSC) proliferation and osteogenic differentiation. High-throughput miRNA sequencing showed that miR181b-5p was upregulated in Exosome-MS compared to the exosomes derived from MLO-Y4 cells lacking MS. The luciferase reporter assay demonstrated that miR-181b-5p may target Phosphatase tension homolog deletion (PTEN). In addition, PTEN was negatively regulated by overexpressing miR-181b-5p. PCR and western blot analyses verified that miR‐181b-5p enhanced the protein kinase B (AKT) activity and improved downstream factor transcription. Furthermore, miR-181b-5p effectively ameliorated the inhibition of HPDLSC proliferation and osteogenesis induced by inflammation. Conclusions: This study concluded that exosomes induced by mechanical strain promote HPDLSC proliferation and osteogenic differentiation via the miR-181b-5p/PTEN/AKT signaling pathway, suggesting a potential mechanism for maintaining periodontal homeostasis.

MicroRNA-212-3p Attenuates Neuropathic Pain via Targeting Sodium Voltage-gated Channel Alpha Subunit 3 (NaV 1.3)

Current Neurovascular Research ◽

10.2174/1567202616666191111104145 ◽

2020 ◽

Vol 16 (5) ◽

pp. 465-472

Author(s):

Yingda Li ◽

Xizhe Zhang ◽

Zhimei Fu ◽

Qi Zhou

Keyword(s):

Neuropathic Pain ◽

Western Blot ◽

Intrathecal Injection ◽

Luciferase Reporter Assay ◽

Male Rats ◽

Luciferase Reporter ◽

Reporter Assay ◽

Expression Levels ◽

Tnf Α ◽

Purpose: To explore the role and potential mechanism of miR-212-3p in neuropathic pain regulation. Methods: Adult male rats were used to establish chronic constriction injury (CCI) model to mimic the neuropathic pain. Then, paw withdrawal threshold (PWT) and paw withdrawal thermal latency (PWL) were determined. The concentrations of interleukin 1 beta (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were measured with enzyme-linked immune sorbent assay (ELISA) kit and the expression of miR-212-3p was measured by real time quantitative PCR (RTqPCR). Besides, miR-212-3p agomir was intrathecally injected into CCI rats and the expression of key apoptotic proteins was determined by western blot. Furthermore, dual-luciferase reporter assay was used to determine the binding of miR-212-3p and 3’ untranslated regions (3’UTR) of NaV1.3 and the expression levels of NaV1.3 were measured by western blot and RT-qPCR. Results: In the CCI group, the PWT and PWL were significantly decreased and IL-1β, IL-6 and TNF-α were increased. miR-212-3p was decreased in response to CCI. The intrathecal injection of miR-212-3p agomir into CCI rats improved the PWT and PWL, decreased the IL-1β, IL-6 and TNF-α, decreased the expression levels of BCL2 associated X, apoptosis regulator (Bax), cleaved caspase-3 and increased the expression levels of BCL2 apoptosis regulator (Bcl-2). The results of dual--luciferase reporter assay showed that miR-212-3p could directly bind with 3’UTR of NaV1.3. The expression of NaV1.3 was up-regulated in CCI rats who were intrathecally injected with miRctrl, whereas it decreased in CCI rats intrathecally injected with miR-212-3p agomir. Conclusion: The expression of miR-212a-3p attenuates neuropathic pain by targeting NaV1.3.