scholarly journals MiR-382-5p’s Function as a Suppressor in Osteosarcoma (OS) by Targeting PDPK1

2021 ◽  
Vol 21 (04) ◽  
Author(s):  
Jilu Liu
Keyword(s):  
Cell Viability ◽  
Regulatory System ◽  
Functional Results ◽  
Luciferase Reporter ◽  
Flow Cytometric Method ◽  
Flow Cytometric ◽  
Cell Metastasis ◽  
Normal Human ◽  
Normal Human Osteoblast ◽  
Dual Luciferase Reporter Assay

ABSTRACT miR-382-5p engages in development of osteosarcoma (OS). However, the regulatory system of miR-382-5p in osteosarcoma remains to be revealed. This research studied the interplay between PDPK1 and miR- 382-5p in OS. RT-PCR was used to evaluate miR-382-5p and PDPK1 expression in OS cells and normal human osteoblast cells. Dual-luciferase reporter assay validated PDPK1 as a miR-382-5p target. CCK-8 evaluated the cell viability. Flow cytometric method determined cell apoptosis rate. Transwell and Scratch assays estimated the cell metastasis. miR-382-5p was inhibited in OS cells. Further functional results showed miR-382-5p upregulation reduced cell viability, and mobility by mediating PDPK1 in OS cells

The Clinical Significance of miR-135b-5p and Its Role in the Proliferation and Apoptosis of Hippocampus Neurons in Children with Temporal Lobe Epilepsy

2020 ◽  
Vol 42 (5-6) ◽  
pp. 187-194
Author(s):  
Ruixiang Li ◽  
Jiahua Hu ◽  
Sue Cao
Keyword(s):  
Temporal Lobe Epilepsy ◽  
Cell Viability ◽  
Temporal Lobe ◽  
Luciferase Reporter ◽  
Diagnostic Biomarker ◽  
Flow Cytometric ◽  
Proliferation And Apoptosis ◽  
Apoptosis Assay ◽  
Polymerase Chain ◽  
Adverse Influence

Temporal lobe epilepsy (TLE) is the most familiar localized epilepsy in children. MicroRNAs (miRNAs) are essential for the inhibition or promotion of numerous diseases. This study aimed to detect the expression of miR-135b-5p and primarily uncover its underlying function and mechanism in children with TLE. Quantitative real-time polymerase chain reaction was used to evaluate the expression of miR-135b-5p in children with TLE and in a rat model of epilepsy. MTT assay and flow cytometric apoptosis assay were conducted to evaluate the effects of miR-135b-5p on cell viability and apoptosis. Additionally, the dual luciferase reporter assay was performed to confirm the direct target of miR-135b-5p. Our data showed that the expression of miR-135b-5p was significantly decreased in children with TLE and in the epileptic rat neuron model. The dysregulation of miR-135b-5p could serve as a promising diagnostic biomarker for children with TLE. The overexpression of miR-135b-5p moderated the adverse influence on cell viability and apoptosis induced by magnesium-free medium. SIRT1 was identified as a target gene of miR-135b-5p. These results proved that miR-135b-5p might serve as a potential diagnostic biomarker in children with TLE. Overexpression of miR-135b-5p alleviates the postepileptic influence on cell viability and apoptosis by targeting SIRT1.

Silencing of lncRNA MEG8 Represses the Viability, Migration, and Invasion of Wilms’ Tumor Cells through Mediating miR-23a-3p/CRK Axis

2021 ◽  
pp. 1-13
Author(s):  
Jing Shen ◽  
Qiang Shu
Keyword(s):  
Cell Viability ◽  
Wilms Tumor ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Transwell Assay ◽  
Reaction Cell ◽  
Tumor Study ◽  
Polymerase Chain ◽  
Dual Luciferase Reporter Assay

<b><i>Purpose:</i></b> Compelling evidence has unveiled the importance of long noncoding RNAs (lncRNAs) in malignant behavior of Wilms’ tumor (WT). Hereon, we intend to assess the function and associated molecular mechanism of lncRNA maternally expressed gene 8 (MEG8) in WT cells. <b><i>Methods:</i></b> Expression levels of MEG8, miR-23a-3p, and CT10 regulator of kinase (CRK) were determined by quantitative real-time polymerase chain reaction. Cell viability was assessed by MTT assay. Besides, wound healing assay and transwell assay were applied to examine abilities of cell migration and invasion, respectively. Dual-luciferase reporter assay was employed to test the interplay among MEG8, miR-23a-3p, and CRK. Western blot was used to detect relative protein expression of CRK. <b><i>Results:</i></b> MEG8 and CRK expression was elevated, while miR-23a-3p expression was decreased in WT tissues and cells. The histologic type, lymphatic metastasis, and National Wilms Tumor Study (NWTS) stage were associated with the expression of MEG8, miR-23a-3p, and CRK in WT patients. MEG8 knockdown or miR-23a-3p overexpression restrained WT cells in cell viability, migration, and invasiveness in vitro. As to mechanism exploration, MEG8 could directly bind to miR-23a-3p and then miR-23a-3p targeted CRK. MEG8 was inversely correlated with miR-23a-3p and positively correlated with CRK in WT tissues. Meantime, miR-23a-3p was inversely correlated with CRK in WT tissues. Additionally, MEG8 knockdown-mediated suppressive impacts on cell viability, migration, and invasiveness were reversed by overexpression of CRK or repression of miR-23a-3p in WT cells. <b><i>Conclusions:</i></b> The cell viability, migration, and invasiveness of WT cells were repressed by MEG8 knockdown via targeting the miR-23a-3p/CRK axis.

scholarly journals MiR-34b-5p Mediates the Proliferation and Differentiation of Myoblasts by Targeting IGFBP2

Cells ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 360 ◽  
Author(s):  
Wang ◽  
Zhang ◽  
Li ◽  
Abdalla ◽  
Chen ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cell Cycle Progression ◽  
Muscle Development ◽  
Muscle Growth ◽  
Flow Cytometric Analysis ◽  
Luciferase Reporter ◽  
Cycle Progression ◽  
Flow Cytometric ◽  
Proliferation And Differentiation ◽  
Dual Luciferase Reporter Assay

As key post-transcriptional regulators, microRNAs (miRNAs) play an indispensable role in skeletal muscle development. Our previous study suggested that miR-34b-5p and IGFBP2 could have a potential role in skeletal muscle growth. Our goal in this study is to explore the function and regulatory mechanism of miR-34b-5p and IGFBP2 in myogenesis. In this study, the dual-luciferase reporter assay and Western blot analysis showed that IGFBP2 is a direct target of miR-34b-5p. Flow cytometric analysis and EdU assay showed that miR-34b-5p could repress the cell cycle progression of myoblasts, and miR-34b-5p could promote the formation of myotubes by promoting the expression of MyHC. On the contrary, the overexpression of IGFBP2 significantly facilitated the proliferation of myoblasts and hampered the formation of myotubes. Together, our results indicate that miR-34b-5p could mediate the proliferation and differentiation of myoblasts by targeting IGFBP2.

Adhesion and Cell Viability of Normal Human Osteoblasts (NHOst) on Scaffolds of Poly (3-hydroxybutyrate)

2015 ◽  
Vol 1721 ◽  
Author(s):  
Maraolina Domínguez-Díaz ◽  
Angelica Meneses-Acosta ◽  
Angel Romo-Uribe
Keyword(s):  
Contact Angle ◽  
Cell Adhesion ◽  
Cell Viability ◽  
Human Osteoblast ◽  
Cell Nuclei ◽  
Electrospun Membrane ◽  
Osteoblast Cells ◽  
Cast Film ◽  
Normal Human ◽  
Normal Human Osteoblast

ABSTRACTBiodegradable Normal Human Osteoblast (NHOst) cells were inoculated into the polymer scaffolds of poly(β-hydroxybutyrate) (PHB) obtained from a specially developed strain of Azotobacter vinelandii. Cell adhesion is essential to promote growth on scaffolds for tissue engineering. Thus, in this research we focused on the adhesion of osteoblast cells to PHB scaffolds produced by solution casting and electrospinning. Cell viability was also investigated up to 168 hrs. Water contact angle on the PHB scaffolds was determined prior to the cells inoculation. The contact angle is usually related to the ability of different cell strains to adhere to a given material. The as cast film exhibited a contact angle α=72° whereas for the electrospun membrane α=102°, thus in theory cell adhesion would be greater for the cast film. Biological testing was carried out on plates of 24 wells; cell viability was determined by Trypan Blue, cell morphology by optical microscopy, and cell nuclei integrity by staining with Acridine orange. Parallel studies were carried out on control (empty) wells. Microscopy observations 168 hrs after cell inoculation showed larger quantities of osteoblast cells in the wells containing PHB scaffolds and the cell nuclei were still active. Moreover, it was found that the cells grew inside the PHB scaffolds and the cell viability was slightly greater for the electrospun scaffold. Interestingly, the time to remove the cells from the scaffolds (film and membranes) was increasing function of the cell culture time, therefore suggesting that PHB promotes adhesion of Normal Human Osteoblast cells to its surface.

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

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 ◽  
Dual Luciferase Reporter Assay

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.

scholarly journals miR-4319 Reduces Tumorigenicity of Cervical Cancer Cells by Targeting TUFT1

2021 ◽  
Author(s):  
Lijun Zheng ◽  
Qiongzhen Ren ◽  
Weipei Zhu ◽  
Xiaomin Tao ◽  
Liangsheng Guo
Keyword(s):  
Cell Viability ◽  
Cell Apoptosis ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Tumor Suppressor Function ◽  
Over Expression ◽  
Anti Cancer ◽  
Cervical Cancer Cells ◽  
Direct Target ◽  
Dual Luciferase Reporter Assay

Abstract Background: In the present study, a new tumor suppressor function of miR-4319 was disclosed in CC. Methods: Up-regulation of miR-4319 suppressed cell viability, proliferation, migration, invasion, and induced cell apoptosis in CC cells were measured by cell transfection, CCK-8, colony formation, EdU, flow cytometer, wound healing, transwell migration and invasion and western blot assays. Moreover, Tuftelin 1 (TUFT1) was verified as a direct target of miR-4319 by binding its 3’-UTR, confirmed by dual-luciferase reporter assay. Result: The expression of miR-4319 was obviously decreased in clinical CC tissues and CC cell lines.TUFT1 was remarkably increased in clinical CC tissues and CC cell lines, and was negatively associated with miR-4319 expression. Furthermore, over-expression of TUFT1 partially restored the effects of miR-4319 mimic on cell viability, proliferation, migration, invasion, and cell apoptosis in CC cells. Conclusion: miR-4319 played an anti-cancer role in the occurrence and development of CC, which might be achieved by targeting TUFT1.

MiR-196a promoted cell migration, invasion and the epithelial-mesenchymal transition by targeting HOXA5 in osteosarcoma

2020 ◽  
Vol 29 (2) ◽  
pp. 291-298
Author(s):  
Xiaoli Wang ◽  
Lili Zhang ◽  
Xingfeng Zhang ◽  
Cuihong Xing ◽  
Ruidong Liu ◽  
...  
Keyword(s):  
Cell Migration ◽  
Epithelial Mesenchymal Transition ◽  
Bone Cancer ◽  
Luciferase Reporter ◽  
Mesenchymal Transition ◽  
Tumor Tissues ◽  
Cell Metastasis ◽  
Primary Bone ◽  
Polymerase Chain ◽  
Dual Luciferase Reporter Assay

INTRODUCTION: Osteosarcoma (OS), aggressive neoplasms of the bone, is the most common primary bone cancer in children. MiR-196a usually low expressed in several tumors and its functions in osteosarcoma still unclear. MATERIALS AND METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to assess the expression of miR-196a and the HOXA5. Cell metastasis and epithelial-mesenchymal transition (EMT) abilities were assessed using Transwell and western blot. The dual luciferase reporter assay was carried out to verify whether miR-196a directly targeted the 3’-untranslated region (UTR) of HOXA5 mRNA. RESULTS: MiR-196a was overexpressed and HOXA5 was low expressed in osteosarcoma versus the non-tumor tissues and normal cell lines. Upregulation of miR-196a or downregulation of HOXA5 was associated with worse outcome of osteosarcoma patients. MiR-196a enhanced cell migration, invasion and EMT by regulating the expression of HOXA5 through directly targeting the 3’-UTR of its mRNA in osteosarcoma. HOXA5 partially reversed roles of miR-196a on metastasis and EMT in osteosarcoma. CONCLUSIONS: MiR-196a promoted cell metastasis and EMT by targeting the 3’-UTR of HOXA5 mRNA in osteosarcoma. The newly identified miR-196a/HOXA5 axis provides novel insight into the pathogenesis of osteosarcoma.

scholarly journals MicroRNA miR-212-3p targets NFIA to suppress HBV replication and tumor progression in hepatocellular carcinoma via repressing Enhancer I activity

2021 ◽  
Author(s):  
Hui Tian ◽  
Zhenkun He
Keyword(s):  
Hepatocellular Carcinoma ◽  
Hepatitis B ◽  
Cell Viability ◽  
Hbv Dna ◽  
Hbv Infection ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Hbv Replication ◽  
Dual Luciferase Reporter Assay

IntroductionEmerging evidence identifies that microRNAs (miRNAs) are associated with hepatitis B virus (HBV) infection. In the current study, we mainly focus on the functions and underlying mechanisms of miR-212-3p in HBV replication in hepatocellular carcinoma (HCC).Material and methodsThe levels of miR-212-3p, nuclear factor I A (NFIA) and HBV DNA copies were measured by qRT-PCR. The level of core particle-associated HBV DNA, the productions of hepatitis B surface antigen (HBsAg) and hepatitis B e-antigen (HBeAg), and the expression of NFIA were detected via southern blot assay, ELISA and western blot assay, respectively. The putative target of miR-212-3p was predicted by TargetScan and Pictar, followed by the dual luciferase reporter assay and RNA immunoprecipitation (RIP) assay to validate the interaction. The interaction between miR-212-3p and Enhancer I/X promoter (EnI/Xp) reporter was also verified by dual luciferase reporter assay. In addition, the cell viability and apoptotic rate were detected by MTT and flow cytometry, respectively.ResultsmiR-212-3p mimics or NFIA knockdown inhibited HBV expression and replication in HepG2.2.15 cells, while miR-212-3p inhibitor or NFIA overexpression showed the opposite trend. NFIA was confirmed as a direct target of miR-212-3p. Furthermore, miR-212-3p impeded HBV expression and replication by suppressing NFIA. Besides, miR-212-3p lowered EnI/Xp activity by regulating NFIA. In addition, miR-212-3p retarded cell viability and induced apoptosis through targeting NFIA.ConclusionsmiR-212-3p targets NFIA to down-regulate its expression, thereby inhibiting HBV replication and tumorigenesis in HCC. Our finding might provide a promising therapeutic target for HBV infection.

scholarly journals Long Noncoding RNA LINC01133 Functions as an miR-422a Sponge to Aggravate the Tumorigenesis of Human Osteosarcoma

2018 ◽  
Vol 26 (3) ◽  
pp. 335-343 ◽  
Author(s):  
Hai-Feng Zeng ◽  
Hai-Yan Qiu ◽  
Fa-Bo Feng
Keyword(s):  
Noncoding Rna ◽  
Malignant Tumors ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Mirna Sponge ◽  
Primary Bone Tumor ◽  
Human Osteosarcoma ◽  
Normal Human ◽  
Primary Bone ◽  
Dual Luciferase Reporter Assay

Long noncoding RNAs (lncRNAs) have been verified to participate in various types of malignant tumors, including osteosarcoma (OS), which is the most common primary bone tumor with outstanding morbidity. Although an increasing number of lncRNAs have been reported to mediate the occurrence of OS, the potential mechanisms are still unclear. This study intends to uncover the mechanism by which lncRNA LINC01133 functions as an miRNA sponge to mediate OS tumorigenicity. In this study, we found that the expression level of LINC01133 was statistically upregulated in OS tumor tissue and cell lines compared to noncancerous tissues and a normal human osteoplastic cell line. LINC01133 silencing could also observably suppress the proliferation, migration, and invasion of OS cells (HOS and U2-OS). Bioinformatics analysis predicted that LINC01133 specifically targeted miR-422a, which was validated by dual-luciferase reporter assay. Furthermore, functional experiments revealed that miR-422a played a tumor-suppressive role in OS progression and could effectively reverse the function of LINC01133. In summary, our study discovered that lncRNA LINC01133 aggravates the proliferation, migration, and invasion of OS by sponging miR-422a, which provides a novel insight in the tumorigenesis of OS.

microRNA-138-5p Participates in Psoriasis Progress Through Regulating the Growth of Keratinocytes and Inflammatory Responses by Targeting Sirtuin 1

2019 ◽  
Vol 9 (11) ◽  
pp. 1575-1582
Author(s):  
Shuyue Chen ◽  
Mengyun Zhou ◽  
Weifeng Zha ◽  
Yunyun Shan
Keyword(s):  
Cell Viability ◽  
Inflammatory Cytokines ◽  
Cell Apoptosis ◽  
Inflammatory Responses ◽  
Sirtuin 1 ◽  
Luciferase Reporter ◽  
Down Regulation ◽  
Novel Target ◽  
Crucial Part ◽  
Dual Luciferase Reporter Assay

Accumulating evidence has shown that microRNAs (miRNAs) are involved in the pathophysiology of psoriasis. The present study aimed to identify the effect and mechanism of miR-138-5p in psoriasis. In the present study, we observed that miR-138-5p expressed higher levels in psoriasis tissues compared to the control. Results from biological software and dual-luciferase reporter assay illustrated that miR-138-5p directly targeted sirtuin 1 (SIRT1). Moreover, we observed that SIRT1 was down-regulated in psoriasis tissues. Therefore, we supposed that miR-138-5p and SIRT1 might regulate the progress of psoriasis. Additionally, the functions of miR-138-5p and SIRT1 on cell viability, cell apoptosis, inflammatory cytokines expression as well as signaling pathways were evaluated. The findings demonstrated that down-regulation of miR-138-5p significantly suppressed cell viability and promoted cell apoptosis accompanied with the decreased expression of Bcl-2 and increased expression of Bax. However, these effects were reversed by SIRT1-siRNA. It was also revealed that the mRNA expression levels of inflammatory cytokines including TNF-α, IFN-γ, and IL-22 were suppressed by miR-138-5p down-regulation in HaCaT cells, while the effects were overturned by SIRT1-siRNA co-transfection. Eventually, we found that miR-138-5p inhibitor suppressed the expression of p-p65 in NF-kB pathway at protein level, and the result was reversed by SIRT1-siRNA in a similar way. In general, our results elucidated that miR-138-5p played a crucial part in psoriasis progress through regulating the growth of keratinocytes and inflammatory responses by targeting SIRT1. Therefore, miR-138-5p might be considered as a novel target for the therapeutic strategies in psoriasis.

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