scholarly journals LncRNA GAS5 Represses Osteosarcoma Cells Growth and Metastasis via Sponging MiR-203a

2018 ◽  
Vol 45 (2) ◽  
pp. 844-855 ◽  
Author(s):  
Yang Wang ◽  
Daliang Kong
Keyword(s):  
Flow Cytometry ◽  
Luciferase Activity ◽  
Cyclin B1 ◽  
Migration And Invasion ◽  
Activity Assay ◽  
Osteosarcoma Cells ◽  
Transwell Assay ◽  
Luciferase Activity Assay ◽  
Lncrna Gas5 ◽  
Growth Suppressor

Background/Aims: LncRNA GAS5, a growth suppressor, has been reported to exert anti-tumor actions in various cancers, whereas the exact mechanism underling the anti-tumor action is still unclear. This study was aimed to investigate the effect of lncRNA GAS5 on osteosarcoma and tried to decode the underling mechanisms. Methods: Expressions of lncRNA GAS5 in MG-63 cells were silenced by shRNA transfection, while were overexpressed by vector transfection. Cell viability, migration, invasion and apoptosis were respectively assessed by MTT, Transwell assay and flow cytometry. Regulations between lncRNA GAS5 and miR-203a, as well as between miR-203a and TIMP2 were detected by qPCR, western blot and dual luciferase activity assay. Results: LncRNA GAS5 was down-regulated in MG-63 and OS-732 cells compared to hFOB1.19 cells. Silence of lncRNA GAS5 significantly promoted MG-63 cells viability, migration and invasion, and up-regulated Cyclin D1, Cyclin B1, CDK1 and CDK4 expressions. miR-203a was negatively regulated by lncRNA GAS5. The promoting activities of lncRNA GAS5 silence on MG-63 cells growth and metastasis were reversed by miR-203a suppression. TIMP2 was a target of miR-203a and the anti-growth and anti-metastasis actions of miR-203a suppression were reversed by TIMP2 silence. Further, lncRNA GAS5 silence, miR-203a overexpression, and TIMP2 silence could activate PI3K/AKT/GSK3β signaling while block NF-κB signaling. Conclusion: LncRNA GAS5 might be a tumor suppressor in osteosarcoma via sponging miR-203a, sequestering miR-203a away from TIMP2.

miR-628-5p promotes growth and migration of osteosarcoma by targeting IFI44L

2020 ◽  
Vol 98 (2) ◽  
pp. 99-105 ◽  
Author(s):  
Ju-Yong Wang ◽  
Ju-Qiang Wang ◽  
Shi-Bao Lu
Keyword(s):  
Untranslated Region ◽  
Poor Prognosis ◽  
Luciferase Activity ◽  
Gene Expression Omnibus ◽  
Migration And Invasion ◽  
Activity Assay ◽  
Luciferase Activity Assay ◽  
And Migration ◽  
Geo Database

This study investigated the role of miR-628-5p and interferon-induced protein 44-like (IFI44L) in osteosarcoma (OS) and determined whether miR-628-5p modulated OS growth by regulating IFI44L. Based on the data downloaded from Gene Expression Omnibus (GEO) database, we revealed that the expression of IFI44L was downregulated in OS and low expression of IFI44L was correlated with better prognosis of patients with OS. Biological prediction of its upstream regulatory miRNAs on the miRWalk website found that miR-628-5p is a possible upstream regulatory miRNA of IFI44L. Luciferase activity assay demonstrated that miR-628-5p could bind to the 3′ untranslated region (UTR) of IFI44L, which proved the above prediction. The expression of miR-628-5p is upregulated in OS and high expression of miR-628-5p is correlated with poor prognosis of patients with OS. The results of RT-qPCR showed that the expression of miR-628-5p in MG-63, U2OS, Saos-2, and SW1353 cells was significantly higher than that in the hFOB1.19 cells. Downregulation of miR-628-5p by miR-628-5p inhibitor significantly inhibited the proliferation, migration, and invasion of MG-63 cells. By rescue assay, we found that knockdown of IFI44L rescued the proliferation and motility of miR-628-5p depleted MG-63 cells. Collectively, our present data illustrated that miR-628-5p promoted the growth and motility of OS at least partly by targeting IFI44L. Moreover, miR-628-5p and IFI44L might be proposed as promising biomarkers in OS diagnosis and treatment.

scholarly journals The tumor-suppressive function of miR-1296-5p by targeting EGFR and CDK6 in gastric cancer

2019 ◽  
Vol 39 (1) ◽  
Author(s):  
Yan Jia ◽  
Lian-Mei Zhao ◽  
Han-Yu Bai ◽  
Cong Zhang ◽  
Su-Li Dai ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Lines ◽  
Target Genes ◽  
Luciferase Activity ◽  
Clinical Stage ◽  
Migration And Invasion ◽  
Cancer Tissue ◽  
Activity Assay ◽  
Luciferase Activity Assay

AbstractWe aimed to confirm the role of miR-1296-5p in gastric cancer and to identify its target genes. The expression of miR-1296-5p was measured in gastric cancer tissues and cell lines. The function of miR-1296-5p was examined by the overexpression and inhibition of its expression in typical gastric cell lines as well as SGC-7901 and MGC-803 cells. The targets of miR-1296-5p were identified by a luciferase activity assay. We found that miR-1296-5p was down-regulated in gastric cancer tissue and cell lines, and low expression levels of miR-1296-5p were associated with advanced clinical stage. Moreover, miR-1296-5p inhibited cell proliferation, migration, and invasion in SGC-7901 and MGC-803 cells. Then, we identified CDK6 and EGFR as novel targets of miR-1296-5p by a luciferase activity assay. Furthermore, the overexpression of miR-1296-5p suppressed the expression of CDK6 and EGFR. Our results indicated a tumor-suppressive role of miR-1296-5p through the translational repression of oncogenic CDK6 and EGFR in gastric cancer.

scholarly journals LncRNA NKILA Promotes Cardiomyocytes Apoptosis by Targeting miR22-3p-TXNIP Signal Axis to Inhibit Proliferation, Migration, and Invasion of Cardiomyocytes under High Glucose-Induced Condition

2021 ◽  
Vol 2021 ◽  
pp. 1-14
Author(s):  
Yan Zhao ◽  
Wenjuan Ming ◽  
Xiaohuan Ma ◽  
Yuchan Zheng ◽  
Yanli Yan
Keyword(s):  
Cell Proliferation ◽  
Molecular Mechanism ◽  
Diabetic Cardiomyopathy ◽  
High Glucose ◽  
Cell Apoptosis ◽  
Luciferase Activity ◽  
Migration And Invasion ◽  
Activity Assay ◽  
Human Cardiomyocytes ◽  
Luciferase Activity Assay

This study aims to investigate the molecular mechanism of LncRNA NKILA underlying promoting cardiomyocytes apoptosis and relevant diabetic cardiomyopathy. We utilized high concentration of glucose to induce human cardiomyocytes cell line AC16 to imitate diabetic cardiomyopathy. And then, we performed high-throughput big data analysis, RT-PCR, and western blot assays to evaluate the expression levels of associated mRNA and protein. Cell apoptosis was tested by Annexin V-FITC. The proliferation, migration, and invasion of AC16 cells were examined by CCK8 assay, colony formation assay, EdU assay, wound healing test, and transwell chamber assay. We utilized statistical analysis and luciferase activity assay to analyze the interaction of relevant genes. LncRNA NKILA was highly expressed in AC16 cells induced by high glucose and inhibited AC16 cell proliferation, migration, and invasion by inducing cell apoptosis. Luciferase activity assay demonstrated that LncRNA NKILA binds to miR22-3p. The influence of LncRNA NKILA on AC16 cell proliferation, migration, and invasion could be reversed by miR22-3p. Luciferase activity assay demonstrated that TXNIP was a target of miR-22-3p in AC16 cells, and all the effects of TXNIP on AC16 cell proliferation, migration, and invasion could be abolished by miR22-3p. These results provided comprehensive data about a novel molecular mechanism of LncRNA NKILA promoting cardiomyocytes apoptosis: LncRNA NKILA performed its function in AC16 cells under high glucose-induced condition by targeting mir-22-3p-TXNIP signal axis, which indicated that LncRNA NKILA may play a crucial role in diabetic cardiomyopathy.

scholarly journals Alkannin Inhibits the Development of Ovarian Cancer by Affecting miR-4461

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Yaowen Wang ◽  
Jingfang Zhang ◽  
Feipeng Wang ◽  
Wenping Chen ◽  
Jie Ma ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Flow Cytometry ◽  
Cell Viability ◽  
Migration And Invasion ◽  
Flow Cytometry Analysis ◽  
Antibacterial Effects ◽  
Transwell Assay ◽  
Ovarian Cells ◽  
Mrna And Protein Expression ◽  
Inhibit Cell Proliferation

Background. Previous studies have shown that alkannin has anticancer, anti-inflammatory, and antibacterial effects. However, the effect of alkannin in the development of ovarian cancer (OC) remains unknown. Therefore, this study aims to elucidate the function of alkannin in OC progression. Methods. RT-qPCR and western blot analysis were used to measure mRNA and protein expression. Cell viability and metastasis were detected by the CCK-8 assay, flow cytometry analysis, and transwell assay. Results. Alkannin had no cytotoxicity toward normal ovarian cells, but alkannin can inhibit cell proliferation and induce apoptosis in OC cells. In addition, alkannin inhibited cell migration and invasion and blocked EMT in OC. Besides, upregulation of miR-4461 was found in OC tissues and cells, which was regulated by alkannin. More importantly, miR-4461 can inverse the effects of alkannin on cell viability and metastasis in OC cells. Conclusion. Alkannin restrains cell viability, metastasis, and EMT in OC by downregulating miR-4461 expression.

scholarly journals ZNF139 increases multidrug resistance in gastric cancer cells by inhibiting miR-185

2018 ◽  
Vol 38 (5) ◽  
Author(s):  
Bibo Tan ◽  
Yong Li ◽  
Qun Zhao ◽  
Liqiao Fan ◽  
Dong Wang
Keyword(s):  
Gastric Cancer ◽  
Drug Resistance ◽  
Multidrug Resistance ◽  
Cell Lines ◽  
Zinc Finger Protein ◽  
Luciferase Activity ◽  
Cell Activity ◽  
Expression Level ◽  
Activity Assay ◽  
Luciferase Activity Assay

It has been reported that the expression of zinc finger protein 139 (ZNF139) and microRNA-185 (miR-185) were associated with proliferation, drug resistance of gastric cancer (GC) cells. However, the detailed mechanisms have not been fully investigated. The expression of ZNF139 in both GC tissues and cell lines was tested, then SGC7901/ADR or SGC7901 cells were transfected with ZNF139-siRNA, miR-185 analog, or pcDNA-ZNF139. Cell activity was determined by MTT assay. Real-time PCR and Western blot were utilized to detect ZNF139, miR-185, and multidrug resistance (MDR) related genes including MDR1/P-gp, GST-π, MRP-1, Bcl-2, TS and Bax. ChIP and dual luciferase activity assay were used to investigate regulation between ZNF139 and miR-185. Increased ZNF139 and decreased miR-185 expression were detected in GC tissues and cell lines. Transfection with ZNF139-siRNA into SGC7901/ADR cells markedly increased expression of miR-185, and treating with chemotherapeutic drugs ADR, 5-FU, L-OHP, the survival rate of SGC7901/ADR cells obviously decreased after ZNF139-siRNA transfection. On the other hand, transfection with pcDNA-ZNF139 in GC cell line SGC7901 resulted in an increased expression level of ZNF139 and a decline in the expression level of miR-185, meanwhile drug resistance of GC cells was clearly enhanced to ADR, 5-FU, L-OHP. Dual luciferase activity assay demonstrated that ZNF139 inhibited transcriptional activities of miR-185’s promoter in cells transfected with the reporter plasmid encompassing the upstream promoter region of miR-185 along with pcDNA-ZNF139. Our data reveal that ZNF139 might promote MDR gene MDR1/P-gp, MRP-1 and Bcl-2 by prohibiting miR-185.

scholarly journals miR-451a Inhibits the Growth and Invasion of Osteosarcoma via Targeting TRIM66

2019 ◽  
Vol 18 ◽  
pp. 153303381987020 ◽  
Author(s):  
Xiao Ma ◽  
Dan Li ◽  
Yan Gao ◽  
Cheng Liu
Keyword(s):  
Cell Lines ◽  
Luciferase Activity ◽  
Cell Counting ◽  
Osteosarcoma Cell ◽  
Reporter Assay ◽  
Osteosarcoma Cells ◽  
Transwell Assay ◽  
Tripartite Motif ◽  
Regulatory Effects ◽  
Novel Therapeutic

The importance of microRNAs in regulating osteosarcoma development has been studied in recent years. However, the function of microRNA-451a in osteosarcoma growth is rarely investigated. Here, we explored the expression of microRNA-451a in osteosarcoma cell lines. Bioinformatic software, luciferase activity reporter assay, and Western blot were conducted to determine the association between microRNA-451a and tripartite motif-containing 66. Cell Counting Kit-8 assay and transwell assay were used to explore the regulatory effects of microRNA-451a on osteosarcoma cells. Moreover, we explored whether microRNA-451a modulates osteosarcoma cell biological activity by regulating tripartite motif-containing 66. The expression of microRNA-451a was found to be downregulated in osteosarcoma and negatively regulated the expression of tripartite motif-containing 66. Tripartite motif-containing 66 was further validated as a target of microRNA-451a. MicroRNA-451a inhibits the growth and invasion of osteosarcoma cell lines through targeting tripartite motif-containing 66. The miR-451a targets tripartite motif-containing 66 may provide novel therapeutic targets for the treatment of osteosarcoma.

scholarly journals Circular RNA hsa_circ_0081343 promotes trophoblast cell migration and invasion and inhibits trophoblast apoptosis by regulating miR-210-5p/DLX3 axis

2021 ◽  
Author(s):  
Jiansheng Xie ◽  
Hui Wang ◽  
Caiqun Luo ◽  
Xiaoxia Wu ◽  
Jianming Zhang ◽  
...  
Keyword(s):  
Luciferase Activity ◽  
Circular Rna ◽  
Extravillous Trophoblast ◽  
Migration And Invasion ◽  
Circular Rnas ◽  
Protein Levels ◽  
Apoptosis Protein ◽  
Luciferase Activity Assay ◽  
Rna Immunoprecipitation

Abstract Background: Various circular RNAs (circRNAs) are dysregulated in the placenta of fetal growth restriction fetuses, but their role and regulatory mechanisms have not been fully elucidated. Herein, we aimed to elucidate the role of hsa_circ_0081343 in regulating the migration, invasion, and apoptosis in the human extravillous trophoblast HTR-8 cells.Methods: circRNA and miRNA levels were examined using quantitative real-time PCR (RT-PCR). Overexpression plasmid constructs and siRNA were used to overexpress and knockdown hsa_circ_0081343, respectively. Transwell assay and flow cytometry analysis were performed to evaluate the effect of hsa_circ_0081343 or miR-210-5p on migration, invasion, and apoptosis. Protein levels were analyzed using western blot. Dual luciferase activity assay and anti-AGO2 RNA immunoprecipitation (RIP) assays were performed to identify the relationship between miR-210-5p and hsa_circ_0081343.Results: Hsa_circ_0081343 expression was significantly downregulated in 37 FGR placental tissues as compared to healthy placental control tissues. Hsa_circ_0081343 overexpression possibly inhibits apoptosis by downregulating the expression of cleaved caspase 3 and caspase 9 and alleviates the migration and invasion of HTR-8 cells by inducing the expression of MMP2 and MMP9. The dual luciferase activity and anti-AGO2 RIP assay results showed that hsa_circ_0081343 binds to miR-210-5p. miR-210-5p overexpression eliminated the effect of hsa_circ_0081343 overexpression in HTR-8 cells. Finally, DLX3 was identified as a direct target of miR-210-5p. Conclusions: Hsa_circ_0081343 regulates the migration, invasion, and apoptosis of HTR-8 cells via the hsa-miR-210-5p/DLX3 axis. Thus, hsa_circ_0081343 plays a key role in the etiology and pathogenesis of FGR implicating its importance as a novel candidate for targeted FGR therapy.

scholarly journals MicroRNA-19 contributes to the malignant phenotypes of osteosarcoma in vitro by targeting Pax6

Tumor Biology ◽  
2018 ◽  
Vol 40 (1) ◽  
pp. 101042831774470 ◽  
Author(s):  
Qingbing Meng ◽  
Ming Dai ◽  
Xuejun Nie ◽  
Wensheng Zhang ◽  
Xingli Xu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Biological Behavior ◽  
Luciferase Reporter ◽  
Osteosarcoma Cell ◽  
Osteosarcoma Cells ◽  
Transwell Assay ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Diphenyltetrazolium Bromide

This study was conducted to detect the expression of miR-19 and Pax6 (Paired box protein 6) in human osteosarcoma cells and the effects on biological characteristics of osteosarcoma cells. Quantitative real-time polymerase chain reaction was used to detect the expression of Pax6 and miR-19 in normal human osteoblasts (hFOB 1.19) and osteosarcoma cell lines (U2OS, Saos-2, and MG-63). Results showed that miR-19 was significantly upregulated in osteosarcoma cell lines compared with that in hFOB 1.19 cells, while the expression of Pax6 messenger RNA was significantly downregulated. Pax6 was defined as the target gene of miR-19 which was validated by luciferase reporter gene analysis. Results indicated that miR-19 had an interaction with Pax6 3′-untranslated region. At the same time, the protein expression of Pax6 was significantly decreased in the MG-63 cells transfected with miR-19 mimic and was notably enhanced in osteosarcoma MG-63 cells transfected with miR-19 inhibitor. These data suggested that Pax6 was a target of miR-19 in osteosarcoma MG-63 cells. The effects of miR-19 on the biological behavior of MG-63 cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, and Transwell assay. Results showed that the downregulation of miR-19 inhibited cell viability, reduced the percentage of cells in S phase and the number of cells passing through the Transwell chamber, and increased the number of apoptotic cells. Western blot analysis showed that the inhibition of miR-19 significantly increased the expression of epithelial proteins (E-cadherin and β-catenin) and decreased the expression of mesenchymal protein (Vimentin), extracellular signal–regulated kinase, and phosphorylated extracellular signal–regulated kinase in MG-63 cells. MiR-19 inhibitor and Pax6 small interfering RNA were simultaneously transfected into MG-63 cells. Results from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, and Transwell assay demonstrated that the inhibition of Pax6 expression in MG-63 cells could reverse the cell biological effects induced by the inhibition of miR-19 expression. Based on these findings, it was suggested that miR-19, upregulated in osteosarcoma cells, negatively regulated the expression of Pax6, which can promote the malignant phenotypes of osteosarcoma cells via activation of the extracellular signal–regulated kinase signaling pathways. Therefore, miR-19/Pax6 may offer potential for use as a target for the treatment of osteosarcoma.

scholarly journals miR-101-3p Contributes to α-Synuclein Aggregation in Neural Cells through the miR-101-3p/SKP1/PLK2 Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Min Zhang ◽  
Wei Liu ◽  
Qingan Zhang ◽  
Hongfeng Hu
Keyword(s):  
Substantia Nigra ◽  
Target Gene ◽  
Luciferase Activity ◽  
Substantia Nigra Pars Compacta ◽  
Activity Assay ◽  
Protein Ubiquitination ◽  
Ubiquitination Assay ◽  
Luciferase Activity Assay ◽  
Vitro Protein

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive neuronal loss in different brain regions, including the dopaminergic (DA) neurons of the substantia nigra pars compacta (SNc). The aggregation of α-synuclein (α-Syn) plays an essential role in the progression of PD-related neuron toxicity. In this study, bioinformatic analysis was used to confirm differentially expressed genes between patients with PD and healthy donors. Immunofluorescence was used to study the aggregation of α-Syn. Flow cytometry was used to confirm the apoptosis of neurons. Western blot was used to investigate the underlying mechanism. Coimmunoprecipitation (co-IP) was used to verify the interaction between proteins. Luciferase activity assay was used to confirm the target gene of miRNA. In vitro protein ubiquitination assay was used to ascertain the role of S-phase kinase-associated protein 1 (SKP1) on the ubiquitination processes of polo-like kinase 2 (PLK2). The result indicated that miR-101-3p was overexpressed in the substantia nigra of the postmortem brains of patients with PD. The underlying role was investigated in the SH-SY5Y cell line. The overexpression of α-Syn did not result in toxicity or aggregation. However, the co-overexpression of miR-101-3p and α-Syn promoted aggregation and neuron toxicity. Luciferase activity assay indicated that SKP1 is a target gene of miR-101-3p. The co-IP experiment confirmed that SKP1 could directly interact with PLK2. In vitro protein ubiquitination assay confirmed that SKP1 could promote the ubiquitination and subsequent protein degradation of PLK2. We also observed that the cotransfection of short hairpin RNA that targets PLK2 and α-Syn overexpression plasmid results in the endoplasmic reticulum stress of neurons. Our results collectively provide evidence that miR-101-3p contributes to α-Syn aggregation in neurons through the miR-101-3p/SKP1/PLK2 pathway.

scholarly journals Expression of SASH1 in Preeclampsia and Its Effects on Human Trophoblast

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Shisan Liu ◽  
Sijia Jiang ◽  
Liping Huang ◽  
Yanhong Yu
Keyword(s):  
Flow Cytometry ◽  
Matched Control ◽  
Migration And Invasion ◽  
Flow Cytometry Analysis ◽  
Vector Group ◽  
Transwell Assay ◽  
Human Trophoblast ◽  
Empty Vector ◽  
Group Flow

Aim. To explore the involvement of SASH1 in preeclampsia. Methods. Expression of SASH1 was determined by qPCR, WB, and immunohistochemistry in the placenta of both normal and preeclamptic pregnancies. The SASH1 gene of human HTR-8/SVneo cells was overexpressed by transfection of pEZ-Lv206-SASH1. After that, the CCK-8 assay, EdU assay, transwell assay, and flow cytometry were used to examine the cell proliferation, migration, invasion, and apoptosis. Results. Higher expression of SASH1 was detected in placental tissues collected from patients with preeclampsia, compared with those from gestational age-matched control samples. The expression of SASH1 was significantly enhanced by transfection with pEZ-Lv206-SASH1 in HTR-8/SVneo cells. In addition, the HTR-8/SVneo cells transfected with pEZ-Lv206-SASH1 exhibited significantly reduced proliferation, migration, and invasion ability compared to the cells in the empty vector group and normal group. Flow cytometry analysis demonstrated that the apoptosis rate of cells transfected with pEZ-Lv206-SASH1 was significantly higher than that of cells transfected with empty vector and untreated cells. Conclusions. SASH1 is significantly upregulated in the placenta of preeclampsia, and overexpression of SASH1 can inhibit the proliferation, migration, and invasion, but induce apoptosis of trophoblast cells in vitro.

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