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 ◽  
Reporter Assay ◽  
Drg Neurons ◽  
New Evidence ◽  
Dual Luciferase Reporter Assay

Abstract Background: The aim of the study was to explore the function and mechanism of lincRNA PADNA in bupivacaine-induced neurotoxicity. Methods: Mouse DRG neurons were cultured in vitro and treated with bupivacaine to establish a neurotoxicity model. Caspase3 activity, cell viability, and TUNEL assays were analyzed to assess the role of lincRNA PADNA. A dual-luciferase reporter assay was used to determine the binding target of lincRNA PANDA. Results: The expression of lincRNA PADNA was significantly increased with increasing concentrations of bupivacaine. Functional analysis revealed that knockdown of lincRNA PADNA increased caspase3 activity and inhibited cell viability. Western blot analysis showed that knockdown of lincRNA PADNA promoted cleaved caspase3 levels. We also revealed that lincRNA PADNA may bind with miR-194. Knockdown of miR-194 rescued the function of lincRNA PADNA, suggesting that lincRNA PADNA may sponge miR-194. In addition, we provided new evidence that the lincRNA PADNA/miR-194/FBXW7 axis plays an important role in the neurotoxicity process. Conclusion: We performed comprehensive experiments to verify the function and mechanism of lincRNA PADNA in bupivacaine-induced neurotoxicity. Our study provides new evidence and clues for the prevention of neurotoxicity.

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 CircRNA_100290 Promotes GC Cell Proliferation And Invasion Via miR-29b-3p/ITGA11 Axis And Is Regulated By EIF4A3

2021 ◽  
Author(s):  
Gang Wang ◽  
Dan Sun ◽  
Wenhui Li ◽  
Yan Xin
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Qrt Pcr ◽  
Node Metastasis ◽  
Dual Luciferase Reporter Assay

Abstract Background: Circular RNA (circRNA) has been reported as an important regulator in the development and progression of various carcinomas. However, the role of circRNA_100290 in gastric cancer (GC) is still unclear. This study aimed to investigate the role of circRNA_100290 in GC invasion and metastasis and its possible mechanism.Methods: The expression of circRNA_100290 in GC cells and tissues were examined using quantitative real-time polymerase chain reaction (qRT-PCR). The role of circRNA_100290 in cell proliferation, migration, and invasion was evaluated on AGS and HGC-27 cell lines in vitro. Bioinformatics tools, dual-luciferase reporter assay, Western blot assay and qRT-PCR were used to explore the downstream pathways of circRNA_100290. The mechanism underlying the regulation of the expression of circRNA_100290 was explored using RNA immunoprecipitation, qRT-PCR, and Western blot assays.Results: The expression of circRNA_100290 was found significantly upregulated in GC cells and 102 GC tissues, high expression of circRNA_100290 in GC was closely related to Borrmann’s types, lymph node metastasis and tumor-node-metastasis staging. In vitro, knockdown of circRNA_100290 in AGS and HGC-27 cells significantly inhibited cell proliferation, migration, and invasion. Mechanistically, dual-luciferase reporter assay confirmed a direct binding between circRNA_100290 and miR-29b-3p, which targets ITGA11, an oncogene which is closely related to epithelial–mesenchymal transition (EMT). In addition, EIF4A3, one of RNA binding proteins (RBPs), could inhibit the formation of circRNA_100290 via enriching flanking sites of circRNA_100290. Low expression of EIF4A3 in GC was related to a worse prognosis.Conclusions: Elevated circRNA_100290 in GC promotes cell proliferation, invasion and EMT via miR-29b-3p/ITGA11 axi and might be regulated by EIF4A3. CircRNA_100290 might be a promising biomarker and target for GC therapy.

scholarly journals EIF4A3-Mediated CircRNA_100290 Promotes GC Cell Proliferation, Invasion and EMT via miR-29b-3p/ITGA11 Axis

2020 ◽  
Author(s):  
Gang Wang ◽  
Dan Sun ◽  
Wenhui Li ◽  
Yan Xin
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Qrt Pcr ◽  
Node Metastasis ◽  
Dual Luciferase Reporter Assay

Abstract Background Circular RNA (circRNA) has been reported as an important regulator in the development and progression of various carcinomas. However, the role of circRNA_100290 in gastric cancer (GC) is still unclear. This study aimed to investigate the role of circRNA_100290 in GC invasion and metastasis and its possible mechanism.Methods The expression of circRNA_100290 in GC cells and tissues were examined using quantitative real-time polymerase chain reaction (qRT-PCR). The role of circRNA_100290 in cell proliferation, migration, and invasion was evaluated on AGS and HGC-27 cell lines in vitro. Bioinformatics tools, dual-luciferase reporter assay, Western blot assay and qRT-PCR were used to explore the downstream pathways of circRNA_100290. The mechanism underlying the regulation of the expression of circRNA_100290 was explored using RNA immunoprecipitation, qRT-PCR, and Western blot assays.Results The expression of circRNA_100290 was found significantly upregulated in GC cells and 102 GC tissues, high expression of circRNA_100290 in GC was closely related to Borrmann’s types, lymph node metastasis and tumor-node-metastasis staging. In vitro, knockdown of circRNA_100290 in AGS and HGC-27 cells significantly inhibited cell proliferation, migration, and invasion. Mechanistically, dual-luciferase reporter assay confirmed a direct binding between circRNA_100290 and miR-29b-3p, which targets ITGA11, an oncogene which is closely related to epithelial–mesenchymal transition (EMT). In addition, EIF4A3, one of RNA binding proteins (RBPs), could inhibit the formation of circRNA_100290 via enriching flanking sites of circRNA_100290. Low expression of EIF4A3 in GC was related to a worse prognosis.Conclusions Elevated circRNA_100290 in GC promotes cell proliferation, invasion and EMT via miR-29b-3p/ITGA11 axi and might be regulated by EIF4A3. CircRNA_100290 might be a promising biomarker and target for GC therapy.

scholarly journals MiR-624-5p enhances cell resistance against cisplatin via PDGFRA/Stat3/PI3K axis in ovarian cancer

2020 ◽  
Vol 19 (4) ◽  
pp. 691-698
Author(s):  
Lin I-Ju ◽  
Tian YongJie
Keyword(s):  
Ovarian Cancer ◽  
Cell Viability ◽  
Cell Line ◽  
Cell Lines ◽  
Clinical Stage ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay

Purpose: The purpose of this study was to evaluate the role of miR-624-5p in ovarian cancer.Methods: MiR-624-5p expression in ovarian cancer {OC) cell lines and normal cells (NCs) was evaluated and compared the differential miR-624-5p in OC A2780 cells and cisplatin-resistant OC cell line (A2780/DDP). CCK-8 was used to evaluate changes in cell viability of the A2780 and A2780/DDP cell lines as well as silenced miR-624-5p. Western Blot examined the Stat3 and phosphorylated Pi3k. The binding between PDGFRA and miR-624-5p was predicted on Targetscan and verified through Luciferase Reporter Assay. The role of PDGFRA in A2780/DDP by overexpressing PDGFRA was evaluated by RT-qPCR and CCK-8 assays. RT-qPCR assay also measured miR-624-5p expression responsive to different dosages of cisplatin and CCK8 examined viability levels correspondingly. In addition, the interplay of PDGFRA and miR-624-5p by combined downregulation of both miR-624-5pand PDGFRA were evaluated.Results: OC cells had higher miR-624-5p expression than NCs but lower compared to cisplatinresistant A2780/DDP cells. A2780/DDP cells had higher viability than OC cell line A2780. Stat3 and phosphorylated PI3K were activated in A2780/DDP cells. Silencing miR-624-5p led to lower viability inA2780/DDP cells. miR-624-5p expression dropped as the cisplatin concentration increased, resulting in decreasing viability respectively. Luciferase Reporter assay validated the binding of miR-624-5p and PDGFRA in A2780/DDP cells. Overexpressed PDGFRA induced lower cell viability in A2780/DDP cells. Downregulation of PDGFRA partially restored the lowered viability and inhibited Stat3 as well as phosphorylated Pi3k induced by miR-624-5p inhibitor.Conclusion: MiR-624-5p could add to the cellular resistance to cisplatin in OC in-vitro model, which indicated that it might help unveil the mystery of drug-resistance in clinical stage of ovarian cancer. Keywords: MiR-624-5p, resistance, cisplatin, PDGFRA/Stat3/PI3K, ovarian cancer

miR-205 Suppresses Pulmonary Fibrosis by Targeting GATA3 Through Inhibition of Endoplasmic Reticulum Stress

2020 ◽  
Vol 21 (8) ◽  
pp. 720-726 ◽  
Author(s):  
Bingke Sun ◽  
Shumin Xu ◽  
Yanli Yan ◽  
Yusheng Li ◽  
Hongqiang Li ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Collagen I ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Sd Rats ◽  
Dual Luciferase Reporter Assay

Objective: To investigate the role of miR-205 and GATA3 in Pulmonary Fibrosis (PF). Methods: Bleomycin (BLM) was used to induce PF in SD rats and in vitro PF model was established by using TGFβ1-induced RLE-6TN cells. miR-205 mimics were used for the overexpression of miR- 205. The expression of miR-205, GATA3, α-SMA, Collagen I, CHOP and GRP78 were measured using RT-qPCR or western blotting. Dual-luciferase reporter assay was used to confirm binding between GATA3 3’-UTR and miR-205. Results: The expression of miR-205 was significantly down-regulated, while the expression of GATA3 was remarkably up-regulated in the model rats. GATA3 levels were remarkably decreased when miR-205 was overexpressed. When miR-205 was overexpressed, the lung injury by BLM-induced fibrosis was improved. The expression of α-SMA, Collagen I, as well as GRP78 and CHOP, was significantly up-regulated in both in vivo and in vitro PF models, and overexpression of miR-205 remarkably reversed the effects. Dual-luciferase reporter assay showed that miR-205 directly targeted and negatively regulated GATA3. Conclusion: miR-205 improved pulmonary fibrosis through inhibiting ER-stress by targeting GATA3.

scholarly journals Sensitization of Chrysosplenetin against Artemisinin Resistant Plasmodium berghei K173 via Blocking Host ABC Transporters Potentially Mediated by NF-κBB p52 or PXR/CAR Signaling Pathway

2022 ◽  
Author(s):  
Jing Chen ◽  
Xuesong Zhao ◽  
Shanhong Ni ◽  
Yuanyuan Zhang ◽  
Xiuli Wu ◽  
...  
Keyword(s):  
Western Blot ◽  
Abc Transporters ◽  
Plasmodium Berghei ◽  
In Vitro Transcription ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Protein Expressions ◽  
Dual Luciferase Reporter Assay

This study investigated if artemisinin-chrysosplenetin combination (ART-CHR) improved ART antimalarial efficacy against resistant Plasmodium berghei K173 via depressing host ABC transporter and potential molecular mechanism. Parasitaemia% and inhibition% were calculated and gene/protein expressions of ABC transporters or PXR/CAR/NF-κB p52 were detected by Western-blot and RT-qPCR. In vitro transcription of PXR/CAR was studied by dual-luciferase reporter assay. Our data indicated that ART-CHR improved ART efficacy against resistant parasites. P-gp inhibitor verapamil and CHR showed a stronger effect in killing resistant parasites while vehicle and Bcrp inhibitor novobiocin did not. ART activated intestinal ABCB1/ABCG2 and CHR inhibited them. ART decreased Bcrp protein whereas CHR increased it. ART ascended ABCC1/ABCC4/ABCC5 mRNA but ART-CHR descended them. CHR as well as rifampin (RIF) or 5-fluorouracil (5-FU) increased transcription levels of PXR/CAR while showed a versatile regulation on in vivo hepatic and enternal PXR/CAR in Mdr1a+/+ (WT) or Mdr1a-/- (KO) mice infected with sensitive or resistant parasites. Oppositely, hepatic and enteric N-7κB p52 mRNA was conformably decreased in WT but increased in KO-resistant mice. NF-κB pathway should potentially involved in the mechanism of CHR on inhibiting ABC transporters and ART resistance while PXR/CAR play a more complicated role in this mechanism.

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

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 ◽  
Dual Luciferase Reporter Assay

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

scholarly journals FENDRR Sponges miR-424-5p to Inhibit Cell Proliferation, Migration and Invasion in Colorectal Cancer

2020 ◽  
Vol 19 ◽  
pp. 153303382098010
Author(s):  
Chuan Cheng ◽  
Huixia Li ◽  
Jiujian Zheng ◽  
Jie Xu ◽  
Peng Gao ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Differential Analysis ◽  
Inhibit Cell Proliferation ◽  
Dual Luciferase Reporter Assay

Objective: LncRNAs are non-coding RNAs exerting vital roles in the occurrence and development of various cancer types. This study tended to describe the expression pattern of FENDRR in colorectal cancer (CRC), and further investigate the role of FENDRR in CRC cell biological behaviors. Methods: Gene expression profile of colon cancer was accessed from the TCGA database, and then processed for differential analysis for identification of differentially expressed lncRNAs and miRNAs. Some in vitro experiments like qRT-PCR, MTT, colony formation assay, wound healing assay and Transwell assay were performed to assess the effect of FENDRR on cell biological behaviors. Dual-luciferase reporter assay was conducted to further validate the targeting relationship between FENDRR and miR-424-5p, and rescue experiments were carried out for determining the mechanism of FENDRR/miR-424-5p underlying the proliferation, migration and invasion of CRC cells. Results: Bioinformatics analysis suggested that FENDRR was significantly down-regulated in CRC tissue, and low FENDRR was intimately correlated to poor prognosis. FENDRR overexpression could greatly inhibit cell proliferation, migration and invasion. Besides, there was a negative correlation between FENDRR and miR-424-5p. Dual-luciferase reporter assay indicated that miR-424-5p was a direct target of FENDRR. Rescue experiments discovered that FENDRR exerted its role in cell proliferation, migration and invasion in CRC via targeting miR-424-5p. Conclusion: FENDRR is poorly expressed in CRC tissue and cells, and low FENDRR is responsible for the inhibition of cell proliferation, migration and invasion of CRC by means of targeting miR-424-5p.

scholarly journals LncRNA LINC01303 Promotes the Progression of Oral Squamous Cell Carcinomas via the miR-429/ZEB1/EMT Axis

2021 ◽  
Vol 2021 ◽  
pp. 1-15
Author(s):  
Bo Sun ◽  
Xianyu Zheng ◽  
Weilong Ye ◽  
Pengcheng Zhao ◽  
Guowu Ma
Keyword(s):  
Squamous Cell ◽  
Luciferase Reporter ◽  
Real Time Quantitative Pcr ◽  
Qrt Pcr ◽  
Cytoplasmic Rna ◽  
Transwell Invasion Assay ◽  
Dual Luciferase Reporter Assay

Objectives. The aim of this research was to uncover the biological role and mechanisms of LINC01303 in oral squamous cell carcinoma (OSCC). Materials and Methods. Real-time quantitative PCR (qRT-PCR) was used to determine LINC01303 expression in OSCC tissues. Subcellular distribution of LINC01303 was examined by nuclear/cytoplasmic RNA fractionation and FISH experiments. The role of LINC01303 in the growth of TSCCA and SCC-25 was examined by CCK-8 assay, colony formation, transwell invasion assay in vitro, and xenograft tumor experiment in vivo. Dual-luciferase reporter assay was used to verify the interaction between LINC01303 and miR-429. RNA pull‐down assay was used to discover miR-429‐interacted protein, which was further examined by qRT-PCR, western blot, and rescue experiments. Results. LINC01303 expression was higher in OSCC tissues compared with adjacent nontumor tissues. LINC01303 was found to be localized in the cytoplasm of OSCC cells. Knockdown of LINC01303 inhibited OSCC cell proliferation and invasion, whereas increasing the expression of LINC01303 showed the opposite effects. Furthermore, LINC01303 served as a miR-429 “sponge” and positively regulated ZEB1 expression. Moreover, LINC01303 promoted OSCC through miR-429/ZEB1 axis both in vivo and in vitro. Conclusions. LINC01303 plays an oncogenic role in OSCC and is a promising biomarker for OSCC patients.

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