Protective Effect of miR-340-5p against Brain Injury after Intracerebral Hemorrhage by Targeting PDCD4

2020 ◽  
Vol 49 (6) ◽  
pp. 593-600
Author(s):  
Wei Zhou ◽  
Guandong Huang ◽  
Jueming Ye ◽  
Jiamei Jiang ◽  
Qing Xu
Keyword(s):  
Brain Injury ◽  
Intracerebral Hemorrhage ◽  
Water Content ◽  
Target Gene ◽  
Neurological Function ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Tnf Α ◽  
Gene Assay ◽  
The Brain

<b><i>Objective:</i></b> Intracerebral hemorrhage (ICH) is a common cerebrovascular disease. Increasing evidence has documented the crucial role of microRNAs in ICH. The present study aimed to investigate the role and underlying mechanism of miR-340-5p in ICH. <b><i>Methods:</i></b> The collagenase-induced ICH rat model was established. The neurological function of rats and the cerebral water content of rat brain tissue were measured to assess the brain injury. BV-2 cells were recruited and treated by LPS to mimic ICH-induced inflammatory response. qRT-PCR was used for the measurement of miR-340-5p. The protein levels of TNF-α, IL-6, and IL-1β were detected using ELISA. Luciferase reporter gene assay was performed to confirm the target gene. <b><i>Results:</i></b> Downregulation of miR-340-5p was detected in the serum of ICH patients and the brain tissues of ICH rats. Overexpression of miR-340-5p reversed the influence of ICH on the neurological function score and cerebral water content and inhibited the production of proinflammatory cytokines (TNF-α, IL-6, and IL-1β), which were induced by ICH in vivo. In in vitro study, levels of TNF-α, IL-6, and IL-1β were significantly enhanced in cells after LPS treatment, but these increases were eliminated by overexpression of miR-340-5p. PDCD4 was a direct target gene of miR-340-5p. <b><i>Conclusion:</i></b> miR-340-5p protects against brain injury after ICH. miR-340-5p might exert an anti-inflammatory effect during the occurrence of ICH via targeting PDCD4.

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 Oxymatrine inhibits proliferation and migration of breast cancer cells by inhibiting miRNA-188 and upregulating its target gene, PTEN

2021 ◽  
Vol 20 (11) ◽  
pp. 2267-2272
Author(s):  
Xiaoying Ma ◽  
Zijiang Sang ◽  
Qinghua Zhang ◽  
Wenbiao Ma
Keyword(s):  
Breast Cancer ◽  
Reporter Gene ◽  
Target Gene ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Pten Gene ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
And Migration ◽  
Regulatory Loop

Purpose: To explore the potential biological functions of oxymatrine on breast cancer (BCa) cells and the underlying molecular mechanism.Methods: Relative levels of microRNA-188 (miRNA-188) and PTEN (gene of phosphate and tension homology deleted on chromosome ten) in BCa cells, MDA-MB-231 and TB549, were determined. The influence of oxymatrine treatment, miRNA-188 and PTEN on proliferative and migratory abilities in BCa cells were assessed by 3-(4,5-imethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), cell counting kit-8 (CCK-8) and Transwell assay, respectively. The binding relationship between miRNA-188 and PTEN was evaluated by dual-luciferase reporter gene assay.Results: Oxymatrine downregulated miRNA-188 and upregulated PTEN in BCa cells. Proliferative and migratory activities in BCa were inhibited by treatment of oxymatrine (p < 0.05). Dual-luciferase reporter gene assay results indicated that PTEN was the target gene of miRNA-188. Furthermore, rescue experiments demonstrated that the regulatory loop, oxymatrine/miRNA-188/PTEN, was involved in the regulation of the migration and proliferation of BCa.Conclusion: Oxymatrine treatment inhibits BCa progression by downregulating miRNA-188, leading to the upregulation of PTEN. The results of the current study may provide new insight into the diagnosis and treatment of BCa.

MicroRNA-448 targets SATB1 to reverse the cisplatin resistance in lung cancer via mediating Wnt/β-catenin signalling pathway

2020 ◽  
Vol 168 (1) ◽  
pp. 41-51
Author(s):  
Mei-Ying Ning ◽  
Zhao-Lin Cheng ◽  
Jing Zhao
Keyword(s):  
Lung Cancer ◽  
Target Gene ◽  
A549 Cells ◽  
Resistance Index ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Lung Cancer Patients ◽  
Qrt Pcr ◽  
Gene Assay

Abstract This study aims to examine whether miR-448 reverses the cisplatin (DDP) resistance in lung cancer by modulating SATB1. QRT-PCR and immunohistochemistry were used to examine the miR-448 and SATB1 expressions in DDP-sensitive and -resistant lung cancer patients. A microarray was used to investigate the cytoplasmic/nucleic ratio (C/N ratios) of genes in A549 cells targeted by miR-448, followed by Dual-luciferase reporter gene assay. A549/DDP cells were transfected with miR-448 mimics/inhibitors with or without SATB1 siRNA followed by MTT assay, Edu staining, flow cytometry, qRT-PCR and western blotting. MiR-448 was lower but SATB1 was increased in DDP-resistant patients and A549/DDP cells. And the patients showed low miR-448 expression or SATB1 positive expression had poor prognosis. SATB1, as a target gene with higher C/N ratios (&gt;1), was found negatively regulated by miR-448. Besides, miR-448 inhibitors increased resistance index of A549/DDP cells, promoted cell proliferation, increased cell distribution in S phrase, declined cell apoptosis and activated Wnt/β-catenin pathway. However, SATB1 siRNA could reverse the above effect caused by miR-448 inhibitors. MiR-448 targeting SATB1 to counteract the DDP resistance of lung cancer cells via Wnt/β-catenin pathway.

Circular RNA CCDC66 Regulates Osteoarthritis Progression by Targeting miR-3622b-5p

Gerontology ◽  
2022 ◽  
pp. 1-11
Author(s):  
Chengyuan Zhang ◽  
Ye Lu ◽  
Feng Yuan ◽  
Shilin Jiang
Keyword(s):  
Reporter Gene ◽  
Bioinformatics Analysis ◽  
Reporter Gene Assay ◽  
Luciferase Reporter ◽  
Chondrocyte Apoptosis ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Tnf Α ◽  
Gene Assay ◽  
Sirt3 Expression

<b><i>Objective:</i></b> CircCCDC66 is involved in cancer progression, but its role in osteoarthritis (OA) remains unknown. This study was carried out to explore the biological role of circCCDC66 in OA and its underlying mechanism. <b><i>Methods:</i></b> The expression levels of miR-3622b-5p and circCCDC66 in OA cartilage tissues were detected by qRT-PCR. Cell Counting Kit-8 (CCK8) and flow cytometry were used to detect the chondrocyte viability and apoptosis. The expression of chondrocyte inflammatory factors (IL-6 and TNF-α) was measured by ELISA. The target genes of circCCDC66 and miR-3622b-5p were analyzed by bioinformatics analysis and luciferase reporter gene assay. The relationship between circCCDC66 and miR-3622b-5p was analyzed by bioinformatics analysis and luciferase reporter gene assay. <b><i>Results:</i></b> It was found that circCCDC66 expression in OA cartilage tissues was upregulated. CircCCDC66 overexpression inhibited proliferation and promoted apoptosis of chondrocytes and increased IL-6 and TNF-α levels in chondrocytes. miR-3622b-5p was predicted to be a downstream target gene of circCCDC66, and circCCDC66 overexpression inhibited miR-3622b-5p expression in chondrocytes. Moreover, miR-3622b-5p expression was downregulated in OA cartilage tissues. miR-3622b-5p overexpression increased chondrocyte proliferation, inhibited chondrocyte apoptosis, and enhanced the expression of IL-6 and TNF-α in chondrocytes. In addition, circCCDC66 overexpression enhanced SIRT3 expression in chondrocytes, while miR-3622b-5p overexpression inhibited SIRT3 expression in chondrocytes. <b><i>Conclusion:</i></b> CircCCDC66 promoted OA chondrocyte apoptosis by regulating the miR-3622b-5p/SIRT3 axis. CircCCDC66 may be a new therapeutic target of OA.

scholarly journals lncRNA H19 Aggravates Brain Injury in Rats following Experimental Intracerebral Hemorrhage via NF-κB Pathway

2022 ◽  
Vol 2022 ◽  
pp. 1-7
Author(s):  
Shuaidong Mao ◽  
Huan Huang ◽  
Xianzheng Chen
Keyword(s):  
Oxidative Stress ◽  
Brain Injury ◽  
Intracerebral Hemorrhage ◽  
Western Blot ◽  
Water Content ◽  
Brain Tissue ◽  
Sham Group ◽  
Neuron Specific Enolase ◽  
Neurological Function ◽  
And Oxidative Stress

Objective. To explore the effect of long noncoding RNA H19 (lncRNA H19) on brain injury in rats following experimental intracerebral hemorrhage (ICH). Methods. Rat ICH model was established with type IV collagenase. The neurological function scores were evaluated, and the water content in brain tissue was measured. The nerve injury indexes, inflammatory factors, and oxidative stress indexes were also measured. Moreover, the expression of lncRNA H19 was determined by qRT-PCR, and Western blot detected NF-κB pathway-related protein expression. Results. Compared with the sham group, the neurological function scores, the water content in brain tissue, and levels of injury indicators myelin basic protein (MBP), S-100B, and neuron-specific enolase (NSE) in the ICH rats were significantly increased. Meanwhile, the levels of TNF-α, IL-6, IL-1β, ROS, and MDA were significantly increased, but the levels of SOD were significantly decreased. In addition, the expression of lncRNA H19 in the brain tissue in the ICH group was significantly higher than that in the sham group. After further interference with lncRNA H19 expression (sh-H19 group), the levels of all the above indicators were reversed and the neurological damage was improved. Western blot results showed that the expression of NF-κBp65 and IKKβ was significantly higher, and IκBα expression was lower in the perivascular hematoma tissue in the ICH group compared with the sham group. Compared with the sh-NC group, NF-κBp65 and IKKβ expression were significantly lower and IκBα was significantly higher in the sh-H19 group. Conclusion. lncRNA H19 exacerbated brain injury in rats with ICH by promoting neurological impairment, brain edema, and releasing inflammatory responses and oxidative stress. This may be related to the activation of NF-κB signaling pathway.

scholarly journals Efficacy of Danhong injection on serum concentration of TNF-α, IL-6 and NF-κB in rats with intracerebral hemorrhage

2018 ◽  
Vol 13 (1) ◽  
pp. 77-81
Author(s):  
Chen Peng ◽  
Shibo Duan ◽  
Lou Gang
Keyword(s):  
Intracerebral Hemorrhage ◽  
Water Content ◽  
Control Group ◽  
Brain Water Content ◽  
Model Group ◽  
Danhong Injection ◽  
Tnf Α ◽  
The Brain ◽  
Brain Water ◽  
Time Point

AbstractObjectiveTo investigate the efficacy of Danhong injection on the serum concentration of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) in rats with intracerebral hemorrhage (ICH) and evaluate its therapeutic effects on inflammation and cerebral edema.MethodsSixty male Wistar rats were randomly divided into control, model and Danhong groups with 25 rats in each group. Intracerebral injection of autologous arterial blood was performed on model and Danhong groups in order to establish intracerebral hemorrhage model. Rats in the control group were given the same operation procedure without blood injection. After successfully establishing the intracerebral hemorrhage model, the rats were given Danhong (2ml/kg/d) through intraperitoneal injection. Rats in the control and model groups were given the same amount of normal saline respectively. The brain water content (BWC) and serum level of TNF-α, IL-6 and NF-κB were measured in all groups at the time points of day 1, 3, 5, 7 and 9.ResultsThe neurological deficit score (NDS) were not statistical different in days 1, 3 and 5 between the model and Danhong group (P>0.05); However, on day 7 and 9 after modeling, the NDS in the Danhong group was significant lower than that of the Model group (P<0.05). The brain water content in the model and Danhong groups were significantly elevated compared to control group (P<0.05). The brain water content was significant elevated after modeling in the model and Danhong groups on day 3 and gradually decreased over the next 6 days.The brain water content was significantly higher in the model group for days 3 to 9 compared to the Danhong group (P<0.05). Compared to the model group, the serum NF-κb was significantly lower in the Danhong group for the time point of day 3 and 5 (P<0.05); However, compared to the model group, the serum TNF-α and IL-6 levels in the Danhong group were significantly lower for each time point (P<0.05). Conclusion Danhong injection can reduce cerebral edema in rats with cerebral hemorrhage, and protect the brain nerve function. These effects may be related to its function of regulating serum TNF-α, NF-κB and IL-6 expression.

scholarly journals Linkage of lncRNA CRNDE sponging miR-181a-5p with aggravated inflammation underlying sepsis

2019 ◽  
Vol 26 (2) ◽  
pp. 152-161 ◽  
Author(s):  
Yijun Wang ◽  
Ziqiang Xu ◽  
Dongyou Yue ◽  
Zhenhua Zeng ◽  
Weijie Yuan ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Inflammatory Cytokines ◽  
Peripheral Blood ◽  
Reporter Gene Assay ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Sepsis Diagnosis ◽  
Tnf Α ◽  
Gene Assay

This investigation was performed to verify whether lncRNA CRNDE sponging miR-181a-5p was involved with sepsis-relevant inflammatory dysfunctions. Aggregately 136 sepsis patients and 151 healthy people were recruited, and their fasting peripheral blood was gathered to detect expressions of CRNDE and miR-181a-5p. In addition, THP-1 cells were transfected with si-CRNDE, miR-181a-5p mimic, pcDNA3.1-TLR4 and si-TLR4, and then sepsis-specific inflammatory cytokines within the cells were quantified. The sponging relationships between CRNDE and miR-181a-5p, as well as between miR-181a-5p and TLR4, were ascertained by means of luciferase reporter gene assay. The experimental results revealed that over-expressed CRNDE and under-expressed miR-181a-5p were associated with shortened lifespan of sepsis patients. Mechanically, si-CRNDE-1 and miR-181a-5p mimic were able to reverse the promoting effects of LPS on production of NF-kB, TNF-α, IL-1β and IL-6 by THP-1 cells. Moreover, the expressional change of miR-181a-5p in THP-1 cells was in part owing to its being sponged by CRNDE. Lastly, TLR4, subjected to targeted modification of miR-181a-5p, was capable of disturbing the contribution of CRNDE and miR-181a-5p to THP-1 cells’ release of NF-kB, TNF-α, IL-1β and IL-6. Collectively, the CRNDE/miR-181a-5p/TLR4 axis seemed to have potential in modifying sepsis-related inflammatory pathogenesis, which offered a direction for sepsis diagnosis and treatment.

scholarly journals CyclinD1 is a new target gene of tumor suppressor miR-520e in breast cancer

Open Medicine ◽  
2019 ◽  
Vol 14 (1) ◽  
pp. 913-919
Author(s):  
Quan Liang ◽  
Qingjuan Yao ◽  
GuoYing Hu
Keyword(s):  
Breast Cancer ◽  
Target Gene ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Gene Assay ◽  
Clinical Breast ◽  
Polymerase Chain ◽  
Cancer Tissues ◽  
Luciferase Reporters

AbstractObjectiveTo investigate the involvement of miR-520e in the modulation of cancer-promoting cyclinD1 in breast cancer.MethodsA reverse transcription-polymerase chain reaction (RT-PCR) was applied to test the regulation of miR-520e on cyclinD1. The binding of miR-520e to 3’-untranslated region (3’UTR) of cyclinD1 mRNA was predicted by an online bioinformatics website. The effect of miR-520e on the luciferase reporters with binding sites of miR-520e and 3’UTR of cyclinD1 mRNA was revealed using a luciferase reporter gene assay. The correlation between miR-520e and cyclinD1 in clinical breast cancer samples was detected through quantitative real-time PCR.ResultsThe expression of cyclinD1 was gradually reduced as the dose of miR-520e increased. Anti-miR-520e obviously induced cyclinD1 in breast cancer cells. After anti-miR-520e was introduced into the cells, the inhibition of cyclinD1 expression mediated by miR-520e was reversed. The binding of miR-520e with cyclinD1 was revealed via bioinformatics. Under the treatment of dose-increasing miR-520e or anti-miR-520e, the luciferase activities of cyclinD1 3’UTR vector were lower or higher by degrees. However, the activity of the mutant vector was not affected at all. Finally, in clinical breast cancer tissues the negative correlation of miR-520e with cyclinD1 was revealed.ConclusionIn conclusion, cyclinD1 is a new target of miR-520e in breast cancer.

circ-NOTCH1 acts as a sponge of miR-637 and affects the expression of its target gene Apelin to regulate gastric cancer cell growth

2020 ◽  
Vol 98 (2) ◽  
pp. 164-170 ◽  
Author(s):  
Encui Guan ◽  
Xiaoguang Xu ◽  
Fangxi Xue
Keyword(s):  
Gastric Cancer ◽  
Cell Proliferation ◽  
Target Gene ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
High Recurrence Rate ◽  
Cure Rate ◽  
Luciferase Reporter Gene ◽  
Cancer Cell Growth ◽  
Gene Assay

Gastric cancer (GC) is a major cause of cancer-related deaths worldwide, and has a low survival rate, low cure rate, high recurrence rate, and poor prognosis. Recent studies have indicated that circular RNAs (circRNAs) have important functions in the occurrence and progression of GC. Studies on circ-NOTCH1, which was shown to be highly expressed in GC, have indicated that miR-637 binds to circ-NOTCH1 at multiple sites, and a dual-luciferase reporter gene assay further confirmed that miR-637 indeed targeted circ-NOTCH1 and Apelin. Circ-NOTCH1 and Apelin are highly expressed in GC cells and tissues, whereas the expression of miR-637 is reduced. Circ-NOTCH1 and miR-637 do not regulate each other’s expression levels, but circ-NOTCH1significantly upregulates the expression of the miR-637 target gene Apelin, whereas miR-637 inhibites the expression of Apelin. Examination of GC cells showed that circ-NOTCH1 enhances cell proliferation and invasiveness, and reduces cell apoptosis; these effects were reversed by miR-637, which could terminate the above effects of circ-NOTCH1. When co-transfected with the circ-NOTCH1 overexpression plasmid and Apelin siRNAs, there were no obvious changes to the levels of cell proliferation, apoptosis, or invasiveness. Therefore, in GC cells, circ-NOTCH1 inhibits the transcriptional activity of miR-637, thereby upregulating the expression of its target gene Apelin and regulating cell proliferation, apoptosis, and invasiveness. This finding provides more experimental evidence for the function of circRNA in GC.

scholarly journals Role of MiR-215 in Hirschsprung's Disease Pathogenesis by Targeting SIGLEC-8

2016 ◽  
Vol 40 (6) ◽  
pp. 1646-1655 ◽  
Author(s):  
Hao Lei ◽  
Hongxing Li ◽  
Hua Xie ◽  
Chunxia Du ◽  
Yankai Xia ◽  
...  
Keyword(s):  
Cell Migration ◽  
Target Gene ◽  
Reporter Gene Assay ◽  
Host Gene ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Gene Assay ◽  
Cell Migration And Proliferation

Background/Aims: Hirschsprung's disease (HSCR), known as aganglionosis, is an infrequent congenital gut motility disorder characterized by absence of enteric neurons. In this study, we focus on the role of the intronic miR-215 and its host gene isoleucyl-tRNA synthetase 2 (IARS2) in the pathogenesis of HSCR. Methods: Quantitative real time PCR and Western blot were used to detect the miRNA, mRNAs, and proteins levels. The dual-luciferase reporter gene assay confirmed the direct regulation of the specific mRNA and miRNAs in cell lines. Transwell assays, CCK8 assay, and flow cytometry were used to measure cell function of the human 293T and SH-SY5Y cells. Results: Expression levels of miR-215 in HSCR patient colon tissues were outstandingly lower than controls, which was positively correlated with expression of the host gene IARS2 and negatively correlated with predicted target gene: sialic acid binding Ig-like lectin 8 (SIGLEC-8). The loss of miR-215 inhibited cell migration and proliferation, which was consistent with the reduction of IARS2. The dual-luciferase reporter gene assay confirmed that miR-215 resulted in the inhibition of SIGLEC-8 by directly binding to the 3'-UTR of SIGLEC-8. Moreover, knocking-down SIGLEC-8 rescued the extent of suppressed cell migration and proliferation that resulted from the diminishment of miR-215. Conclusions: Our findings indicate that miR-215 acts in concert with the host gene IARS2 to affect neuron migration and proliferation through the target gene SIGLEC-8. We infer that the IARS2-miR-215-SIGLEC-8 pathway may play a crucial role in the pathogenesis of HSCR.

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