scholarly journals PiRNA MW557525 Promotes the Vitality and Pluripotency of Piwil2-iCSCs by Regulating NOP56.

2022 ◽  
Author(s):  
Liming Jin ◽  
Zhaoxia Zhang ◽  
Zhang Wang ◽  
Xiaojun Tan ◽  
Zhaoying Wang ◽  
...  
Keyword(s):  
Target Gene ◽  
Opposite Effect ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Tumor Development ◽  
Luciferase Reporter ◽  
Transwell Assay ◽  
Kegg Analysis ◽  
Detailed Function ◽  
Dual Luciferase Reporter Assay

Abstract Background: CSCs play an important role in tumor development. Some studies have demonstrated that piRNAs participate in the progression of various cancers. However, the detailed function of piRNAs in CSCs requires further investigation. This study aimed to investigate the significance of some piRNAs in Piwil2-iCSCs. Methods and Results: Differentially expressed piRNAs in Piwil2-iCSCs were screened by high-throughput sequencing. Target genes were predicted by the miRanda algorithm and subjected to GO and KEGG analysis. One of the differential piRNAs, novel piRNA MW557525, was transfected and its target gene NOP56 was silenced in Piwil2-iCSCs, respectively. RT-qPCR, western blot and dual luciferase reporter assay were used to investigate the interaction of piRNA MW557525 and NOP56. We identified the effect of piRNA MW557525 and NOP56 knockdown on cell proliferation, migration, invasion, and apoptosis via CCK-8, transwell assay, and flow cytometry. The expressions of CD24, CD133, KLF4, and SOX2 were detected via WB. The results showed that piRNA MW557525 was negatively correlated with NOP56, and it promoted the proliferation, migration, invasion, and inhibited apoptosis in Piwil2-iCSCs, and it also promoted the expressions of CD24, CD133, KLF4, and SOX2, while NOP56 showed the opposite effect. Conclusions: These findings suggested that novel piRNA MW557525 might be a novel therapeutic target in Piwil2-iCSCs.

scholarly journals PiRNA MW557525 Promotes the Vitality and Pluripotency of Piwil2-iCSCs by Regulating NOP56.

2021 ◽  
Author(s):  
Zhaoxia Zhang ◽  
Zhang Wang ◽  
Xiaojun Tan ◽  
Liming Jin ◽  
Zhaoying Wang ◽  
...  
Keyword(s):  
Target Gene ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Tumor Development ◽  
High Mobility ◽  
P Element ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Go Analysis ◽  
Protein Levels

Abstract Objective Cancer stem cells (CSCs) play an important role in tumor development. Some studies have demonstrated that P-element–induced wimpy testis (Piwi)–interacting ribonucleic acids (piRNAs) participate in the progression of various cancers. However, the detailed function of piRNAs in CSCs requires further investigation. The aim of the present study was to investigate the effect of the uknown upregulated piRNA MW557525 and its predicted target gene nucleolar protein 56 (NOP56) inPiwi-like protein 2 (Piwil2)–induced CSCs (Piwil2-iCSCs).Methods We screened differential piRNAs of Piwil2-iCSCs using high-throughput sequencing (HTS). Target genes were predicted by the miRanda algorithm and subjected to Gene Ontology (GO) analysis. One of the differential piRNAs, MW557525, and its target gene NOP56 were transfected and silenced in Piwil2-iCSCs, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to detect expression levels of piRNA MW557525 and NOP56 in Piwil2-iCSCs after transfection. We measured protein levels of NOP56 in different groups via Western blot (WB), verified interactions using a dual luciferase reporter assay (LRA) and investigated the effect of piRNA MW557525 and NOP56 on Piwil2-iCSC proliferation using a Cell Counting Kit-8 (CCK-8). In addition, we evaluated cell migratory and invasive abilities via transwell assay and detected cell apoptotic ability via flow cytometry (FCM) assay. Protein levels of Cluster of Differentiation 24 (CD24), CD133, Krüppel-like factor 4 (KLF4) and sex-determining region Y–related high-mobility group (HMG) box 12 (SOX2) were measured to evaluate the change in Piwil2-iCSC pluripotency after transfection.Results Via HTS, we screened out 204 differential piRNAs, and miRanda predicted 77 target genes. GO analysis showed that the biological processes (BPs) of these target genes were mainly involved in regulating the calcium concentration of cells and their molecular functions (MFs) were mainly involved in ATPase activity.The expression of piRNA MW557525 and NOP56 were significantly upregulated,and piRNA MW557525 was negatively associated with NOP56 in Piwil2-iCSCs. PiRNA MW557525 promoted proliferation, migration, invasion and pluripotency and inhibited apoptosis, while NOP56 suppressed proliferation, migration, invasion and pluripotency and induced apoptosis, in Piwil2-iCSCs.Conclusion Taken together, these findings suggested that piRNA MW557525 promoted and maintained the vitality and pluripotency of Piwil2-iCSCs, while NOP56 inhibited these characteristics. Therefore, piRNA MW557525 might be a novel therapeutic target in Piwil2-iCSCs.

scholarly journals miR-17~92 Promotes Progression of ABC-DLBCL Lymphoma via Regulation of Canonical NF-kB Signaling

2021 ◽  
Author(s):  
Xianhuo Wang ◽  
Huaqing Wang ◽  
Xiaoyan Zhang ◽  
Chengfeng Bi ◽  
Timothy W. McKeithan ◽  
...  
Keyword(s):  
B Cell ◽  
Therapeutic Strategy ◽  
Target Gene ◽  
Target Genes ◽  
B Cell Lymphoma ◽  
Aggressive Lymphoma ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Over Expression ◽  
Dual Luciferase Reporter Assay

Abstract Background: Activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive lymphoma characterized by constitutive NF-κB activation. Nevertheless, the role and mechanisms of miR-17~92 in contributing to the NF-κB activation in ABC-DLBCL are still elusive. Methods: The expression of miR-17~92 primary transcript (MIR17HG) and NF-κB target genes was determined using RNA-sequencing. The expression of miR-17~92 was performed using microarray analysis. Plasmids carrying conditional over-expression and loss-of-function of miR-17~92 were respectively constructed and dual-luciferase reporter assay was used to validate the target gene of miR-17~92. Immunoprecipitation and polyubiquitination were further used to the study of potential mechanisms.Results: Expression of MIR17HG was positively correlated with NF-κB activity, miR-17~92 activated the NF-κB signaling in ABC-DLBCL, and its over-expression promoted ABC-DLBCL cell growth, accelerated cell G1 to S phase transition and enhanced cell resistance to NF-κB inhibitor. Importantly, miR-17~92 promoted NF-κB activation through directly targeting multiple ubiquitin-editing regulators to lead to increase the K63-linked polyubiquitination and decrease the K48-linked polyubiquitination of RIP1 complex in ABC-DLBCL. We further found that miR-17~92 selectively activated IκB-α and NF-κB p65 but not NF-κB p52/p100, and high miR-17~92 expression was also associated with poorer outcome in ABC-DLBCL patients. Conclusions: Taken together, miR-17~92 selectively activate the canonical NF-κB signaling via targeting ubiquitin-editing regulators to lead to constitutively NF-κB activation and poorer outcome, which is an innovative function of miR-17~92 and previously unappreciated regulatory mechanism of NF-κB activation in ABC-DLBCL. Targeting miR-17~92 may thus provide a novel bio-therapeutic strategy for ABC-DLBCL patients.

scholarly journals miR-199a Targeting PNRC1 to Promote Keratinocyte Proliferation and Invasion in Cholesteatoma

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Lihui Yao ◽  
Wenjing Zhang ◽  
Jian Zheng ◽  
Xing Lu ◽  
Fan Zhang
Keyword(s):  
Target Gene ◽  
Target Genes ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Keratinocyte Proliferation ◽  
Qrt Pcr ◽  
Candidate Target ◽  
Candidate Target Gene ◽  
Proliferation And Invasion ◽  
Dual Luciferase Reporter Assay

Introduction. miR-199a has been reported as an oncogene of various cancers. However, the biological function and regulatory mechanism of miR-199a in keratinocytes of cholesteatoma are still unclear. Methods. Detection by qRT-PCR was conducted on miR-199a’s expression in both thirty pairs of cholesteatoma tissues and normal skins. For characterizing the function of miR-199a, this research adopted transwell assay, wound healing assay, and CCK8 assays. Under the support of qRT-PCR, efforts were made to investigate the relative expression of candidate target genes. Moreover, the evaluation of the targeting relationship between miR-199a and the candidate target gene was conducted with the dual-luciferase reporter assay. Results. The upregulation of miR-199a was found in cholesteatoma tissues, which facilitated the proliferation, migration, and invasion of HaCaT cells, while its downregulation caused opposite results. Conclusions. The findings of the present research offer more insights into the molecular mechanism of cholesteatoma progression.

scholarly journals Hsa-miR-494-3p attenuates gene HtrA3 transcription to increase inflammatory response in hypoxia/reoxygenation HK2 Cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Qian Gong ◽  
Zhi-ming Shen ◽  
Zhe Sheng ◽  
Shi Jiang ◽  
Sheng-lin Ge
Keyword(s):  
Cardiac Surgery ◽  
Inflammatory Response ◽  
Target Gene ◽  
Kidney Injury ◽  
Human Kidney ◽  
Luciferase Reporter ◽  
The Relationship ◽  
Hk2 Cells ◽  
Dual Luciferase Reporter Assay ◽  
Hypoxia Reoxygenation

AbstractThe occurrence of cardiac surgery-associated acute kidney injury (CSA-AKI) increases hospital stay and mortality. MicroRNAs has a crucial role in AKI. This objective of the current study is to explore the function of hsa-miR-494-3p in inflammatory response in human kidney tubular epithelial (HK2) cells with hypoxia/reoxygenation. According to KDIGO standard, patients after cardiac surgery with cardiopulmonary bypass were divided into two groups: AKI (n = 10) and non-AKI patients (n = 8). HK2 were raised in the normal and hypoxia/reoxygenation circumstances and mainly treated by overexpression ofmiR-494-3p and HtrA3. The relationship between miR-494-3p and HtrA3 was determined by dual-luciferase reporter assay. Our result showed that Hsa-miR-494-3p was elevated in the serum of patients with CSA-AKI, and also induced in hypoxic reoxygenated HK2 cells. Hsa-miR-494-3p also increased a hypoxia-reoxygenation induced inflammatory response in HK2 cells. Moreover, as a target gene of miR-494-3p, overexpression of HtrA3 downregulated the hypoxia-reoxygenation induced inflammatory response in HK2 cells. Overexpression of hsa-miR-494-3p-induced inflammatory response was inhibited by overexpression of HtrA3. Collectively, we identified that hsa-miR-494-3p, a miRNA induced in both circulation of AKI patients and hypoxia-reoxygenation-treated HK2 cells, enhanced renal inflammation by targeting HtrA3, which may suggest a possible role as a new therapeutic target for CSA-AKI.

scholarly journals MiR-196b Promotes the Invasion and Migration of Lung Adenocarcinoma Cells by Targeting AQP4

2021 ◽  
Vol 20 ◽  
pp. 153303382098586
Author(s):  
Xuhui Wu ◽  
Gongzhi Wu ◽  
Huaizhong Zhang ◽  
Xuyang Peng ◽  
Bin Huang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Cell Invasion ◽  
Target Gene ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Invasion And Migration ◽  
And Migration ◽  
Dual Luciferase Reporter Assay

Objective: We aimed to investigate the mechanism of the regulatory axis of miR-196b/AQP4 underlying the invasion and migration of lung adenocarcinoma (LUAD) cells. Methods: LUAD miRNA and mRNA expression profiles were downloaded from TCGA database and then differential analysis was used to identify the target miRNA. Target gene for the miRNA was obtained via prediction using 3 bioinformatics databases and intersection with the differentially expressed mRNAs searched from TCGA-LUAD. Then, qRT-PCR and western blot were used to validate the expression of miR-196b and AQP4. Dual-luciferase reporter assay was performed to confirm the targeting relationship between miR-196b and AQP4. Transwell assay was used to investigate the migration and invasion of LUAD cells. Results: MiR-196b was screened out by differential and survival analyses, and the downstream target gene AQP4 was identified. In LUAD, miR-196b was highly expressed while AQP4 was poorly expressed. Besides, overexpression of miR-196b promoted cell invasion and migration, while overexpression of AQP4 had negative effects. Moreover, the results of the dual-luciferase reporter assay suggested that AQP4 was a direct target of miR-196b. In addition, we also found that overexpressing AQP4 could suppress the promotive effect of miR-196b on cancer cell invasion and migration. Conclusion: MiR-196b promotes the invasion and migration of LUAD cells by down-regulating AQP4, which helps us find new molecular targeted therapies for LUAD.

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.

Screening candidate microR-15a- IRAK2 regulatory pairs for predicting the response to Staphylococcus aureus-induced mastitis in dairy cows

2019 ◽  
Vol 86 (4) ◽  
pp. 425-431 ◽  
Author(s):  
Zhi Chen ◽  
Jingpeng Zhou ◽  
Xiaolong Wang ◽  
Yang Zhang ◽  
Xubin Lu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Differential Expression ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Interleukin 1 ◽  
Differentially Expressed ◽  
Luciferase Reporter ◽  
Expression Of Genes ◽  
Chinese Holstein Cows ◽  
Reporter Assays

AbstractWe established a mastitis model using exogenous infection of the mammary gland of Chinese Holstein cows with Staphylococcus aureus and extracted total RNA from S. aureus-infected and healthy mammary quarters. Differential expression of genes due to mastitis was evaluated using Affymetrix technology and results revealed a total of 1230 differentially expressed mRNAs. A subset of affected genes was verified via Q-PCR and pathway analysis. In addition, Solexa high-throughput sequencing technology was used to analyze profiles of miRNA in infected and healthy quarters. These analyses revealed a total of 52 differentially expressed miRNAs. A subset of those results was verified via Q-PCR. Bioinformatics techniques were used to predict and analyze the correlations among differentially expressed miRNA and mRNA. Results revealed a total of 329 pairs of negatively associated miRNA/mRNA, with 31 upregulated pairs of mRNA and 298 downregulated pairs of mRNA. Differential expression of miR-15a and interleukin-1 receptor-associated kinase-like 2 (IRAK2), were evaluated by western blot and luciferase reporter assays. We conclude that miR-15a and miR-15a target genes (IRAK2) constitute potential miRNA–mRNA regulatory pairs for use as biomarkers to predict a mastitis response.

scholarly journals Downregulated KIF3B Induced by miR-605-3p Inhibits the Progression of Colon Cancer via Inactivating Wnt/β-Catenin

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Qilong Wang ◽  
Xiaomin Hao ◽  
Gang Xu ◽  
Tiesheng Lv
Keyword(s):  
Colon Cancer ◽  
Tumor Cells ◽  
Malignant Disease ◽  
Luciferase Reporter ◽  
High Morbidity ◽  
Transwell Assay ◽  
Flow Cytometry Assay ◽  
Colon Cells ◽  
Proliferation And Invasion ◽  
Dual Luciferase Reporter Assay

Colon cancer is a common malignant disease with high morbidity and mortality, and miRNA dysfunction has been confirmed as an important reason for cancer development. Several studies have verified miR-605-3p as a tumor inhibitor while its roles in colon cancer remain uncertain. In this study, the specimen of the patients and the cell lines of colon cancer were used to observe the expression of miR-605-3p, and the CCK-8, Transwell assay, and flow cytometry assay were used to observe the functions of miR-605-3p in colon cancer cells. The downstream factors of miR-605-3p were predicted by TargetScan and then were verified by dual-luciferase reporter assay. Moreover, western blot was used to investigate the effect of miR-605-3p on Wnt/β-catenin signal pathway. The result showed that miR-605-3p was extremely downregulated in the pathological tissues and tumor cells, and miR-605-3p could effectively induce the apoptosis and impede the proliferation and invasion of the tumor cells. It was found that KIF3B was a target of KIF3B; decreased KIF3B could reverse the effects of miR-605-3p on colon cancer. Besides, the inactivated Wnt/β-catenin pathway was also observed in colon cells when miR-605-3p was upregulated, and the phenomenon could be rescued by KIF3B upregulation. In conclusion, miR-605-3p could inactivate the Wnt/β-catenin pathway induced via promoting KIF3B expression.

scholarly journals Circular RNA CircITGA7 Promotes Tumorigenesis of Osteosarcoma via miR-370/PIM1 Axis

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Chuanwu Fang ◽  
Xiaohong Wang ◽  
Dongliang Guo ◽  
Run Fang ◽  
Ting Zhu
Keyword(s):  
Circular Rna ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Functional Assay ◽  
Transwell Assay ◽  
Qrt Pcr ◽  
Proliferation And Metastasis ◽  
And Migration ◽  
Dual Luciferase Reporter Assay ◽  
Proliferation And Migration

Many studies have shown that there are many circular RNA (circRNA) expression abnormalities in osteosarcoma (OS), and this abnormality is related to the development of osteosarcoma. But at present, it is unclear as to what circITGA7 has in the OS and what it does. In this study, qRT-PCR was used to detect the expression of circITGA7, miR-370, and PIM1 mRNA in OS tissues and cells. The CCK-8 assay was used to detect the effect of circITGA7 on cell proliferation. Later, the transwell assay was used to detect cell migration and invasion. The dual-luciferase reporter assay confirmed the existence of the targeting relationship between circITGA7 and miR-370, and miR-370 and PIM1. We found that circITGA7 was upregulated in OS tissues and cell lines. Knockdown of circITGA7 weakened the cell’s ability to proliferate and metastasize. Furthermore, we observed that miR-370 was negatively regulated by circITGA7, while PIM1 was positively regulated by it. A functional assay validated that circITGA7 promoted OS progression via suppressing miR-370 and miR-370 affected OS proliferation and migration via PIM6 in OS. In summary, this study shows that circITGA7 promotes OS proliferation and metastasis via miR-370/PIM1.

scholarly journals The Expression of microRNA in Adult Rat Heart with Isoproterenol-Induced Cardiac Hypertrophy

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1173 ◽  
Author(s):  
Mailin Gan ◽  
Shunhua Zhang ◽  
Yuan Fan ◽  
Ya Tan ◽  
Zhixian Guo ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Signaling Pathway ◽  
Natriuretic Peptide ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Pathological Condition ◽  
Wnt Signaling Pathway ◽  
Differentially Expressed ◽  
Mitogen Activated Protein Kinase ◽  
Luciferase Reporter

Cardiac hypertrophy is a common pathological condition and an independent risk factor that triggers cardiovascular morbidity. As an important epigenetic regulator, miRNA is widely involved in many biological processes. In this study, miRNAs expressed in rat hearts that underwent isoprenaline-induced cardiac hypertrophy were identified using high-throughput sequencing, and functional verification of typical miRNAs was performed using rat primary cardiomyocytes. A total of 623 miRNAs were identified, of which 33 were specifically expressed in cardiac hypertrophy rats. The enriched pathways of target genes of differentially expressed miRNAs included the FoxO signaling pathway, dopaminergic synapse, Wnt signaling pathway, MAPK (mitogen-activated protein kinase) signaling pathway, and Hippo signaling pathway. Subsequently, miR-144 was the most differentially expressed miRNA and was subsequently selected for in vitro validation. Inhibition of miR-144 expression in primary myocardial cells caused up-regulation of cardiac hypertrophy markers atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). The dual luciferase reporter system showed that ANP may be a target gene of miR-144. Long non-coding RNA myocardial infarction associated transcript (LncMIAT) is closely related to heart disease, and here, we were the first to discover that LncMIAT may act as an miR-144 sponge in isoproterenol-induced cardiac hypertrophy. Taken together, these results enriched the understanding of miRNA in regulating cardiac hypertrophy and provided a reference for preventing and treating cardiac hypertrophy.

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