Chromatin RNA Immunoprecipitation (ChRIP)

Abstract Background Aberrant expressions of long noncoding RNAs (lncRNAs) have been demonstrated to be related to the progress of HCC. The mechanisms that SNHG14 has participated in the development of HCC are obscure. Methods Quantitative real-time PCR (qRT-PCR) was used to measure the lncRNA, microRNA and mRNA expression level. Cell migration, invasion and proliferation ability were evaluated by transwell and CCK8 assays. The ceRNA regulatory mechanism of SNHG14 was evaluated by RNA immunoprecipitation (RIP) and dual luciferase reporter assay. Tumorigenesis mouse model was used to explore the roles of miR-876-5p in vivo. The protein levels of SSR2 were measured by western blot assay. Results In this study, we demonstrated that SNHG14 was highly expressed in HCC tissues, meanwhile, the elevated expression of SNHG14 predicted poor prognosis in patients with HCC. SNHG14 promoted proliferation and metastasis of HCC cells. We further revealed that SNHG14 functioned as a competing endogenous RNA (ceRNA) for miR-876-5p and that SSR2 was a downstream target of miR-876-5p in HCC. Transwell, CCK8 and animal experiments exhibited miR-876-5p inhibited HCC progression in vitro and in vivo. By conducting rescue experiments, we found the overexpression of SSR2 or knocking down the level of miR-876-5p could reverse the suppressive roles of SNHG14 depletion in HCC. Conclusion SNHG14 promotes HCC progress by acting as a sponge of miR-876-5p to regulate the expression of SSR2 in HCC.

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Long Non-Coding RNA ARAP1-AS1 Facilitates the Progression of Cervical Cancer by Regulating miR-149-3p and POU2F2

Pathobiology ◽

10.1159/000507830 ◽

2021 ◽

pp. 1-12

Author(s):

Ling Zhou ◽

Xiao-li Xu

Keyword(s):

Cervical Cancer ◽

Tumor Model ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Non Coding Rna ◽

Injection Model ◽

Rna Immunoprecipitation ◽

Long Non Coding Rna

Background: Emerging research has demonstrated that long non-coding RNAs (lncRNAs) attach great importance to the progression of cervical cancer (CC). LncRNA ARAP1-AS1 was involved in the development of several cancers; however, its role in CC is far from being elucidated. Methods: Real-time PCR (RT-PCR) was employed to detect ARAP1-AS1 and miR-149-3p expression in CC samples. CC cell lines (HeLa and C33A cells) were regarded as the cell models. The biological effect of ARAP1-AS1 on cancer cells was measured using CCK-8 assay, colony formation assay, flow cytometry, Transwell assay and wound healing assay in vitro, and subcutaneous xenotransplanted tumor model and tail vein injection model in vivo. Furthermore, interactions between ARAP1-AS1 and miR-149-3p, miR-149-3p and POU class 2 homeobox 2 (POU2F2) were determined by bioinformatics analysis, qRT-PCR, Western blot, luciferase reporter and RNA immunoprecipitation assay, respectively. Results: The expression of ARAP1-AS1 was enhanced in CC samples, while miR-149-3p was markedly suppressed. Additionally, ARAP1-AS1 overexpression enhanced the viability, migration, and invasion of CC cells. ARAP1-AS1 downregulated miR-149-3p via sponging it. ARAP1-AS1 and miR-149-3p exhibited a negative correlation in CC samples. On the other hand, ARAP1-AS1 enhanced the expression of POU2F2, which was validated as a target gene of miR-149-3p. Conclusion: ARAP1-AS1 was abnormally upregulated in CC tissues and indirectly modulated the POU2F2 expression via reducing miR-149-3p expression. Our study identified a novel axis, ARAP1-AS1/miR-149-3p/POU2F2, in CC tumorigenesis.

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Warburg effect-promoted exosomal circ_0072083 releasing up-regulates NANGO expression through multiple pathways and enhances temozolomide resistance in glioma

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01942-6 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Chenyu Ding ◽

Xuehan Yi ◽

Xiangrong Chen ◽

Zanyi Wu ◽

Honghai You ◽

...

Keyword(s):

Warburg Effect ◽

Lactate Production ◽

Xenograft Model ◽

Cell Counting ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Circular Rnas ◽

Rna Immunoprecipitation ◽

Resistant Cells

Abstract Background Temozolomide (TMZ) resistance limits its application in glioma. Exosome can carry circular RNAs (circRNAs) to regulate drug resistance via sponging microRNAs (miRNAs). miRNAs can control mRNA expression by regulate the interaction with 3’UTR and methylation. Nanog homeobox (NANOG) is an important biomarker for TMZ resistance. Hitherto, it is unknown about the role of exosomal hsa_circ_0072083 (circ_0072083) in TMZ resistance in glioma, and whether it is associated with NANOG via regulating miRNA sponge and methylation. Methods TMZ-resistant (n = 36) and sensitive (n = 33) patients were recruited. The sensitive cells and constructed resistant cells were cultured and exposed to TMZ. circ_0072083, miR-1252-5p, AlkB homolog H5 (ALKBH5) and NANOG levels were examined via quantitative reverse transcription polymerase chain reaction and western blot. The half maximal inhibitory concentration (IC50) of TMZ, cell proliferation, apoptosis, migration and invasion were analyzed via Cell Counting Kit-8, colony formation, flow cytometry, wound healing and transwell assays. The in vivo function was assessed using xenograft model. The N6-methyladenosine (m6A) level was analyzed via methylated RNA immunoprecipitation (MeRIP). Target relationship was investigated via dual-luciferase reporter assay and RNA immunoprecipitation. Warburg effect was investigated via lactate production, glucose uptake and key enzymes expression. Exosome was isolated and confirmed via transmission electron microscopy and specific protein expression. Results circ_0072083 expression was increased in TMZ-resistant glioma tissues and cells. circ_0072083 knockdown restrained the resistance of resistant cells via decreasing IC50 of TMZ, proliferation, migration, invasion and xenograft tumor growth and increasing apoptosis. circ_0072083 silence reduced NANOG expression via blocking ALKBH5-mediated demethylation. circ_0072083 could regulate NANOG and ALKBH5 via targeting miR-1252-5p to control TMZ resistance. Warburg effect promoted the release of exosomal circ_0072083 in resistant cells. Exosomal circ_0072083 from resistant cells increased the resistance of sensitive cells to TMZ in vitro and xenograft model. Exosomal circ_0072083 level was enhanced in resistant patients, and it had a diagnostic value and indicated a lower overall survival in glioma. Conclusion Exosomal circ_0072083 promoted TMZ resistance via increasing NANOG via regulating miR-1252-5p-mediated degradation and demethylation in glioma.

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LncRNA SNHG16 promotes pulmonary fibrosis by targeting miR-455-3p to regulate the Notch2 pathway

Respiratory Research ◽

10.1186/s12931-021-01632-z ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Panpan Liu ◽

Lei Zhao ◽

Yuxia Gu ◽

Meilan Zhang ◽

Hongchang Gao ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Lung Fibrosis ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Interstitial Lung Diseases ◽

Luciferase Reporter ◽

Qrt Pcr ◽

Rna Immunoprecipitation ◽

And Migration

Abstract Background Idiopathic pulmonary fibrosis (IPF) is the most common interstitial lung diseases with a poor prognosis. Long non-coding RNAs (lncRNAs) have been reported to be involved in IPF in several studies. However, the role of lncRNA SNHG16 in IPF is largely unknown. Methods Firstly, experimental pulmonary fibrosis model was established by using bleomycin (BML). Histology and Western blotting assays were used to determine the different stages of fibrosis and expression of several fibrosis biomarkers. The expression of SNHG16 was detected by quantitative real-time polymerase chain reaction (qRT‐PCR). EdU staining and wound-healing assay were utilized to analyze proliferation and migration of lung fibroblast cells. Molecular mechanism of SNHG16 was explored by bioinformatics, dual-luciferase reporter assay, RNA immunoprecipitation assay (RIP), and qRT-PCR. Results The expression of SNHG16 was significantly up-regulated in bleomycin-(BLM) induced lung fibrosis and transforming growth factor-β (TGF-β)-induced fibroblast. Knockdown of SNHG16 could attenuate fibrogenesis. Mechanistically, SNHG16 was able to bind and regulate the expression of miR-455-3p. Moreover, SNHG16 also regulated the expression of Notch2 by targeting miR-455-3p. Finally, SNHG16 could promote fibrogenesis by regulating the expression of Notch2. Conclusion Taken together, our study demonstrated that SNHG16 promoted pulmonary fibrosis by targeting miR-455-3p to regulate the Notch2 pathway. These findings might provide a novel insight into pathologic process of lung fibrosis and may provide prevention strategies in the future.

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MiR-489-3p inhibits cell proliferation, migration, and invasion, and induces apoptosis, by targeting the BDNF-mediated PI3K/AKT pathway in glioblastoma

Open Life Sciences ◽

10.1515/biol-2020-0024 ◽

2020 ◽

Vol 15 (1) ◽

pp. 274-283

Author(s):

Bo Zheng ◽

Tao Chen

Keyword(s):

Cell Proliferation ◽

Luciferase Reporter ◽

Akt Pathway ◽

Migration And Invasion ◽

Bdnf Mrna ◽

Protein Levels ◽

Rna Immunoprecipitation ◽

Underlying Mechanisms ◽

Induced Apoptosis ◽

Protein Cell

AbstractAmong astrocyte tumors, glioblastoma (GBM) is the most malignant glioma, highly aggressive and invasive, with extremely poor prognosis. Previous research has reported that microRNAs (miRNAs) participate in the progression of many cancers. Thus, this study aimed to explore the role and the underlying mechanisms of microRNA (miR)-489-3p in GBM progression. The expression of miR-489-3p and brain-derived neurotrophic factor (BDNF) mRNA was measured by quantitative real-time polymerase chain reaction. Western blot analysis was used to detect BDNF protein and the PI3K/AKT pathway-related protein. Cell proliferation, apoptosis, migration, and invasion were analyzed using CKK-8 assay, flow cytometry, and transwell assay, respectively. The interaction between BDNF and miR-489-3p was explored by luciferase reporter assay and RNA immunoprecipitation (RIP) assay. MiR-489-3p was down-regulated and BDNF was up-regulated in GBM tissues and cells. MiR-489-3p re-expression or BDNF knockdown inhibited GBM cell proliferation, migration, and invasion, and promoted apoptosis. BDNF was a target of miR-489-3p, and BDNF up-regulation reversed the effects of miR-489-3p on GBM cells. The protein levels of p-AKT and p-PI3K were notably reduced in GBM cells by overexpression of miR-489-3p, but were rescued following BDNF up-regulation. Therefore, miR-489-3p inhibited proliferation, migration, and invasion, and induced apoptosis, by targeting the BDNF-mediated PI3K/AKT pathway in GBM, providing new strategies for clinical treatment of GBM.

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LncRNA NEAT1 Enhances Glioma Progression via Regulating the miR-128-3p/ITGA5 Axis

Molecular Neurobiology ◽

10.1007/s12035-021-02474-y ◽

2021 ◽

Author(s):

Jiakai Chen ◽

Handong Wang ◽

Junjun Wang ◽

Wenhao Niu ◽

Chulei Deng ◽

...

Keyword(s):

Flow Cytometric Analysis ◽

Biological Functions ◽

Quantitative Real Time Pcr ◽

Flow Cytometric ◽

Non Coding Rna ◽

Rna Immunoprecipitation ◽

Human Gbm ◽

Novel Strategy ◽

Lncrna Neat1 ◽

Long Non Coding Rna

AbstractAccumulating evidences indicate that long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) promotes the progression of glioma. In this study, we postulated that NEAT1 may act as a miR-128-3p sponge. Relative levels of NEAT1 and miR-128-3p expression in human glioma samples and GBM cells were detected using quantitative real-time PCR. By means of CCK-8 assays, transwell assays, and flow cytometric analysis, the biological functions of miR-128-3p and NEAT1 were investigated in U87MG and U251MG human GBM cell lines with stable miR-128-3p and NEAT1 knockdown or overexpression. The luciferase reports, RNA pull-down assay, and RNA immunoprecipitation assay were conducted to determine the relevance of NEAT1 and miR-128-3p in glioma. As a result, high expression of NEAT1 and lack of miR-128-3p were observed in glioma specimens and cells. By binding to anti-oncogene miR-128-3p in the nucleus, NEAT1 enhanced tumorigenesis and glioma development. Further experiments suggested that ITGA5 expression was increased in glioma tissues and was found to be connected with miR-128-3p. Additionally, NEAT1 facilitated ITGA5 expression via competitively binding to miR-128-3p. For this reason, ITGA5 would not be decomposed by miR-128-3p and could activate FAK signaling pathway, thereby promoting cell growth. Collectively, these results indicated that the NEAT1/miR-128-3p/ITGA5 axis was involved in glioma initiation and progression, and might offer a potential novel strategy for treatment of glioma.

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Hypoxic TAM-derived exosomal miR-155-5p promotes RCC progression through HuR-dependent IGF1R/AKT/PI3K pathway

Cell Death Discovery ◽

10.1038/s41420-021-00525-w ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Wenyu Gu ◽

Linjing Gong ◽

Xu Wu ◽

Xudong Yao

Keyword(s):

Mrna Stability ◽

Xenograft Model ◽

Pi3k Pathway ◽

Clinicopathological Parameters ◽

Loss Of Function ◽

Cell Renal Cell Carcinoma ◽

Rna Immunoprecipitation ◽

Hypoxic Tumor ◽

Lipid Bilayer Vesicles

AbstractHypoxic tumor-associated macrophages (TAMs) are related to poor prognosis of patients with clear cell renal cell carcinoma (ccRCC). Exosomes are small lipid-bilayer vesicles that implicated in tumor progression and metastasis. However, whether hypoxic TAM-derived exosomes affect RCC progression within the hypoxic tumor microenvironment has not been elucidated. GSE analysis identified miR-155-5p was upregulated in RCC. Moreover, we quantified levels of miR-155-5p using RT-qPCR, performed immunohistochemical staining in 79 pairs of primary RCC specimens and related them to clinicopathological parameters. Higher miR-155-5p levels were related to more CD163 + TAM infiltration and elevated HIF-1a expression in our cohort. In the in vitro studies, we initially purified and characterized the exosomes from the supernatant of TAMs subjected to normoxia or hypoxia, and then transfected antagomiR-155-5p or control into these TAMs to produce corresponding exosomes. Gain and loss-of-function studies further investigated the effect of transferred hypoxic exosomal miR-155-5p on the cross-talk between TAMs and RCC cells in xenograft model and in vitro co-culture experiments. The results of RNA immunoprecipitation analyses elucidated that miR-155-5p could directly interact with human antigen R (HuR), thus increasing IGF1R mRNA stability. Mechanistically, hypoxic TAM-Exo transferred miR-155-5p promoted RCC progression partially through activating IGF1R/PI3K/AKT cascades. Taken together, transfer of miR-155-5p from hypoxic TAMs by exosomes to renal cancer cells explains the oncogenic manner, in which M2 macrophages confer the malignant phenotype to RCC cells by enhancing HuR-mediated mRNA stability of IGF1R.

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Quantitative profiling of N6-methyladenosine at single-base resolution in stem-differentiating xylem of Populus trichocarpa using Nanopore direct RNA sequencing

Genome Biology ◽

10.1186/s13059-020-02241-7 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Yubang Gao ◽

Xuqing Liu ◽

Bizhi Wu ◽

Huihui Wang ◽

Feihu Xi ◽

...

Keyword(s):

Rna Sequencing ◽

Populus Trichocarpa ◽

Alternative Polyadenylation ◽

High Accuracy ◽

Single Base ◽

Rna Immunoprecipitation ◽

Single Base Resolution ◽

Single Transcript ◽

Quantitative Profiling ◽

Identification And Quantification

AbstractThere are no comprehensive methods to identify N6-methyladenosine (m6A) at single-base resolution for every single transcript, which is necessary for the estimation of m6A abundance. We develop a new pipeline called Nanom6A for the identification and quantification of m6A modification at single-base resolution using Nanopore direct RNA sequencing based on an XGBoost model. We validate our method using methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and m6A-sensitive RNA-endoribonuclease–facilitated sequencing (m6A-REF-seq), confirming high accuracy. Using this method, we provide a transcriptome-wide quantification of m6A modification in stem-differentiating xylem and reveal that different alternative polyadenylation (APA) usage shows a different ratio of m6A.

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Circular RNA circCSPP1 knockdown attenuates doxorubicin resistance and suppresses tumor progression of colorectal cancer via miR-944/FZD7 axis

Cancer Cell International ◽

10.1186/s12935-021-01855-6 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Lanlan Xi ◽

Quanlin Liu ◽

Wei Zhang ◽

Linshan Luo ◽

Jingfeng Song ◽

...

Keyword(s):

Colorectal Cancer ◽

Tumor Growth ◽

Circular Rna ◽

Luciferase Reporter ◽

Circular Rnas ◽

Protein Levels ◽

Cell Progression ◽

Rna Immunoprecipitation ◽

Induced Apoptosis

Abstract Background Circular RNAs (circRNAs) have been reported to play vital roles in colorectal cancer (CRC). However, only a few circRNAs have been experimentally validated and functionally described. In this research, we aimed to reveal the functional mechanism of circCSPP1 in CRC. Methods 36 DOX sensitive and 36 resistant CRC cases participated in this study. The expression of circCSPP1, miR-944 and FZD7 were detected by quantitative real time polymerase chain reaction (qRT-PCR) and the protein levels of FZD7, MRP1, P-gp and LRP were detected by western blot. Cell proliferation, migration, invasion, and apoptosis were assessed by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay, transwell assay, or flow cytometry analysis, respectively. The interaction between miR-944 and circCSPP1 or frizzled-7 (FZD7) was predicted by Starbase 3.0 and verified by the dual luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull down assay. Xenograft tumor assay was performed to examine the effect of circCSPP1 on tumor growth in vivo. Results The expression of circCSPP1 and FZD7 was upregulated while miR-944 expression was downregulated in doxorubicin (DOX)-resistant CRC tissues and cells. CircCSPP1 knockdown significantly downregulated enhanced doxorubicin sensitivity, suppressed proliferation, migration, invasion, and induced apoptosis in DOX-resistant CRC cells. Interestingly, we found that circCSPP1 directly downregulated miR-944 expression and miR-944 decreased FZD7 level through targeting to 3′ untranslated region (UTR) of FZD7. Furthermore, circCSPP1 mediated DOX-resistant CRC cell progression and doxorubicin sensitivity by regulating miR-944/FZD7 axis. Besides, circCSPP1 downregulation dramatically repressed CRC tumor growth in vivo. Conclusion Our data indicated that circCSPP1 knockdown inhibited DOX-resistant CRC cell growth and enhanced doxorubicin sensitivity by miR-944/FZD7 axis, providing a potential target for CRC therapy.

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LncRNA OIP5-AS1 accelerates ox-LDL-treated HUVECs injury by NF-κB pathway via miR-30c-5p

Clinical Hemorheology and Microcirculation ◽

10.3233/ch-211130 ◽

2021 ◽

pp. 1-12

Author(s):

Lei Zhang ◽

Qiulai Li ◽

Yanxia Chen ◽

Qiao Zhu

Keyword(s):

Oxidative Stress ◽

Vascular Endothelial Cells ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Vital Role ◽

Cell Counting ◽

Luciferase Reporter ◽

Sod Activity ◽

Interacting Protein ◽

Rna Immunoprecipitation

BACKGROUND: Oxidized low-density lipoprotein (ox-LDL) could induce endothelial injury and played a vital role in the progression and development of atherosclerosis. This study aimed to investigate the role of Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) in ox-LDL-induced human umbilical vascular endothelial cells (HUVECs) injury and the potential mechanisms. METHODS: Cell proliferation and apoptosis were evaluated by Cell Counting Kit-8 (CCK-8) assay and flow cytometry assay, respectively. The levels of lactate dehydrogenase (LDH), reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and nitric oxide (NO) were detected by corresponding detection kits, respectively. Quantitative real-time PCR (qRT-PCR) was conducted to measure the expression of OIP5-AS1 or microRNA-30c-5p (miR-30c-5p) in HUVECs. Binding between OIP5-AS1 and miR-30c-5p was predicted through bioinformatics analysis and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP). Western blot was used to analyze p-IκB, IκB, p-p65 and p65 levels. RESULTS: In HUVECs, exposure to ox-LDL led to a decrease in cell viability and an increase in LDH release and apoptosis with concomitant enhancement of oxidative stress, as evidenced by increased ROS and MDA generation, as well as decreased SOD activity and NO levels, while OIP5-AS1 knockdown or miR-30c-5p upregulation could rescue these effects above. Mechanically, OIP5-AS1 functioned as a sponge of miR-30c-5p. OIP5-AS1-induced injury and apoptosis, oxidative stress and activation of NF-κB pathway were reversed by miR-30c-5p in ox-LDL-treated HUVECs. CONCLUSION: OIP5-AS1 contributed to ox-LDL-treated HUVECs injury by activation of NF-κB pathway via miR-30c-5p.

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