RNA Binding Protein Immunoprecipitation Assay Research Articles

MiR-302a-3p Promotes Radiotherapy Sensitivity of Hepatocellular Carcinoma by Regulating Cell Cycle via MCL1.

Background The relationship between tumor suppressor gene miR-302a-3p and radiotherapy for hepatocellular carcinoma (HCC) remains unclear. This study intended to illustrate the molecular mechanism how miR-302a-3p regulated radiotherapy sensitivity of HCC. Methods miR-302a-3p expression in HCC tissues and cells was examined by qRT-PCR. The effect of miR-302a-3p on HCC radiotherapy sensitivity were detected by CCK-8, colony formation, and flow cytometry assays. The expression levels of cell cycle-related proteins were detected by Western blot. The influence of miR-302a-3p on radiotherapy sensitivity of HCC was further investigated via cell cycle inhibitor (Caudatin) treatment. The target gene (MCL1) of miR-302a-3p was obtained by bioinformatics analysis, and their binding relationship was confirmed by RNA-binding protein immunoprecipitation assay. The mechanisms of miR-302a-3p regulating cell cycle and affecting radiotherapy sensitivity of HCC cells through MCL1 were further explored through the rescue experiments. Results miR-302a-3p expression was remarkably reduced in radiotherapy-resistant tissues and cells of HCC. miR-302a-3p overexpression restored sensitivity of radiotherapy-resistant HCC cells to radiotherapy. Treatment with cell cycle inhibitor Caudatin could reverse suppressive effect of miR-302a-3p downregulation on sensitivity of HCC to radiotherapy. Additionally, miR-302a-3p could restrain MCL1 expression. In vitro cell assays further revealed that miR-302a-3p/MCL1 axis could enhance radiotherapy sensitivity of HCC cells by inducing G0/G1 arrest. Conclusions miR-302a-3p facilitated radiotherapy sensitivity of HCC cells by regulating cell cycle via MCL1, which provided a new underlying target for radiotherapy resistance of HCC patients.

MiR-654-3p, reduced by the excessive ALKBH5, Alleviated the Inflammation in OA by targeting TNFRSF9, the trigger of the NF-κB pathway

MicroRNA (miRNA) is one of the most potent therapeutic targets for osteoarthritis (OA). We identified that miR-654-3p protected the phenotype of chondrocytes. We demonstrated that TNF receptor superfamily member 9 (TNFRSF9) was the target of miR-654-3p by binding to its 3′UTR regions, based on a dual-luciferase reporter assay and an RNA binding protein immunoprecipitation (RIP) assay. In addition, further experiments proved that TNFRSF9, as a trigger of the NF-κB pathway, correlated with the inflammation in chondrocytes. MiR-654-3p overexpressed in the knee of mice alleviated the OA in vivo. Moreover, we examined the m6A enzyme level in OA, proving that the abnormal expression of α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) contributed to the miR-654-3p decrease. Our research illustrated the significant role of miR-654-3p in OA, including its maturation and the mechanism in protecting the phenotype of chondrocytes, which could be a new treatment target for OA.

A novel long non-coding RNA, lnc-RNU12, influences the T-cell cycle via c-JUN and CCNL2 in rheumatoid arthritis.

Long non-coding RNAs (lncRNAs) play important roles in RA pathogenesis. However, specific lncRNAs that regulate gene expression in RA pathogenesis are poorly known. This study was undertaken to characterize a novel lncRNA (lnc-RNU12) that has a lower-than-normal expression level in RA patients. We performed initial genome-wide lncRNA microarray screening in peripheral blood mononuclear cells from 28 RA cases and 18 controls. Multiple methods were used to validate the detected associations between lncRNAs and RA. Furthermore, we identified the source and characteristics of the highlighted lncRNAs, detected the target genes, and determined the functional effect on immune cells through lncRNA knock-down in Jurkat T cell lines. lnc-RNU12 was downregulated in peripheral blood mononuclear cells and T cell subtypes of RA patients and was genetically associated with RA risk. lnc-RNU12 mediates the effect of microbiome alterations on RA risk. Activation of T cells caused low expression of lnc-RNU12. Knock-down of lnc-RNU12 in Jurkat T cells caused cell cycle S-phase arrest and altered the expression of protein-coding genes related to the cell cycle and apoptosis (e.g. c-JUN, CCNL2, CDK6, MYC, RNF40, PKM, VPS35, DNAJB6 and FLCN). Finally, c-JUN and CCNL2 were identified as target genes of lnc-RNU12 at the mRNA and protein expression levels. RNA-binding protein immunoprecipitation assays verified the interaction between lnc-RNU12 and the two proteins (c-Jun and cyclin L2) in Jurkat cells. Our study suggested that lnc-RNU12 was involved in the pathogenesis of RA by influencing the T cell cycle by targeting c-JUN and CCNL2.

Long noncoding RNA-LINC00478 promotes the progression of clear cell renal cell carcinoma through PBX3.

Long noncoding RNAs play an important regulatory role in the development and progression of tumors. Our study found that LINC00478 was upregulated in clear cell renal cell carcinoma (ccRCC), so we made an in-depth exploration into its mechanism. In Caki-2 cells, we established the oe-LINC00478 cell line overexpressing LINC00478, and established underexpressing sh-LINC00478 cell line by short hairpinRNA silencing. The abilities of oe-LINC00478 cell invasion and metastasis were significantly enhanced, and the cell proliferative potential was also improved. The cellular expressions of PBX3, CDCA8, and CDK2 were upregulated, while in the sh-LINC00478 cells, the proliferative potential and metastatic and invasive abilities were weakened. Similarly, we established the PBX3-overexpressing oe-PBX3 cell line and the PBX3-underexpressing sh-PBX3 cell line, finding that the PBX3 overexpression enhanced the metastatic and invasive abilities of Caki-2 cells. When we overexpressed LINC00478 in PBX3-knockout Caki-2-PBX3- / - cells, no significant changes were noted in the metastatic or invasive ability. Through RNA pull-down and RNA-binding protein immunoprecipitation assays, we found that LINC00478 could facilitate the transcription-translation processes of PBX3 by binding to it, thus further promoting the expression of downstream cyclins to exert its action. In animal experimentation, the oe-LINC00478 and sh-LINC00478 Caki-2 cells were separately seeded, revealing that the tumor volume was significantly larger in the oe-LINC00478 group than in the sh-LINC00478 group. This study finds that by promoting the PBX3 transcription, LINC00478 can further regulate the expressions of downstream cyclins, thereby facilitating the metastasis and invasion of ccRCC.

Long Non-Coding RNA AC008972.1 as a Novel Therapeutic Target for Prostate Cancer.

Background: Prostate cancer is a common male malignancy and the leading cause of cancer death in men. Long non-coding RNAs (lncRNAs), microRNA (miRNAs) and mRNAs networks mediate prostate cancer progression. Herein, we investigated the functions of lncRNA AC008972.1 and its regulatory mechanism in prostate cancer. Materials and Methods: The expression levels of lncRNA AC008972.1, miR-143-3p, and TAOK2 were detected in prostate cancer tissues and cell lines by reverse transcription-quantitative polymerase chain reaction. PC3 and LNCaP cells were used to establish lncRNA AC008972.1-knockdown, miR-143-3p-overexpressing, and thousand-and-one-amino acid 2 kinase (TAOK2)-downregulated cells. Cell viability was examined by MTT assays and cell proliferation was detected by clone formation assay. Cell migration and invasion were detected by wound scratch assay and transwell chamber assay. The apoptosis rate was analyzed by flow cytometry. The protein expression was detected by Western blot assay. The RNA interaction was explored and validated by RNA binding protein immunoprecipitation (RIP) assay and dual luciferase activity assay. A mouse xenograft model was established to investigate the effect of lncRNA AC008972.1 on prostate cancer progression. Results: High expression of lncRNA AC008972.1 was associated with low overall survival in prostate cancer patients. Downregulation of lncRNA AC008972.1 suppressed prostate cancer progression by inhibiting cell viability, proliferation, migration, and invasion, in addition to the EMT process, whereas cell apoptosis was significantly promoted. LncRNA AC008972.1 bound with miR-143-3p and negatively regulated miR-143-3p expression. MiR-143-3p overexpression suppressed prostate cancer malignant behaviors in vitro. TAOK2 expression was decreased by miR-143-3p through the complementary targeting of TAOK2 mRNA. Downregulation of lncRNA AC008972.1 mitigated prostate cancer malignant behaviors in vitro based on miR-143-3p/TAOK2 node. Furthermore, the data of xenograft model experiment showed that inhibition of lncRNA AC008972.1 suppressed tumor growth in vivo. Conclusions: Knockdown of lncRNA AC008972.1 inhibits prostate cancer cell growth via downregulation of TAOK2 induced by miR-143-3p. LncRNA AC008972.1 acts as an oncogene in the progression of prostate cancer and may provide a novel therapeutic target for prostate cancer.

LncRNA OIP5-AS1 Mitigates Bupivacaine-Induced Neurotoxicity in Dorsal Root Ganglion Neurons Through Regulating NFAT5 Expression via Sponging miR-34b.

Bupivacaine (BUP), which is widely used in anesthesia, can cause neurotoxicity and neurological abnormalities. This work intended to study the function of long non-coding RNA (lncRNA) OIP5 antisense RNA 1 (OIP5-AS1) in BUP-triggered neurotoxicity. OIP5-AS1, microRNA (miR)-34b, and nuclear factor of activated T cells 5 (NFAT5) levels were examined via real-time quantitative PCR (RT-qPCR). Cell proliferation, caspase-3 activity, and apoptosis were assessed via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), caspase-3 activity, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. The regulatory relationships between miR-34b and OIP5-AS1 or NFAT5 were validated via RNA binding protein immunoprecipitation (RIP) and dual-luciferase reporter assays. Our data demonstrated that OIP5-AS1 and NFAT5 levels were downregulated and miR-34b was upregulated upon exposure to BUP. Functional assays implied that the OIP5-AS1 deficiency impeded cell proliferation and enhanced the apoptosis of DRG neurons, while OIP5-AS1 addition reversed these changes. Moreover, OIP5-AS1 could bind to miR-34b and OIP5-AS1 regulated BUP-induced neurotoxicity via miR-34b. Besides, miR-34b could directly interact with NFAT5. Augmentation of miR-34b impeded cell proliferation and expedited the apoptosis and caspase-3 activity, while NFAT5 addition neutralized these impacts. Finally, it was verified that OIP5-AS1 could upregulate NFAT5 through sponging miR-34b. In sum, our results disclosed that OIP5-AS1 ameliorated BUP-caused neurotoxicity via regulating the miR-34b/NFAT5 axis, suggesting that OIP5-AS1 might be a promising therapeutic target for the treatment of BUP-induced neurotoxicity.

Long Noncoding RNA PSMA3 Antisense RNA 1 Promotes Cell Proliferation, Migration, and Invasion in Pancreatic Ductal Adenocarcinoma Via Targeting MicroRNA-154-5p to Positively Modulate Karyopherin Subunit Alpha 4

Pancreatic ductal adenocarcinoma (PDAC), as the most frequent pancreatic tumor, featuring high death rate. The current study intends to explore the biological role of PSMA3 antisense RNA 1 (PSMA3-AS1) and its mechanism underlying PDAC progression. Expression analyses were conducted using quantitative reverse transcription-polymerase chain reaction. Proliferative, apoptotic, migratory, and invasive capacities were determined by functional assays, encompassing 5-ethynyl-2'-deoxyuridine, colony formation, JC-1, terminal deoxynucleotidyl transferase dUTP nick-end labeling, and transwell assays in PDAC cells. The RNA-binding protein immunoprecipitation, RNA pulldown, and luciferase reporter assays uncovered the biological and regulatory role of PSMA3-AS1 in PDAC. Long noncoding RNA PSMA3-AS1 was aberrantly overexpressed in PDAC cells. Downregulated PSMA3-AS1 repressed cell proliferative, migratory, and invasive capacities and propelled cell apoptosis of PDAC. MicroRNA-154-5p (miR-154-5p) was proved to be targeted by PSMA3-AS1 in PDAC cells. Karyopherin subunit alpha 4 (KPNA4) was the downstream target messenger RNA of miR-154-5p. Karyopherin subunit alpha 4 knockdown hindered cell proliferation, migration, and invasion in PDAC. In rescue assays, KPNA4 overexpression or miR-154-5p interference counteracted the inhibitory influence of PSMA3-AS1 ablation on the progression of PDAC cells. Our results suggested that PSMA3-AS1 enhances PDAC cell proliferative, migratory, and invasive capacities via modulating miR-154-5p/KPNA4 axis.

ALKBH5-mediated m6A modification of circCCDC134 facilitates cervical cancer metastasis by enhancing HIF1A transcription

BackgroundMetastasis is the main cause of mortality in cervical cancer (CC). Circular RNAs (circRNAs) have been demonstrated to play a crucial role in carcinoma biology. However, the expression and function of circRNAs in cervical cancer metastasis are still unclear.MethodsIn the present study, we identified a circRNA with an N6-methyladenosine (m6A) modification, circCCDC134, whose expression was increased in CC tissues by circRNA-Seq and qPCR. CircCCDC134 upregulation in CC was fine-tuned by ALKBH5-mediated m6A modification, which enhanced its stability in a YTHDF2-dependent manner. The functional experiments illustrated that circCCDC134 enhanced tumour proliferation and metastasis in vitro and in vivo. For the comprehensive identification of RNA-binding proteins, circRNA pull-down and mass spectrometry (ChIRP-MS), chromatin immunoprecipitation-seq (Chip-seq), RNA binding protein immunoprecipitation (RIP) and luciferase reporter assays were used to perform mechanistic investigations.ResultsThe results revealed that circCCDC134 recruited p65 in the nucleus and acted as a miR-503-5p sponge to regulate the expression of MYB in the cytoplasm, ultimately stimulating HIF1A transcription and facilitating CC growth and metastasis. Conclusion: These findings indicate that circCCDC134 is an important therapeutic target and provide new regulatory model insights for exploring the carcinogenic mechanism of circCCDC134 in CC.

RNA binding protein PUM2 promotes hepatocellular carcinoma proliferation and apoptosis via binding to the 3'UTR of BTG3.

Pumilio homolog 2 (PUM2) is an RNA-binding protein that functions as an oncogene in various types of cancer. However, its role in hepatocellular carcinoma (HCC) has remained to be fully elucidated. In the present study, the role of PUM2 was investigated in HCC and its regulation was assessed by examining its binding to the 3′-untranslated region (UTR) of B-cell translocation gene 3 (BTG3). The expression levels of PUM2 were determined in datasets from the UALCAN and Cancer Cell Line Encyclopedia databases. Furthermore, Gene Expression Profiling Interactive Analysis was used to analyze overall survival in patients with HCC. Reverse transcription-quantitative PCR (RT-qPCR) and western blot analyses were then performed to detect the expression levels of PUM2 and BTG3 in HCC cells. Cell proliferation was assessed using Cell Counting Kit-8 and colony-formation assays. The induction of cell apoptosis was evaluated using TUNEL and western blotting assays. StarBase and RNA-Protein Interaction Prediction were used to determine the possible direct interaction between PUM2 and BTG3. The interaction between PUM2 and BTG3 was then verified by luciferase reporter and RNA-binding protein immunoprecipitation assays. The results indicated that PUM2 expression was upregulated in HCC tissues and cells and that it was associated with the prognosis of patients with HCC. PUM2 silencing inhibited the proliferation and promoted the apoptosis of Huh-7 cells. In addition, PUM2 was confirmed to directly bind to the 3′UTR of BTG3. Downregulation of BTG3 reversed the effects of PUM2 silencing on cell proliferation and apoptosis in Huh-7 cells. Collectively, the results suggested that PUM2 regulated HCC cell proliferation and apoptosis via interacting with BTG3, which may provide a novel therapeutic strategy for the treatment of human HCC.

LncRNA OSTM1-AS1 acts as an oncogenic factor in Wilms' tumor by regulating the miR-514a-3p/MELK axis.

Wilms' tumor (WT) is the most typical basic renal tumor in children and is associated with a high recurrence rate and improper diagnosis. Long noncoding RNAs (lncRNAs) play important roles in WT development. However, the impact of the OSTM1 antisense RNA 1 (OSTM1-AS1) lncRNA on WT remains largely unexplored. Differential expression of OSTM1-AS1, miR-514a-3p and maternal embryonic leucine zipper kinase (MELK) in mice with WT cells was assessed via quantitative reverse transcription-PCR and western blotting. Changes in the proliferation, migration and apoptosis of WT cells after OSTM1-AS1, miR-514a-3p or MELK knockdown were assessed using the cell counting kit-8, Transwell and caspase-3 activity assays, respectively. Additionally, the tumorigenicity of WT cells after OSTM1-AS1 knockdown in vivo was analyzed using a xenograft tumor assay. The association among OSTM1-AS1, MELK and miR-514a-3p was confirmed using the RNA binding protein immunoprecipitation and luciferase reporter assays. OSTM1-AS1 and MELK were upregulated in WT cells, whereas miR-514a-3p was downregulated. OSTM1-AS1 was mostly observed in the cytoplasm, and its knockout suppressed WT cell migration and proliferation in vitro , triggered apoptosis and attenuated tumor development in vivo . MiR-514a-3p was sponged by OSTM1-AS1, and miR-514a-3p interference counteracted the tumoricidal effect of OSTM1-AS1 knockdown. MiR-514a-3p reduced WT progression by downregulating the expression of MELK, which is the target gene of miR-514a-3p. lncRNA OSTM1-AS1 acts as an oncogenic factor in WT by releasing MELK through sponging miR-514a-3p and could be a useful target for WT diagnosis and therapy.

The long noncoding RNA LINC00200 promotes the malignant progression of MYCN-amplified neuroblastoma via binding to insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) to enhance the stability of Zic family member 2 (ZIC2) mRNA

ObjectivesNeuroblastoma (NB) is one of the most common extracranial malignant tumors in children and remarkable heterogeneous tumor of the sympathetic nervous system. Long noncoding RNAs (lncRNAs) have been reported to be vital biological roles in the initiation and malignant progression of tumors. The aim of this study was to explore the biological role and the possible molecular mechanism of LINC00200 in NB. Design & methodsThe expression level of LINC00200 in NB tissues and cell lines were detected by real-time quantitative PCR (qRT-PCR). The biological effects of LINC00200 on NB cell proliferation, migration and invasion were examined by EdU and transwell assays. The molecular mechanism of LINC00200 in NB were explored and verified by bioinformatics analysis, RNA binding protein immunoprecipitation (RIP) assay and RNA pull down assay. ResultsThe results showed that the expression of LINC00200 was significantly higher in NB tissues than in normal tissues. Besides, the expression of LINC00200 was higher in MYCN Amplified NB tissues than in MYCN non-Amplified NB tissues. Moreover, overexpression of LINC00200 could remarkedly promote proliferation, migration and invasion of NB cell. Mechanistically, LINC00200 might bind to RNA binding protein (RBP) IGF2BP3 and promote the expression of ZIC2. ConclusionsOverall, we showed that LINC00200 was upregulated in NB tissues and the LINC00200/IGF2BP3/ZIC2 regulatory axis might be the possible therapeutic target for NB.

Circ_0005699 participates in ox-LDL-induced human umbilical vein endothelial cell injury via targeting the miR-636/TLR4/NF-κB pathway

BackgroundThe vital roles of some circular RNA (circRNA) have been reported to be associated with atherosclerosis (AS). In this paper, we explored the function of circ_0005699 in AS. MethodsThe 3-(4, 5-dimethyl-2-thiazolyl)−2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay and 5-Ethynyl-2′-deoxyuridine (EdU) assay were applied to evaluate cell proliferation. The flow cytometry assay uncovered cell apoptosis. The ELISA and tube formation assay were implemented to assess cell inflammatory response and angiogenesis. The circ_0005699, microRNA-636 (miR-636) and toll-like receptor 4 (TLR4) abundances were measured by qRT-PCR and western blot in serum specimens and HUVECs. The Bcl2-associated X, apoptosis regulator (Bax), Cleaved-caspase-3, p-P65, P65, p-lkBα, and lkBα levels were quantified by western blot. The binding sites of miR-636 and circ_0005699 or TLR4 were uncovered by dual-luciferase reporter assay and RNA Binding Protein Immunoprecipitation (RIP) assay. ResultsCirc_0005699 and TLR4 abundances were up-regulated but miR-636 abundance was down-regulated in AS serum specimens and HUVECs with ox-LDL treatment. Circ_0005699 knockdown impaired ox-LDL-mediated repression of proliferation and angiogenesis, as well as promotion of apoptosis and inflammatory response in HUVECs. Circ_0005699 activated the NF-κB signaling via up-regulating TLR4 by functioning as a miR-636 sponge molecular. ConclusionCirc_0005699 participated in ox-LDL-induced HUVEC injury via miR-636/TLR4/NF-κB axis, which might provide different thinking to cure AS.

LINC00893 inhibits the progression of prostate cancer through miR-3173-5p/SOCS3/JAK2/STAT3 pathway

BackgroundProstate cancer (PCa) is one of the most common malignant tumors in the male urinary system. In recent years, the morbidity and mortality of PCa have been increasing due to the limited effects of existing treatment strategies. Long non-coding RNA (lncRNA) LINC00893 was reported to inhibit the proliferation and metastasis of papillary thyroid cancer cells, but its role in PCa has not been reported. This study aims to investigate the role and underlying mechanism of LINC00893 in regulating the progression of PCa cells.MethodsWe first compared LINC00893 expression levels between PCa tissues and normal prostate tissues through TCGA database. The relative LINC00893 expression levels were further validated in 66 pairs of PCa tissues and para-cancerous normal tissues, as well as in PCa cell lines. Gain-of-function experiment was performed by transfecting PCa cell with LINC00893 expression vector, and CCK (Cell count kit)-8, 5-Ethynyl-2′-deoxyuridine (EdU) incorporation, colony information and transwell assays were conducted to assess the functional phenotypes. Dual-luciferase reporter, RNA-binding protein immunoprecipitation (RIP) and RNA pull-down assays were performed to evaluate the molecular interactions.ResultsLINC00893 was downregulated in PCa tissues and cell lines, and patients with low expression of LINC00893 were associated with a poorer overall survival rate. LINC00893 overexpression hindered the proliferation, epithelial-mesenchymal transition (EMT) as well as the migratory ability of PCa cells, and suppressed the tumorigenesis of PCa cells in nude mice. We further demonstrated that LINC00893 acted as a sponge for miR-3173-5p and inhibited its activity, which in turn regulated the suppressor of cytokine signaling 3 (SOCS3)/Janus Kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling axis.ConclusionsOur study demonstrated that LINC00893 suppresses the progression of PCa cells through targeting miR-3173-5p/SOCS3/JAK2/STAT3 axis. Our data uncovers a novel tumor-suppressor role of LINC00893 in PCa, which may serve as a potential strategy for targeted therapy in PCa.Grapical

KIF26B-AS1 Regulates TLR4 and Activates the TLR4 Signaling Pathway to Promote Malignant Progression of Laryngeal Cancer.

Laryngeal cancer is one of the highest incidence, most prevalently diagnosed head and neck cancers, making it critically necessary to probe effective targets for laryngeal cancer treatment. Here, real-time quantitative reverse transcription PCR (qRT-PCR) and western blot analysis were used to detect gene expression levels in laryngeal cancer cell lines. Fluorescence in situ hybridization (FISH) and subcellular fractionation assays were used to detect the subcellular location. Functional assays encompassing Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), transwell and wound healing assays were performed to examine the effects of target genes on cell proliferation and migration in laryngeal cancer. The in vivo effects were proved by animal experiments. RNA-binding protein immunoprecipitation (RIP), RNA pulldown and luciferase reporter assays were used to investigate the underlying regulatory mechanisms. The results showed that KIF26B antisense RNA 1 (KIF26B-AS1) propels cell proliferation and migration in laryngeal cancer and regulates the toll-like receptor 4 (TLR4) signaling pathway. KIF26B-AS1 also recruits FUS to stabilize TLR4 mRNA, consequently activating the TLR4 signaling pathway. Furthermore, KIF26B-AS1 plays an oncogenic role in laryngeal cancer via upregulating TLR4 expression as well as the FUS/TLR4 pathway axis, findings which offer novel insight for targeted therapies in the treatment of laryngeal cancer patients.

Delta-like factor 1 negatively regulates angiogenesis as a target gene of miR-126-5p after indirect revascularization surgery in patients with moyamoya disease

Promoting endothelial cell (EC) proliferation and angiogenesis after indirect revascularization surgery is crucial for decreasing the stroke rate in moyamoya patients. However, the role of delta-like factor 1 (DLK1) in regulating EC proliferation in chronically ischaemic brains and the specific mechanisms remain unclear. Therefore, we compared the expression levels of DLK1 in the dura mater (DM) tissues of patients with moyamoya disease and patients with aneurysms, and dual luciferase reporter and RNA binding protein immunoprecipitation assays were conducted to determine whether DLK1 is a target gene of miR-126-5p. The effect of DLK1 on EC proliferation and the interaction between DLK1 and miR-126-5p were explored in vitro. Then, we established an animal model of two-vessel occlusion together with encephalo-myo-synangiosis (2VO+EMS). The temporalis muscles (TMs) of the animals were transfected with DLK1 lentiviral RNA (Lv-DLK1) and short hairpin RNA (sh-DLK1) to compare the DLK1 expression, angiogenesis (CD31 count), and numbers of vacuoles and impaired tight junctions in the ECs of TM-covered chronically ischaemic brains as well as the cognitive improvement in each group. DLK1 expression was lower in the DM tissues of moyamoya patients than in those of the aneurysm patients, and DLK1 was identified as a target gene of miR-126-5p. In vitro, DLK1 inhibited EC proliferation, migration and angiogenesis and exerted effects opposite those of miR-126-5p. In 2VO+EMS rats, compared to the control transfection, the TM transfection of Lv-DLK1 induced significantly higher DLK1 expression and worse angiogenesis in the TM-covered ischaemic brain as well as less extensive cognitive improvement, while the transfection of sh-DLK1 into the TM had the opposite effects. In summary, DLK1, a target gene of miR-126-5p, negatively regulates angiogenesis in chronically ischaemic brains and is expected to be a new target to improve the efficacy of indirect revascularization surgery and the prognosis of moyamoya patients.

Circ-FOXM1 promotes the proliferation, migration and EMT process of osteosarcoma cells through FOXM1-mediated Wnt pathway activation

BackgroundOsteosarcoma (OS) is a malignant bone tumor that commonly occurs in adolescents with a high mortality rate and frequent pulmonary metastasis. Emerging evidence has suggested that circular RNAs (circRNAs) are important regulators in multiple biological activities of carcinomas. Nevertheless, the role of circRNAs derived from forkhead box M1 (FOXM1), a well-accepted modulator of OS progression, has not been discussed in OS.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was utilized to test circ-FOXM1 (hsa_circ_0025033) expression in OS cell lines. Cell counting kit-8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), transwell assays and western blot analysis of epithelial-mesenchymal transition (EMT) markers were conducted to evaluate cell proliferation, apoptosis, migration, and EMT process. Luciferase reporter assay and RNA-binding protein immunoprecipitation (RIP) assay were utilized to detect the interaction of circ-FOXM1 and RNAs.ResultsHigh expression of circ-FOXM1 was detected in OS cell lines. Functionally, circ-FOXM1 knockdown inhibited the proliferation, migration and EMT process, whereas induced the apoptosis of OS cells. From the aspect of molecular mechanism, circ-FOXM1 was discovered to upregulate FOXM1 expression via sponging miR-320a and miR-320b, therefore activating Wnt signaling pathway. Besides, rescue experiments elucidated that circ-FOXM1 regulated cellular activities of OS cells via FOXM1. Further, in vivo assays supported that loss of circ-FOXM1 restrained OS tumor growth.ConclusionCirc-FOXM1 facilitated the malignant phenotypes of OS cells through FOXM1-mediated Wnt pathway activation, revealing circ-FOXM1 as a potential biomarker for OS treatment.

Attenuation of Piwil2 induced by hypoxic postconditioning prevents cerebral ischemic injury by inhibiting CREB2 promoter methylation.

Epigenetic modification contributes to the pathogenesis of cerebral ischemia. Piwil2 belongs to the PIWI proteins subfamily and has a key role in the regulation of gene transcription through epigenetics. However, the roles of Piwil2 in cerebral ischemia have not been investigated. In this study, we aim to elucidate the roles and the underlying molecular mechanisms of Piwil2 in ischemic tolerance induced by hypoxic postconditioning (HPC) against transient global cerebral ischemia (tGCI). We found that the expression of Piwil2 in CA1 was downregulated by HPC after tGCI. Silencing Piwil2 with antisense oligodeoxynucleotide (AS-ODN) in CA1 after tGCI decreased the expression of apoptosis-related proteins and exerted neuroprotective effects. Opposite results were observed after overexpression of Piwil2 induced by administration of Piwil2-carried lentivirus. Furthermore, we revealed differentially expressed Piwil2-interacting piRNAs in CA1 between HPC and tGCI groups by RNA binding protein immunoprecipitation (RIP) assay. Moreover, downregulating Piwil2 induced by HPC or AS-ODN after tGCI caused a marked reduction of DNA methyltransferase 3A (DNMT3A), which in turn abolished the tGCI-induced increase in the DNA methylation of cyclic AMP response element-binding 2 (CREB2), thus increasing mRNA and protein of CREB2. Finally, downregulating Piwil2 restored dendritic complexity and length, prevented the loss of dentritic spines, thereby improving cognitive function after tGCI. These data firstly reveal that Piwil2 plays an important part in HPC-mediated neuroprotection against cerebral ischemia through epigenetic regulation of CREB2.

Dihydroartemisinin Attenuates Hypoxia-Induced Pulmonary Hypertension Through the ELAVL2/miR-503/PI3K/AKT Axis.

:Dihydroartemisinin (DHA) is an active form of artemisinin extracted from the traditional Chinese medicine Artemisia annua, which is used to treat malaria. Previous studies have shown that DHA has a therapeutic effect on pulmonary hypertension (PH), but its specific mechanism has not been fully elucidated. In this study, a hypoxia-induced PH mouse model was established and DHA was administered as a therapeutic intervention. We measured hemodynamics and right ventricular hypertrophy and observed hematoxylin and eosin staining of lung tissue sections, proving the therapeutic effect of DHA on PH. Furthermore, cell counting kit-8 and 5-ethynyl-2′-deoxyuridine (EdU) cell proliferation assay kit were performed to examine cell proliferation of pulmonary artery smooth muscle cells cultured in hypoxia or in normoxia. Transwell migration chamber assay was performed to examine cell migration of the same cell model. Consistent with the therapeutic effect in vivo, DHA inhibited hypoxia-induced cell proliferation and migration. Through high-throughput sequencing of mouse lung tissue, we screened embryonic lethal abnormal vision-like 2 (ELAVL2) as a key RNA binding protein in PH. Mechanistically, DHA inhibited the proliferation and migration of pulmonary artery smooth muscle cells by promoting the expression of ELAVL2 and regulating the miR-503/PI3K/AKT pathway. The binding relationship between ELAVL2 and pre-miR-503 was verified by RNA binding protein immunoprecipitation assay. In conclusion, we first propose that DHA alleviates PH through the ELAVL2/miR-503/PI3K/AKT pathway, which may provide a basis for new therapeutic strategies of PH.

Withdrawal: "Long Non-coding RNA NEAT1 Drives the Development of Polycystic Ovary Syndrome via Sponging Multiple MicroRNAs" by Xia Sang and Yuzhen Zhang.

A line of evidences have validated that multiple microRNAs (including miR-16, miR-483 and miR-324-3p) were abnormally expressed in granulosa cells, which may be closely related with the pathogenesis of polycystic ovary syndrome (PCOS). Long-chain non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) has been proved to participate in the progression of various human diseases via regulating microRNAs, but its function in PCOS are not yet clear. In this work, RT-PCR was performed to detect NEAT1 expression in PCOS tissues, and human ovarian granulosa cell line KGN were taken as the model to construct the cell line with high expression and low expression of NEAT1. CCK-8 and flow cytometry were carried out to monitor cells proliferation and apoptosis, respectively. Besides, we performed luciferase reporter gene assay and RNA binding protein immunoprecipitation (RIP) assay to verify the interaction between NEAT1, miR-16, miR-483 and miR-324-3p. We demonstrated that NEAT1 was highly expressed in PCOS tissues and cells. Over-expressed NEAT1 promoted the proliferation and inhibited the apoptosis. Moreover, NEAT1 was negatively correlated with the expression levels of miR-16, miR-483 and miR-324-3p. Dual luciferase analysis and RIP assay confirmed that NEAT1 can specifically bind to miR-16, miR-483 and miR-324-3p, by which NEAT1 can reduce their expression. In conclusion, NEAT1 can promote the proliferation of ovarian granulosa cells and arrest apoptosis via impeding expressions of miR-16, miR-483 and miR-324-3p. Our research would shed new light on the molecular mechanism of ovarian granulosa cell disorder. This article is protected by copyright. All rights reserved.

A feedback loop between GATA2-AS1 and GATA2 promotes colorectal cancer cell proliferation, invasion, epithelial-mesenchymal transition and stemness via recruiting DDX3X

BackgroundColorectal cancer (CRC) is a common malignant tumor with a high risk of metastasis. Long non-coding RNAs (lncRNAs) have been reported to be implicated in cancer progression via regulating its nearby gene. Herein, we investigated the function of GATA binding protein 2 (GATA2) and lncRNA GATA2 antisense RNA 1 (GATA2-AS1) in CRC and the mechanism underlying their interaction.MethodsColony formation assay, flow cytometry analysis and transwell assay were implemented to detect cell proliferation, apoptosis and invasion. Western blot analysis and sphere formation assay were conducted to assess epithelial-mesenchymal transition (EMT) and cancer stemness of CRC cells. RNA pull down, RNA-binding protein immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP) and luciferase reporter assays were implemented to investigate the regulatory mechanism between GATA2-AS1 and GATA2.ResultsGATA2-AS1 and GATA2 were highly expressed in CRC cells. Knockdown of GATA2-AS1 and GATA2 impeded CRC cell proliferation, invasion, EMT and cancer stemness, and induced cell apoptosis. GATA2-AS1 expression was positively correlated with GATA2. GATA2-AS1 recruited DEAD-box helicase 3 X-linked (DDX3X) to stabilize GATA2 mRNA. GATA2 combined with GATA2-AS1 promoter to enhance GATA2-AS1 expression.ConclusionOur study confirmed that a feedback loop between GATA2-AS1 and GATA2 promotes CRC progression, which might offer novel targets for CRC treatment.